# Patent

## Title
Downregulation of SINE/ALU retrotransposon transcription to induce or restore proliferative capacity and/or pluripotency to a stem cell

## Abstract

In certain embodiments methods are provided for inducing and/or restoring and/or maintaining a non-senescent phenotype, or aspects thereof (e.g., proliferative capacity and/or pluripotency) in a mammalian cell. The methods typically involve reducing the level or activity of SINE/Alu retrotransposon transcripts in the cell in an amount sufficient to induce or restore proliferative capacity and/or pluripotency to said mammalian cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 National Phase of PCT/US2011/057140, filed on Oct. 20, 2011, which claims priority to and benefit of U.S. Ser. No. 61/406,954, filed on Oct. 26, 2010 and to U.S. Ser. No. 61/455,808, filed on Oct. 26, 2010 both of which are incorporated herein by reference in their entirety for all purposes.


STATEMENT OF GOVERNMENTAL SUPPORT
This invention was made with government support under Grant No: DE019608 awarded by the by the National Institutes of Health. The government has certain rights in this invention.


BACKGROUND
Adult stem cells are extremely important for long-term tissue homeostasis throughout life. Their self-renewing proliferative capacity involves numerous tightly coordinated processes to ensure preservation of genome integrity during cell division. The regulatory mechanisms underlying their aging are less well defined. Nonetheless, global gene expression studies of stem cells purified from young and old mice have implicated the involvement of epigenetic regulation in higher-order chromatin dynamics. These studies have suggested coordinated age-dependent regulation of chromosomal regions, chromatin remodeling activities and lineage specification genes (Chambers et al. (2007) PLoS Biol., 5: e201; Rossi et al. (2007) Exp. Gerontol., 42: 385-390; Rossi et al. (2005) Proc. Natl. Acad. Sci. USA, 102: 9194-9199).
All cells are constantly challenged by exogenous and endogenous sources of DNA damage; depending on the nature of the damage, they activate different DNA damage repair mechanisms (Sinclair et al. (2004) Am. Nat., 164: 396-414). In parallel, cells also activate checkpoint pathways, which delay cell cycle progression until genome integrity has been restored (Shiloh (2001) Curr. Opin. Genet. Dev., 11: 71-77). One aspect of the stem cell hypothesis of aging postulates that the gradual and coordinated age-related loss of DNA damage repair capacity results in DNA damage accumulation over time. This damage would pose a significant threat to adult stem cell survival by altering proliferation and differentiation patterns, ultimately triggering cellular senescence. Therefore, the ability of adult stem cells to monitor and faithfully repair DNA damage is key to the prevention of aging and neoplastic transformations.
Little is known about the precise relationship between chromatin and DNA-repair factors. More than 50% of the human genome consists of retrotransposons (Lander et al. (2001) Nature, 409: 860-921). Their epigenetic makeup is poorly understood and inadequately annotated at the genomic level, due to a high degree of sequence conservation. In fact, many retrotransposons are derived from ancestral RNA genes and might represent genetically active sequences that encode different types of RNA with yet unknown functions (McClintock (1956) Cold Spring Harb. Symp. Quant. Biol., 21: 197-216). However, clear evidence exists that the retrotransposal portion of the genome profoundly influences the organization, integrity, and evolution of the host's genome and transcriptome (Han et al. (2004) Nature, 429: 268-274; Kazazian (2004) Science, 303: 1626-1632). A growing body of evidence demonstrates that, during mammalian evolution, a large number of ancient retroelements acquired regulatory or structural functions.
The majority of retrotransposons are expressed in extraordinarily complex patterns in a cell- or tissue-specific manner, and potentially provide a rich source of non-protein coding RNAs to guide the trajectories of cellular differentiation and multicellular development (Amaral et al. (2008) Science 319: 1787-1789; Birney et al. (2007) Nature, 447: 799-816; Denoeud et al. (2007) Genome Res., 17: 746-759; Dinger et al. (2008) Genome Res., 18: 1433-1445; Dinger et al. (2008) J. Mol. Endocrinol., 40: 151-159; Emanuelsson et al. (2007) Genome Res., 17: 886-897; Faulkner et al. (2009) Nat. Genet., 41: 563-571; Lowe et al. (2007) Proc. Natl. Acad. Sci. USA, 104: 8005-8010; Mattick et al., (2009) Bioessays, 31: 51-59; Mercer et al. (2008) Proc. Natl. Acad. Sci. USA, 105: 716-721; Mikkelsen et al., ed. (2007) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells; Rozowsky et al. (2007) Genome Res., 17: 732-745; Trinklein et al. (2007) Genome Res., 17: 720-731). Recent studies have proven that retrotransposon transcriptional activities trigger and guide the processes of (i) assembly of centromeric chromatin, (ii) gene transcription, (iii) compartmentalization of chromatin and, (iv) nuclear organization of chromatin insulation during X-chromosome inactivation. Retrotransposons also serve a distinct function in non-random chromosomal translocations in tumors (Allen et al. (2004) Nat. Struct. Mol. Biol., 11: 816-821; Chueh et al. (2005) Hum. Mol. Genet., 14: 85-93; Lei and Corces (2006) Cell, 124: 886-888; Lei and Corces (2006) Nat. Genet., 38: 936-941; Lin et al. (2009) Cell, 139: 1069-1083; Lunyak (2008) Curr. Opin. Cell Biol., 20: 281-287; Lunyak et al. (2007) Science, 317: 248-251; Mattick et al. (2009) Bioessays, 31: 51-59; Navarro et al. (2009) Epigenetics Chromatin 2: 8).
There is also a considerable amount of tissue-specific, development-specific, and disease-related variability in DNA methylation and covalent modifications of chromatin within the retrotransposal portion of the genome (Kondo and Issa (2003) J. Biol. Chem. 278: 27658-27662; Estecio et al. (2007) PLoS ONE 2: e399). A genome-wide study by Martens (Martens et al. (2005) EMBO J., 24: 800-812) demonstrates that LINEs, SINE/Alus, and other interspersed retrotransposons have variable degrees of H3K9, H3K27, and H4K20 histone methylation, raising the possibility that posttranscriptional modifications (PTM) of retrotransposal chromatin can influence diverse cellular processes.
SUMMARY
Efficient repair of DNA double-strand breaks and authentic genome maintenance at the chromatin level are fundamental to faithful human adult stem cell self-renewal. Stem cell aging can be linked to deficiencies in these two processes. In one example, we report that  ̃65% of naturally occurring repairable damage in self-renewing adult stem cells occurs in transposable elements. Upregulation of transcriptional activity from SINE/Alu retrotransposons interferes with the recruitment of condensin I and cohesin complexes in pericentric chromatin, resulting in the loss of efficient DNA repair and, in turn, senescence. Stable knockdown of generic SINE/Alu transcripts in senescent human adult stem cells reinstates the cells self-renewing properties and unexpectedly increases their plasticity as manifested by upregulation of Nanog and Oct4. Our results demonstrate the functional significance of SINE/Alu retrotransposons and provide mechanistic insight into their novel role in mediating crosstalk between chromatin, DNA repair and aging of human adult stem cells.
In certain embodiments, methods are provided for restoring a non-senescent phenotype, or aspects of a non-senescent phenotype to a senescent cell (e.g., a senescent adult stem cell). In certain embodiments, methods are provided for maintaining a non-senescent phenotype, or aspects of a non-senescent phenotype in a cell (e.g., a senescent adult stem cell). In certain embodiments methods are provided for inducing and/or restoring and/or maintaining a non-senescent phenotype, or aspects thereof (e.g., proliferative capacity and/or pluripotency) in a mammalian cell. The methods typically involve reducing the level or activity of SINE/Alu retrotransposon transcripts in the cell in an amount sufficient to induce or restore proliferative capacity and/or pluripotency to said mammalian cell.
In certain embodiments methods of transdifferentiating a mammalian cell from a first cell type or lineage into a second cell type or lineage are provided. The method typically involves transforming a differentiated cell into a pluripotent cell (or restoring pluripotency to a stem cell or a progenitor cell) by one of the methods described herein (e.g., by reducing the level or activity of SINE/Alu retrotransposon transcripts in the cell); culturing the cell under conditions that induce or permit differentiation of the cell; selecting cells that have differentiated into the second cell type or lineage; and culturing the cells of said second cell type or lineage. In certain embodiments the first cell type is a mesodermal cell type. In certain embodiments the first cell type is an ectodermal cell type. In certain embodiments the first cell type is an endodermal cell type. In certain embodiments the first cell type or lineage is a mesodermal cell type and the second cell type or lineage is a neuroectodermal cell type. In certain embodiments the first cell type is an ectodermal cell type and the second lineage is a mesodermal or an endodermal cell type. In certain embodiments, the first cell type is an endodermal cell type and the second lineage is an ectodermal or mesodermal cell type. In certain embodiments the first cell type is an adipocyte or a bone marrow cell. In certain embodiments the second cell type is a cell type selected from the group consisting of a blood cell, a fetal cell, an epithelial cell, an adipocyte, a smooth muscle cell, a nerve cell, a pancreatic beta cell, and a cardiomyocyte. In certain embodiments the culturing the cell under conditions that induce or permit differentiation of said cell comprises culturing said cells in a medium lacking or having a reduced quantity of leukemia inhibitory factor (LIF) and/or contacting (or culturing) the cell with one or more reagents (e.g., retinoic acid, PDGF, insulin, Arctigenin, ATRA (vitamin A), boswellic acid, bromelain and other proteolytic enzymes, CAPE, flavonoids (including apigenin, luteolin, quercetin, genistein, and daidzein), emodin, EPA and DHA, monoterpenes, resveratrol, 1,25-D3 (vitamin D3)) that induce differentiation.
DEFINITIONS
Micro-RNAs are single-stranded RNAs of typically 22-nucleotides that are processed from  ̃70-nucleotide hairpin RNA precursors by the Rnase III nuclease, Dicer. Similar to siRNAs, miRNAs can silence gene activity through destruction of homologous mRNA in plants or blocking its translation in plants and animals.
shRNA or short hairpin RNA is an RNA molecule that contains a sense strand, antisense strand, and a short loop sequence between the sense and antisense fragments. Due to the complementarity of the sense and antisense fragments in their sequence, such RNA molecules tend to form hairpin-shaped double-stranded RNA (dsRNA). shRNA is cloned into a vector, allowing for expression by a pol III type promoter. The expressed shRNA is then exported into the cytoplasm where it is processed by dicer into siRNA which then get incorporated into the siRNA induced silencing complex (RISC).
Small Interfering RNA (siRNA) are typically 21-23 nucleotide double-stranded RNA molecules. Once incorporated into the RNA-induced silencing complex (RISC) they facilitate the cleavage and degradation of its recognized mRNA.
Piwi-interacting RNA (piRNA) is class of small non-coding RNA molecules that is expressed in, or can be introduced into animal cells (see, e.g., Seto et al. (2007) Molecular Cell, 26(5): 603-609; Siomi et al. (2011) Nat. Rev. Mol. Cell. Biol., 12:246-258). piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements.
By “pluripotency” and “pluripotent stem cells” it is meant that such cells have the ability to differentiate into all types of cells in an organism. Pluripotent cells are characterized by the expression of one or more pluripotency markers known by one of ordinary skill in the art. Such markers include, but are not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. In certain embodiments pluripontent cells are capable of forming or contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.
The terms “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cell cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture. For example primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary adipose cells of the present invention are maintained for fewer than 10 passages in vitro prior to use.
The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
By “adipose” is meant any fat tissue. The adipose tissue may be brown or white adipose tissue, derived from subcutaneous, omental/visceral, mammary, gonadal, or other adipose tissue site. In certain embodiments the adipose is subcutaneous white adipose tissue or visceral adipose tissue or a lipoaspirate sample. The adipose tissue may be from any organism having fat tissue. Preferably, the adipose tissue is mammalian, most preferably the adipose tissue is human. A convenient source of adipose tissue is from liposuction surgery, however, the source of adipose tissue need not be so limited.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C illustrate features of the ex-vivo aging properties of hADSCs. FIG. 1A: Representative cumulative long term growth curve. Three distinct states are shown: SR—self-renewing (population doubling <17); preSEN—presenescent (population doubling 29-38); SEN—senescent (population doubling >39). FIG. 1B: Immunohistochemical detection of senescence-associated J3-galactosidase (SA-J3-Gal) activity and 5′′-bromo-2′′deoxyuridine (BrdU) incorporation. Examples of hADSCs′ morphological changes (10× magnification) shown in inserts. Bar graphs correspond to percentage of SA-J3-Gal positive cells (left) and BrdU positive cells (right) with progressive ex-vivo hADSC expansion, based on three independent experiments. Error bars are standard deviations from the mean. FIG. 1C: DNA damage response (DDR) in senescent hADSCs. Representative immunostaining for persistent γH2AX (green)/53BP1 (red) foci formation upon senescence of hADSCs. Quantification of γH2AX/53BP1 foci formation upon ex-vivo expansion of hADSCs is given in FIG. 8.
FIGS. 2A-2G illustrate genome-wide location analysis of γH2AX. FIG. 2A: Schematic flow-chart for nucleosomal ChIP-seq analysis using SOLiD (ABI) platform. FIG. 2B: Relative chromosomal distributions of γH2AX tags in self-renewing and senescent cells illustrated for chromosomes 10 and 21. γH2AX tag enrichment levels, calculated as the log 2 ratio of position-specific tag counts normalized by the genomic background, are shown for self-renewing and senescent cells. Relative differences in γH2AX tag enrichment levels between cells, calculated as the absolute values of the differences in cell stage specific enrichment levels, are shown below the individual cell tracks. Below the difference tracks, the chromosomal locations of large clusters of γH2AX modified sites are shown for the self-renewing and senescent cell types. FIG. 2C: Differences in the relative γH2AX enrichment levels for self-renewing (SR) versus senescent (SEN) cells across various genomic features. The absolute values of the normalized differences in γH2AX tag counts between cell types are shown on the y-axis. Blue bars show genomic features that have higher fractions in SR cells, and red bars show genomic features that have higher fractions in SEN cells. FIG. 2D: Scatter plots showing the relationship between gene density and γH2AX tag density for human chromosomes in self-renewing (SR) and senescent cells (SEN). The slopes and intercepts of the linear trends are shown (y-value) along with Spearman's rank correlation coefficients (R-value) and statistical significance levels (P-value). FIG. 2E: Scatter plots showing the relationship between GC content and γH2AX tag density for human chromosomes in self-renewing (SR) and senescent cells (SEN). The slopes and intercepts of the linear trends are shown (y-value) along with Spearman's rank correlation coefficients (R-value) and statistical significance levels (P-value). FIG. 2F: The enrichment levels of γH2AX in promoter regions, surrounding transcriptional start sites (TSS), are shown for self-renewing (blue) and senescent (red) cells. Enrichment levels are calculated as the log 2 ratio of the position-specific tag counts normalized to the genomic background. FIG. 2G: The numbers of genes with γH2AX modified sites in promoter regions, defined as ±2 Kb from transcription start sites, are shown for self-renewing (SR) and senescent (SEN) cells. The number of genes with γH2AX modified promoters found in both cell phenotypes is indicated in the intersection.
FIGS. 3A-3E show peri-telomeric and pericentric accumulation of γH2AX. FIG. 3A: γH2AX enrichment levels in peritelomeric regions for self-renewing (SR—blue) and senescent (SEN—red) cell lines. Chromosome ends (telomeres) are shown at the origin of the x-axis, which then extends into the chromosome arms. Average γH2AX enrichment levels are calculated as the log 2 ratio of the position-specific tag counts normalized to the genomic background averaged over all chromosome arms. FIG. 3B: γH2AX enrichment levels in pericentric regions for self-renewing (SR—blue) and senescent (SEN—red) cell lines. Centromeres are shown as gap centered on the x-axis, which extends into the chromosome arms in either direction. Average γH2AX enrichment levels are calculated as the log 2 ratio of the position-specific tag counts normalized to the genomic background averaged over all chromosomes. FIG. 3C: Statistical significance of γH2AX accumulation for the peri-telomeric regions of human chromosomes, left and right arms, in self-renewing (SR—blue) and senescent (SEN—red) cells. Significance levels (P-values) are calculated by comparing peritelomeric γH2AX levels to the genomic background and are plotted as −ln P, where −ln P=3 (horizontal line) corresponds roughly to the 95% confidence interval. FIG. 3D: Statistical significance of γH2AX accumulation for the pericentric regions of human chromosomes in self-renewing (SR—blue) and senescent (SEN—red) cells. Significance levels (P-values) are calculated by comparing pericentric γH2AX levels to the genomic background and are plotted as −ln P, where −ln P=3 (green line) corresponds roughly to the 95% confidence interval. FIG. 3E: Differences in the numbers of large γH2AX clusters in pericentric regions between senescent versus self-renewing cells. The absolute values of the normalized differences for γH2AX clusters between cell phenotypes are shown on the y-axis. Blue bars show chromosomes that have more large clusters in SR cells, and red bars show chromosomes that have more large clusters in SEN cells.
FIG. 4, panels A-C show centromere-associated persistent DNA damage foci in senescent hADSCs are sites of active transcription. Panel A: Immunofluorescent labeling of self-renewing hADSCs. Cells were seeded on coverslips and co-stained with anti-CENP-A (green) and anti-53BP1 (red) antibodies. DAPI staining is shown in blue. Confocal image of representative interphase nucleus is shown as separate channels and as a merged image. 4 μm z-slice was analyzed by Imaris software and z1, z2 and z3 projections are shown. Self-renewing hADSCs show no focal damage associated sites. Centromeric areas are clearly visible. Panel B: Persistent DNA damage is associated with centromeres. Senescent hADSCs were seeded on coverslips and as in (panel A), immunostaining was performed. An arrow depicts the co-localization of a centromeric region with persistent, senescence-associated γH2AX/53BP1 damage foci. Quantitation of these data and complementary co-staining with γH2AX are shown in FIG. 13. (Scale bar, 4 μm) Panel C: Association of persistent DNA damage sites upon senescence with regions of high transcriptional activity. Co-immunostaining of DNA damage foci depicted by 53BP1 antibodies (green) with PML bodies (blue) and nascent RNA (red) studied by confocal microscopy. Senescent hADSCs were incubated with halogenated precursor FUr for 10 min in vivo, fixed and stained with antibodies. BrdU antibodies were used to detect FUr labeled RNA. Representative image of a single nucleus is shown. Arrow points at the site of DNA damage focus co-localized with RNA. Spatial relationship between FUr incorporation sites, 53BP1 and PML bodies, is shown. Single 5 mm confocal section is shown. Image was analyzed by Imaris software and z1, z2 and z3 planes are shown. Cartoon demonstrates the orientations of z1, z2 and z3 planes within single z-section. Confocal sectioning confirms the tight association of nascent transcripts with persistent DNA damage sites in senescent hADSCs.
FIG. 5 shows that centromere-associated persistent DNA damage in senescent hADSCs correlates with transcriptional up-regulation of SINE/Alu retrotransposons and defects in recruitment of cohesin and condensin I complexes. C) Loss of cohesin and condensin I in the peri-centric location of persistent DNA damage in senescent hADSC. ChIP analysis of the peri-centric repeats on chromosome 10 in self-renewing (blue bars) and senescent (red bars) hADSCs. Repeats were assessed as locations for recruitment of TFIIIC, Eco1 as well as components of cohesin (Rad21) and condensin I (CAP-H) complexes (n=3, ±SEM). Schematic representation of the subunits of the cohesin and condensin I complexes, as well as a cartoon of previously reported function of Eco1, are shown. *p<0.02, **p<0.2.
FIGS. 6A-6G illustrate that stable knockdown of generic SINE/Alu transcript in senescent human adult stem cells restores cell's proliferative properties and produces iPS-like phenotype. FIG. 6A: Model of SINE/Alu retrotransposon. Secondary structure of generic SINE/Alu RNA (SEQ ID NO:1). Regions for shRNA design are shaded. FIG. 6B: Representative example of the efficiency of lentiviral transduction of hADSCs depicted by GFP. FIG. 6C: Northern blot hybridization of the RNA recovered from hADSCs cells stably expressing sh-RNA against SINE/Alu. Senescent hADSCs were infected with lentiGFP sh-193Alu, lentiGFP sh-132Alu or control no shRNA insert lentiGFP. RNA was isolated after 24 hrs post transduction and Northern hybridization was performed with a SINE/Alu specific oligonucleotide. Senescent hADSCs stably expressing sh-132Alu show near complete knockdown of the SINE/Alu transcripts. FIG. 6D: Proliferative properties of senescent hADSCs were reinstated in the cells upon stable knockdown of SINE/Alu transcripts. 3[H] thymidine uptake is shown. Senescent cells (wt) or senescent cells transduced with lentiGFP (control) or lentiGFP sh-132Alu were pulse-labeled with 1 μCi of 3[H] thymidine for 24 hrs either 24 or 96 hrs post infection. Data shown are mean±SEM for triplicate measurements. FIG. 6E: Expression of pluripotency markers Nanog and Oct4 was measured by qPCR analysis in senescent hADSCs (wt) and in hADSC with reversed-senescent phenotype upon stable knockdown of SINE/Alu transcription (lentiGFP sh-132Alu). RNA was isolated from the cells 96 hrs post infection. Results are expressed as relative quantity (DCt). Samples were normalized against β-actin. Data are shown as mean±SEM. (n=3) ***p=6.98e05, *p=0.03. FIG. 6F: Morphological changes in the reversed-senescence hADSCs with stable knockdown of SINE/Alu transcripts. After 7 days in culture without feed cells form GFP-positive cell aggregates stained positive for the pluripotency marker alkaline phosphatase (AP). FIG. 6G: Model of SINE/Alu transcriptional interference in triggering persistent DDR causing senescence of human adult mesenchymal stem cells.
FIGS. 7A-7B show FACS analysis and proliferative properties of hADSCs. FIG. 7A: FACS analysis of hADSCs. Early PD hADSCs were stained with FITC (CD 31, CD44 and CD 45) or AlexaFlour-488 (CD105) conjugated antibodies against cell surface markers and subjected to flow cytometric analysis. The cells were positive for CD 105 and CD 45, and negative for CD 34 and CD 44. The cell populations are shown as fluorescence to side scatter graphs (top), and the histograms (bottom) of stained cells (blue line) compared to un-stained cells (red line); with percentage of positive cells indicated. FIG. 7B: Replication capacity of hADSCs declines with ex-vivo aging. Proliferation in self-renewing (SR), pre-senescent (preSEN) and senescent cells (SEN) hADSCs was measured by 3[H]-thymidine uptake: 1 μCi was added to 10,000 cells in 2 ml DMEM/F12 medium. After a 24-hr incubation, the cells were harvested, DNA was isolated, and radioactivity was measured by liquid scintillation counting. Results are presented as the amount of 3[H]-thymidine (cpm) incorporated during DNA synthesis per 1 μg of isolated DNA. DNA from cells not exposed to 3[H]-thymidine was used as background radiation control.
FIG. 8 shows quantification of accumulation of persistent DNA damage foci with ex-vivo passaging of hADSCs. γH2AX was stained with affinity-purified rabbit polyclonal antibody. Histogram indicates the percentage of the cells with 1, 2, 3 or more than 3 foci. Representative examples are shown below. Foci formation was scored in self-renewing, SR (population doubling less than 17), pre-senescent, preSEN (population doubling more than 29, but less than 38) and senescent, SEN (population doubling more than 39) hADSCs cultures. n=total number of nuclei counted in all 3 independent experiments.
FIG. 9 shows DNA damage response activation in senescent hADSC. Senescent hADSC cultures were immunostained with antibodies against phosphorylated forms of Chk1 (S345), Chk2 (T68) and cdc2 (Tyr15) (brighter) and DAPI (dimmer). 50 mm confocal sections are shown. Chk1 and Chk2 are transducer kinases that act downsteam of ATM/ATR kinase to provide for DNA damage checkpoint control. Contrary to genotoxic stress or irradiation induced DNA damage, senescent hADSCs do not show robust nuclear localization of phosphorylated forms of Chk1 (S345) and Chk2 (T68).
FIG. 10. Fractions of γH2AX modified nucleosomes that map to different genomic features for self-renewing (SR left bars) and senescent (SEN right bars) adult adipose derived mesenchymal stem cells (hADSCs). The right tail of the distribution is enlarged and shown as an inset for clarity.
FIGS. 11A-11C show a comparison of mono-nucleosomal sized γH2AX modified positions to genomic clusters of γH2AX modified nucleosomes. γH2AX modified mono-nucleosomes and γH2AX modified clusters were identified as described in Supplemental Methods. FIG. 11A: Frequency distributions for γH2AX modified genomic positions in self-renewing (SR) and senescent (SEN) hADSCs. Mono-nucleosomal sized positions dominate the distributions. Accordingly, the frequencies of mid-size and large γH2AX modified nucleosome clusters are enlarged and shown as insets for clarity. FIG. 11B: Percentages of genomic features occupied by γH2AX modified mono-nucleosomes and large clusters of γH2AX modified nucleosomes. FIG. 11C: Relative entropy was calculated as a measure of the difference between the SR versus SEN γH2AX cluster size frequency distributions (see panel FIG. 11A). The relationship between relative entropy and cluster lengths was used to calculate a threshold (7,400 nt) between mid-size and large clusters as described in Supplemental Methods.
FIG. 12 shows relative enrichment of γH2AX modified nucleosomes in peri-telomeric versus pericentric genomic regions. Enrichment values were calculated as log 2 normalized ratios of the γH2AX ChIP-seq tag counts per position in each region divided the genomic background tag counts per position. Values for self-renewing (SR) cells are shown in blue and for senescent (SEN) cells in red. Peri-telomeric regions are depleted for γH2AX, whereas pericentric regions are enriched
FIG. 13, panels A-D, show persistent γH2AX/53BP1 foci in senescent hADSCs are associated with centromeric regions. Panel A: Quantification of CENP-A and 53BP1 co-localization in senescence. Senescent hADSCs were stained with antibodies against CENP-A (green) and 53BP1 (red) and DAPI (blue). Total of 200 cells were scored from three independent experiments. Error bars represent +/−SAM. Example of higher magnification of the image is shown Panel B. Scale bar 1 tm. Images were analyzed by IMARIS software with optical sections representation as depicted in FIGS. 4C and 4D. D) Co-localization of senescence-associated persistent γH2AX/53BP1 foci with kinetochore. Double immunostaining of senescent hADSCs with antibodies against of γH2AX (panel C) or 53BP1 (panel D) and antisera to the inner kinetochore from patients with calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia (collectively abbreviated as CREST). Scale bars are shown for each individual image. Black and white images of the separate channels are given in (panel D) for the convenience of visualization.
FIG. 14 illustrates SINE/Alu expression in SR and SEN hADSCs. Northern hybridization of self-renewing (SR) and senescent (SEN) hADSCs with SINE/Alu oligonucleotide probe. SINE/Alu and 7SL are indicated. Total RNA of 2 μg per lane was loaded as described in Experimental Procedures. Ribosomal small RNAs can be seen in the ethidium bromide stained gel for loading comparison. The ssRNA ladder sizes are indicated on the right.
FIGS. 15A and 15B illustrates lentiviral shRNA-mediated knockdown of generic Alu transcripts in ADSCs and the effects of such knock down on cell senescence and proliferation. FIG. 15A schematically illustrates a protocol for stably knocking-down Alu transcripts using an shRNA delivered by a lentiviral vector. FIG. 15B illustrates the delivery vector, transfected cells and a Northern blot showing the results of transfection.
FIG. 16 illustrates the effect of lentiviral shRNA-mediated knockdown of generic Alu transcript in ADSCs. Cell proliferation is show as a function of time. Knockdown of Alu transcript reinstated proliferation of prior senescent cells.
FIG. 17 illustrates the morphology of Alu shRNA mutant ADSCs in culture and their initial characterization.
FIG. 18 shows a comparison between “standard” protocols for the generation of induced pluripotent stem cells (iPSCs) and one of the protocols described herein.
FIGS. 19A and 19B illustrate the subsequent differentiation of Alu shRNA mutant cells. FIG. 19A illustrates the differentiation of ADSCs into neuroblasts. FIG. 19B illustrates the use of the methods described herein to transdifferentate cells into numerous other pathways/lineages.
FIGS. 20A-20E illustrates the transdifferentiation of kidney fibroblasts into neuroblasts. FIG. 20A illustrates HEK 293T cells two days after infection with sh-132Alu. FIG. 20B illustrates colony formation 7 days after single colony isolation and desegregation into individual cells (ES medium supplemented with LIF). FIG. 20C shows the expression of pluripotency markers (nanog, oct4, and alkaline phosphatase). FIG. 20D shows the expression of mesodermal markers two days after the formation of embryoid bodies (EB). FIG. 20E shows the expression of osterogenic (osteopontin), adipogenic (lipoprotein lipase), and glial (GAFP) genes 2 days after formation Embryoid bodies (EB).
FIG. 21, panels A (left) and B (right), illustrate alternative models of Alu shRNA action. Panel A: shRNA against Alu forms a hairpin shRNA, which directs either nuclear PIWI or Dicer machinery to the genomic SINE/Alu repeat location, initiating transcriptional silencing via heterochromatinization involving both DNA methylation and histone modification. Panel B: shRNA against Alu activates the PTGS Dicer-dependent Ago2 pathway, leading to the cytoplasmic degradation of unprocessed Alu RNA transcripts.
FIG. 22A (top) shows a representation of 7SL-conserved region within Alu full-length sequence. FIG. 22A (bottom) shows secondary structure of generic full-length Alu RNAv (SEQ ID NO:2). The horizontal highlighted segment represents the highly conserved 7SL-derived portion, while the vertical highlighted segment represents region used for synthetic RNA affinity assay. FIG. 22B shows sequence conservation of the Alu shRNA sequence compared to the rest of the element. Average percent identity levels among dispersed repeated element copies are compared for the shRNA Alu sequence regions versus other Alu sequence regions across four Alu subfamilies. Significance values for the differences are shown for each comparison. FIG. 22C shows a step-by-step schematic representation of unbiased RNA affinity assay. FIG. 22D shows silver-stained denaturing 4-12% NuPAGE Novex 4-12% Bis-Tris gel loaded with RNA affinity assay precipitants, with excised bands labeled A-E.
FIG. 23 shows a brightness-coded representation of abundance of members in major functional categories. Most abundant proteins within each group are depicted with the brightest color, fading to black for those with an abundance of only 1.
FIG. 24A shows confidence levels for the involvement of isolated complex proteins in various cellular processes, represented Gene Ontology (GO) terms as a −log10(p-value), using p-values calculated by DAVID software. 24B shows confidence levels for the presence of isolated complex proteins in various cellular components, represented as a −log10(p-value), as calculated using DAVID software. FIG. 24C shows an interaction web of isolated proteins produced in STRING 9.0 software. Thicker lines represent higher confidence (experimentally derived) interactions while dotted lines represent inferred (lower confidence) interactions.
FIG. 25 shows a representation of protein complex using Gene Ontology (GO) terms in String 9.0. Each specific cellular process is grouped into one of the following 5 categories: transcription, DNA repair, chromatin modifications, cell cycle regulation, and others.



DETAILED DESCRIPTION
It was discovered that a significant (e.g.,  ̃65% in certain instances) of naturally occurring repairable damage in self-renewing adult stem cells occurs in transposable elements. Upregulation of transcriptional activity from SINE/Alu retrotransposons interferes with the recruitment of condensin I and cohesin complexes in pericentric chromatin, resulting in the loss of efficient DNA repair and, in turn, senescence.
It was demonstrated that stable knockdown of generic SINE/Alu retrotransposon transcripts in senescent human adult stem cells reinstates the cells' self-renewing properties and unexpectedly increases their plasticity (e.g., as manifested by upregulation of the pluripotency markers Nanog and Oct4). The results presented herein demonstrate the functional significance of SINE/Alu retrotransposons and provide mechanistic insight into their novel role in mediating crosstalk between chromatin, DNA repair and aging of human adult stem cells.
In view of this discovery, methods are provided for inducing and/or restoring and/or maintaining proliferative capacity and/or pluripotency in a mammalian cell. More generally, the methods reduce the rate of onset of senescence or prevent senescence, or rescuing a cell from senescence. In various embodiments the methods induce or restore a pluripotent phenotype to a differentiated and/or senescent cell. In various embodiments the methods typically involve downregulating or inhibiting the level or activity of SINE/Alu retrotransposon transcripts in the cell (see, e.g., FIG. 18).
As demonstrated herein, such down regulation/inhibition rescues cells (e.g., adult stem cells) from senescence restoring pluripotency and/or proliferative capacity (see, e.g., FIGS. 6D, 6E 6F, 15, 16, and 17). Such down regulation affords a number of uses. For example, it is believed the viable lifetime of stem cell populations (e.g., stem cell lines) can be extended (perhaps indefinitely) by down regulating (e.g., stably down-regulating) SINE/Alu retrotransposon transcripts in those cells. When/if desired, the stem cells can be induced to differentiate into embryoid bodies, precursors or particular lineages, or terminally differentiated cells according to standard methods well known to those of skill in the art.
Accordingly cells (including previously terminally differentiated mammalian cells, progenitor cells, stem cells, stem cell lines, and induced pluripotent stem cells (iPSCs)) are provided where the cells have SINE/Alu retrotransposon transcripts downregulated. In various embodiments the cells include normal (non-senescent) stem cells, stem cells that have been “rescued” from senescence by downregulating SINE/Alu retrotransposon transcript production or activity, induced pluripotent stem cells (IPSCs) that contain a construct that downregulates SINE/Alu retrotransposon transcript level or activity, embryonic stem cells, and the like. In certain embodiments cells are excluded that have been treated in a manner to form IPSCs (e.g., cells are excluded in which one or more of Nanog, and/or LIN-28, and/or Oct3/4, and/or Sox2, and/or Klf4, and/or c-myc are directly upregulated and/or that contain heterologous constructs that express Oct3/4, and/or Sox2, and/or Klf4, and/or c-myc).
It was also demonstrated that cells in which pluripotency has been induced and/or restored according to the methods described herein (e.g., inhibition of Alu retrotransposon) can be subsequently induced to differentiate using standard methods well known to those of skill in the art (e.g., withdrawal of LIF from culture media) (see, e.g., FIGS. 19A, 19B, and FIGS. 20A-20E). This provides further evidence that the methods described herein induce or restore pluripotency to a cell. Accordingly, in certain embodiments, differentiated cells comprising cells that have been induced to differentiate from pluripotent cells generated according to the methods described herein are contemplated.
In addition, in certain embodiments, methods of transdifferentiating cells are contemplated. The transdifferentiation methods typically comprise inducing or restoring a cell to a pluripotent phenotype according to the methods described herein (e.g., by inhibiting the Alu retrotransposon or other components of the pathway), and then culturing the resulting pluripotent cells under conditions that allow or induce differentiation. Cells that differentiate into a desired cell type (e.g., pancreatic beta cells, motoneurons, hematopoietic progenitor cells, neural cells, dopaminergic neurons, adipocytes, cardiomyocytes, and the like) and/or lineage are then selected and can be subsequently cultured (e.g., expanded in culture) or directly utilized.
In certain embodiments the methods described herein can be used to restore pluripotency and/or proliferative capacity to a cell (e.g., to a cell that has committed to a differentiation pathway). In certain embodiments the methods described herein can be used to restore to a lesser senescent state or to a non-senescent state a cell that shows one or more indications of senescence.
In various embodiments stem cell comprises a cell selected from the group consisting of an embryonic stem cell, a cord blood stem cell, an adult stem cell, and an IPSC. In various embodiments the mammalian stem cell is a stem cell derived from a tissue selected from the group consisting of human adipose tissue, human bone marrow, human neurological tissue, human smooth muscle, human adipose tissue, human cardiomyocytes, human endothelial tissue, human epithelial tissue, human pancreatic tissue, human bone or cartilage and the like.
In certain embodiments the cell is one that has committed to differentiation to a cell type selected from the group consisting of ectoderm, mesoderm, and endoderm. In certain embodiments the cell is one that has committed to differentiation to a cell type selected from the group consisting of human adipose cells, human blood cells, human nerve cells, human smooth muscle cells, human adipocytes, human chondrocytes, human osteoclasts and hosteoblasts, human cardiomyocytes, human endothelial cells, and human epithelial cells. In certain embodiments the cell comprises a non-renewing progenitor cell. In certain embodiments the mammalian cell comprises a terminally differentiated cell.
Downregulating/Inhibiting SINE/Alu Retrotransposon Transcripts
It was a surprising discovery that inhibition of SINE/Alu retrotransposon transcripts can restore proliferative capacity and/or pluripotency to a senescent stem cell or can maintain proliferative capacity and/or pluripotency in a non-senescent stem cell. Any of a variety of methods to inhibit SINE/Alu retrotransposon transcripts can be used.
In various embodiments SINE/Alu retrotransposon transcripts can be reduced/inhibited using inhibitory RNAs. Suitable inhibitory RNAs include, but are not limited to siRNAs, shRNAs, miRNAs, dicer-substrate 27-mer duplexes, single-stranded interfering RNA, and the like.
siRNAs typically refer to a double-stranded interfering RNA unless otherwise noted. In various embodiments, suitable siRNA molecules to inhibit SINE/Alu retrotransposon transcripts include double-stranded ribonucleic acid molecules comprising two nucleotide strands, each strand having about 19 to about 28 nucleotides (i.e. about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). Thus, the phrase “interfering RNA having a length of 19 to 49 nucleotides” when referring to a double-stranded interfering RNA means that the antisense and sense strands independently have a length of about 19 to about 49 nucleotides, including interfering RNA molecules where the sense and antisense strands are connected by a linker molecule.
In addition to siRNA molecules, other interfering RNA molecules and RNA-like molecules can to inhibit SINE/Alu retrotransposon transcripts. Examples of other interfering RNA molecules that can to inhibit SINE/Alu retrotransposon transcripts include, but are not limited to short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs (miRNAs), and dicer-substrate 27-mer duplexes. Examples of RNA-like molecules that can to inhibit SINE/Alu retrotransposon transcripts include, but are not limited to siRNA, single-stranded siRNA, microRNA, piwiRNA, and shRNA molecules containing one or more chemically modified nucleotides, one or more non-nucleotides, one or more deoxyribonucleotides, and/or one or more non-phosphodiester linkages. Typically, all RNA or RNA-like molecules that can interact with SINE/Alu retrotransposon transcripts RISC and participate in RISC-related changes in gene expression can be referred to as “interfering RNAs” or “interfering RNA molecules.” SiRNAs, single-stranded siRNAs, shRNAs, miRNAs, and dicer-substrate 27-mer duplexes are, therefore, subsets of “interfering RNAs” or “interfering RNA molecules.”
Using the known nucleotide sequences for SINE/Alu retrotransposon transcript(s), suitable siRNAs can readily be produced. In various embodiments siRNA that inhibit SINE/Alu retrotransposon transcripts can comprise partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include, for example, addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.
In various embodiments one or both strands of the siRNA can comprise a 3′ overhang. As used herein, a “3′ overhang” refers to at least one unpaired nucleotide extending from the 3′-end of an RNA strand. Thus in one embodiment, the siRNA comprises at least one 3′ overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, from 1 to about 5 nucleotides in length, from 1 to about 4 nucleotides in length, or about 2 to about 4 nucleotides in length.
In an illustrative embodiment in which both strands of the siRNA molecule comprise a 3′ overhang, the length of the overhangs can be the same or different for each strand. In certain embodiments the 3′ overhang is present on both strands of the siRNA, and is one, two, or three nucleotides in length. For example, each strand of the siRNA can comprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).
In order to enhance the stability of the siRNA, the 3′ overhangs can be also stabilized against degradation. In one embodiment, the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. In certain embodiments, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation. In particular, it is believed the absence of a 2′ hydroxyl in the 2′-deoxythymidine can significantly enhance the nuclease resistance of the 3′ overhang.
In certain embodiments, the siRNA comprises the sequence AA(N19)TT (SEQ ID NO:3), AA(N21)TT (SEQ ID NO:4), NA(N21) (SEQ ID NO:5), and the like, where N is any nucleotide. In various embodiments these siRNA comprise approximately 30%-70% GC, and preferably comprise approximately 50% G/C. The sequence of the sense siRNA strand corresponds to (N19)TT (SEQ ID NO:6) or N21 (SEQ ID NO:7) (i.e., positions 3 to 23), respectively. In the latter case, the 3′ end of the sense siRNA is converted to TT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense strand 3′ overhangs. The antisense RNA strand is then synthesized as the complement to positions 1 to 21 of the sense strand.
Because position 1 of the 23-nt sense strand in these embodiments is not recognized in a sequence-specific manner by the antisense strand, the 3′-most nucleotide residue of the antisense strand can be chosen deliberately. However, the penultimate nucleotide of the antisense strand (complementary to position 2 of the 23-nt sense strand in either embodiment) is generally complementary to the targeted sequence.
In another illustrative embodiment, the siRNA comprises the sequence NAR(N17)YNN (SEQ ID NO:8), where R is a purine (e.g., A or G) and Y is a pyrimidine (e.g., C or U/T). The respective 21-nt sense and antisense RNA strands of this embodiment therefore generally begin with a purine nucleotide. Such siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.
In various embodiments the siRNA of the invention can be targeted to any stretch of approximately 10-30, or 15-25, or 19-25 contiguous nucleotides in any of the target mRNA sequences (the “target sequence”). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl et al., “The siRNA User Guide,” revised May 6, 2004. The “siRNA User Guide” is available on the world wide web at a website maintained by Dr. Thomas Tuschl, and can be found by accessing the website of Rockefeller University and searching with the keyword “siRNA.” In addition, the “siRNA User Guide” can be located by performing a google search for “siRNA User Guide” and can also be found at “www.rockefeller.edu/labheads/tusch1/sirna.html. Techniques for selecting target sequences for siRNA and miRNA can also be found in Sioud (2008) siRNA and miRNA Gene Silencing: From Bench to Bedside (Methods in Molecular Biology), Humana Press.
In certain embodiments the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 19 to about 25 nucleotides in the target SINE/Alu retrotransposon transcript(s).
The SINE/Alu retrotransposon transcript silencing siRNAs can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application US 2002/0086356.
In certain embodiments the siRNAs are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. The siRNAs can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK). Custom siRNA can be obtained from commercial suppliers (see, e.g., Thermo Fisher Scientific, Lafayette Colo.; Qiagen, Valencia, Calif.; Applied Biosystems, Foster City, Calif.; and the like).
In certain embodiments siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing siRNA from a plasmid include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant plasmids can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
The siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly at or near the target area(s) in vivo. The use of recombinant plasmids to deliver siRNA to cells in vivo is discussed in more detail below.
siRNA can be expressed from a recombinant plasmid either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Selection of plasmids suitable for expressing siRNAs, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art (see, e.g., Tuschl (2002) Nat. Biotechnol., 20: 446-448; Brummelkamp et al. (2002) Science 296: 550 553; Miyagishi et al. (2002) Nat. Biotechnol. 20: 497-500; Paddison et al. (2002) Genes Dev. 16: 948-958; Lee et al. (2002) Nat. Biotechnol. 20: 500-505; Paul et al. (2002) Nat. Biotechnol. 20: 505-508, and the like).
In one illustrative embodiment, a plasmid comprising nucleic acid sequences for expressing an siRNA for inhibiting SINE/Alu retrotransposon transcripts comprises a sense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter, and an antisense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter. The plasmid is ultimately intended for use in producing a recombinant adeno-associated viral vector comprising the same nucleic acid sequences for expressing the siRNA.
As used herein, “in operable connection with a polyT termination sequence” means that the nucleic acid sequences encoding the sense or antisense strands are adjacent to the polyT termination signal in the 5′ direction or sufficiently close so that during transcription of the sense or antisense sequences from the plasmid, the polyT termination signals act to terminate transcription after the desired product is transcribed.
As used herein, “under the control” of a promoter means that the nucleic acid sequences encoding the sense or antisense strands are located 3′ of the promoter, so that the promoter can initiate transcription of the sense or antisense coding sequences.
In certain embodiments the siRNA can be delivered as a small hairpin RNA or short hairpin RNA (shRNA). shRNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. In typical embodiments, shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA that is bound to it.
In certain embodiments the sense sequence of the shRNA will be from about 19 to about 30, more nucleotides (e.g. about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) in length, more typically from about 19 to about 22 nucleotides in length, the antisense sequence will be from about 19 to about 30, more typically from 19 to about 22 nucleotides (e.g. about 19, 20, 21 or 22 nucleotides), in length, and the loop region will be from about 3 to about 19 nucleotides (e.g., about 3, 4, 5, etc., . . . up to about 19) nucleotides in length. In some embodiments, the sense and antisense sequences are the same length, i.e. the shRNA will form a symmetrical hairpin, but this is not necessarily the case. In some cases, the sense or antisense strand may be shorter than its complementary strand, and an asymmetric hairpin is formed. Further, while in some instances the base pairing between the sense and antisense sequences is exact, this also need not be the case. In other words, some mismatch between the sequences may be tolerated, or even desired, e.g. to decrease the strength of the hydrogen bonding between the two strands. However, in one illustrative embodiment, the sense and antisense sequences are the same length, and the base pairing between the two is exact and does not contain any mismatches. The shRNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. In addition, the loop portion of the shRNA molecule can comprise, for example, nucleotides, non-nucleotides, linker molecules, conjugate molecules, etc.
The shRNA/siRNA/piRNA described herein targets and causes the RNAi-mediated degradation of SINE/Alu retrotransposon transcripts, or alternative splice forms, or participates in genomic silencing via (PIWI RNA pathways). Thus, in certain embodiments, methods are provided for inhibiting SINE/Alu retrotransposon transcripts in a cell, comprising administering an effective amount of an SINE/Alu retrotransposon transcript siRNA/shRNA/piRNA to the cell, such that the target mRNA is degraded.
In various embodiments the siRNA/shRNA/piRNA can be expressed from recombinant viral vectors introduced into the subject cells. The recombinant viral vectors comprise sequences encoding the siRNA/shRNA and any suitable promoter for expressing the siRNA/shRNA/piRNA sequences. Suitable promoters include, but are not limited to, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors can also comprise inducible or regulatable promoters for expression of the siRNA/shRNA in a particular tissue or in a particular intracellular environment.
The siRNA/shRNA/piRNA can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
Any viral vector capable of accepting the coding sequences for the siRNA/shRNA/piRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g. lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of the viral vectors can also be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses. For example, an AAV vector can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
Selection of recombinant viral vectors suitable for use in methods for inserting nucleic acid sequences for expressing the siRNA/shRNA/piRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art (see, e.g., Domburg (1995) Gene Therap. 2: 301-310; Eglitis (1988) Biotechniques 6: 608-614; Miller (1990) Hum. Gene Therap. 1: 5-14; Anderson (1998) Nature 392: 25-30, and the like).
In certain embodiments suitable viral vectors include, but are not limited to lentiviral vectors. In one illustrative embodiment, lentiviral shRNA constructs to knockdown genetic SINE/Alu transcript are designed. The shRNA sense and anti-sense strands are chemically synthesized and the strands are annealed with equal amounts of each other creating restriction site specific overhangs for cloning, and ligated into a vector (e.g., a HindIII and BglII digested, gel purified pENTR/pTER+ vector). Equal amounts of each construct is mixed with pLenti-CMV-GFP DEST vector in LR Clonase reaction to recombine cloned shRNA production elements into a destination vector according to the manufacturer's instructions (Invitrogen). The produced lentiviral plasmid is transformed into E. coli Stbl3 cells (Invitrogen) for amplification.
In certain embodiments suitable viral vectors include those derived from AV and AAV. In one illustrative embodiment, the siRNA/shRNA/piRNA is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. A suitable AV vector for expressing the siRNA, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia et al. (2002) Nat. Biotech. 20: 1006 1010.
Suitable AAV vectors for expressing the siRNA/shRNA, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are also described in Samulski et al. (1987) J. Virol. 61: 3096-3101; Fisher et al. (1996) J. Virol., 70: 520-532; Samulski et al. (1989) J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479 and 5,139,941; International Patent Application Nos. WO 1994/013788; and WO 1993/024641, and the like.
Sources of Cells.
Stem cells (e.g., adult stem cells, embryonic stem cells, cord stem cells, IPSCs, etc.) can be obtained according to standard methods well known to those of skill in the art. In certain embodiments, stem cells are also commercially available.
Differentiating Stem Cells.
In certain embodiments methods are provided for differentiating stem cells in which level or activity of SINE/Alu retrotransposon transcripts is reduced/inhibited. It is believed these stem cells can be differentiated into embryoid bodies or terminally differentiated cells using standard differentiation methods well known to those of skill in the art.
For example, in certain embodiments, removal of leukemia inhibitory factor (LIF) can result in the differentiation of the modified stem cells described herein (e.g., stem cells in which SINE/Alu retrotransposon transcripts are reduced/inhibited) into embryoid bodies.
Methods of differentiating stem cells are well known to those of skill in the art. For example, extract from infarcted myocardium contains biochemical factors that induce cardiomyocyte differentiation from bone marrow-mesenchymal stem cells (BM-MSCs) (see, e.g., Ge et al. (2009) Biochem. Biophys. Res. Commun., 381(3): 317-321). Bone morphogenic factors (BMP4, BMP7, and BMP8b) can increase differentiation of human germ cells from human ES cells (see e.g., Gonsalves et al. (2006) Stem Cells Dev., 15(6): 831-837). Primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose (see, e.g., Aguiari et al. (2008) Proc. Natl. Acad. Sci., USA, 105(4): 1226-1231). Interleukin-27 induces differentiation in hematopoietic stem cells (see, e.g., Seita et al. (2008) Blood, 111(4): 1903-1912).
With respect to neural cell differentiation, the neurotrophin family is one of the most important inducible signals for the differentiation. Among them, nerve growth factor is well known to induce neurogenesis, and neurotrophin 3 is involved in oligodendrocyte development. Another important differentiation signal family is the ciliary neurotrophic factor (CNTF)-leukemia inhibitory factor (LIF) cytokine family, which plays a pivotal role in regulating gliogenesis in the developing mammalian central nervous system. In addition, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside;) induces astroglial differentiation of neural stem cells (see, e.g., Zang et al. (2008) J. Biol. Chem., 283(10): 6201-6208 and references therein).
A number of natural compounds are also known to induce differentiation of stem cells in vitro. Such compounds include, but are not limited to retinoic acid, PDGF, insulin, Arctigenin, ATRA (vitamin A), boswellic acid, bromelain and other proteolytic enzymes, CAPE, flavonoids (including apigenin, luteolin, quercetin, genistein, and daidzein), emodin, EPA and DHA, monoterpenes, resveratrol, 1,25-D3 (vitamin D3), and the like.
It is believed the pluripotent cells produced using the methods described herein can be subsequently induced to differentiate using methods well known to induce differentiation if iPSCs. In this regard it is noted that methods of differentiating induced pluripotent cells into CD34+CD43+ hematopoietic progenitors and CD31+CD43− endothelial cells are described by Choi et al. (2009) Stem Cells 27: 559. These cells can be further separated into phenotypically defined subsets of primitive hematopoietic cells in a pattern of differentiation resembling that of ES cells. Methods of differentiating induced pluripotent cells into pancreatic insulin-producing cells are described by Zhang et al. (2009) Cell Res. 19: 429. After first generating PDX-1 positive progenitor cells, human iPS cells were further differentiated into pancreatic cells expressing MafA, Glut2, insulin, and in some cases, amylase and C-peptide. Functional cardiomyocytes demonstrating sarcomeric organization and expressing cardiac markers including Nkx2.5, cardiac Troponin T, atrial natriuretic factor, and myosin heavy and light chains, have also been derived from human iPS cells and are indistinguishable from those generated from ES cells (see, e.g., Zhang et al. (2009) Circ. Res. 104:e30). Electrophysiology experiments revealed that like ES cells, iPS cells differentiate into nodal-, atrial-, and ventricular-like phenotypes, and are responsive to the canonical cardiomyocyte beta-adrenergic signaling pathway. Both human ES and iPS cells were efficiently converted to neural cells using two inhibitors of TGF-beta/Smad signaling, Noggin and the drug SB431542 (Chambers et al. (2009) Nat. Biotechnol. 27: 275). The synergistic action of these two inhibitors resulted in Pax6+ primitive neural cells that could then be further differentiated into neural crest, anterior CNS, somatic motoneurons, and dopaminergic neurons.
These approaches are intended to be illustrative and not limiting. Other methods of inducing differentiation of stem cells are well known to those of skill in the art.
In certain embodiments cells and compositions comprising cells that have been differentiated into embryoid bodies or further differentiated (e.g., terminally differentiated) are also contemplated. Such differentiated cells include but are not limited to embryoid bodies and/or progenitor cells and/or terminally differentiated cells that are differentiated into lineages for cardiomyocytes, blood cells, epithelial cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, smooth muscle cells, nerve cells (neurons), glial cells, pancreatic β-cells, motoneurons, and the like.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
SINE/Alu Retrotransposon-Dependent Chromatin Changes Induce Ex-Vivo Aging of Human Adult Stem Cells
In this example, we report mechanistic aspects of ex-vivo adult mesenchymal stem cell aging. Using genome-wide comparative analysis of repairable and persistent DNA damage in human adult stem cells as they traverse into the state of cellular senescence, we were able to uncover a new, unexpected functional role of retrotransposons in the complex pathways of cellular aging. We present evidence that a majority of repairable DNA damage in self-renewing human adult stem cells is distributed non-randomly and is enriched at retrotransposal repeats. Our data suggests that the misregulation of transcriptional activity of at least one class of human retrotransposons (SINE/Alu repeats) impairs the assembly of cohesin and condensin complexes in pericentric chromatin sites of γH2AX enrichment, thus interfering with cellular DNA repair machinery. The inability of adult stem cells to resolve pericentric DNA damage results in exit from self-renewal to senescence. We provide evidence that this event is reversible, and suggest a new mechanistic model wherein the loss of appropriate control of transcriptional activity in the retrotransposal portion of the human genome can directly or indirectly influence complex molecular events leading to the cessation of adult stem cell self-renewal and triggering their senescence.
Results
Replicative Senescence of Human Adult Mesenchymal Stem Cells
Human adult adipose derived mesenchymal stem cells (hADSCs) were isolated from subcutaneous abdominal fat as described in Experimental Procedures, and characterized based on cell surface antigens (FIG. 7A). After four weeks in the culture, the cells became more uniform as demonstrated by FACS analysis. More than 99.3% and 99.9% of the cells expressed mesenchymal stem cell MSC-specific cell type markers CD105 and CD44 (Dominici et al. (2006) Cytotherapy 8: 315-317), respectively, but did not express markers of hematopoietic stem cells (CD45) or endothelial progenitor cells (CD31) (FIG. 7A). hADSC can be induced to differentiate along several mesenchymal tissue lineages, including adipocytes, osteoblasts, myocytes, and chondrocytes (Erickson et al. (2002) Biochem. Biophys. Res. Commun. 290: 763-769; Zuk et al. (2001) Tissue Eng., 7: 211-228). Thus, hADSCs in these experiments bore strong resemblance to mesenchymal stem cells (MSCs).
The self-renewing (SR) capacity of hADSCs remained consistent until population doubling (PD17), after which they displayed characteristic phenotypes of “old age” with prolonged ex vivo passages shown in FIG. 1A (and also described by (Bonab et al. (2006) BMC Cell Biol., 7: 14; Fehrer et al. (2007) Aging Cell, 6: 745-757; Kern et al. (2006) Stem Cells, 24: 1294-1301). The culture's morphological abnormalities are typical of the Hayflick model of cellular aging (Juckett (1987) Mech. Ageing Dev., 38: 49-71). By PD37, hADSC cultures accumulated non-dividing giant cells expressing the enzyme lysosomal pH6 senescence-associated J3-galactosidase (SA-J3-Gal) (Dimri et al. (1995) Proc. Natl. Acad. Sci. USA, 92: 9363-9367), as shown in FIG. 1B. Cells self-renewed poorly due to a decrease in the number of dividing cells as determined by incorporation of bromodeoxyuridine (BrdU) and 3[H] thymidine into DNA (FIG. 1B and FIG. 7B).
To determine if the hADSCs limited expansion capacity was due to senescence associated with persistent DNA damage accumulation, we performed immunostaining experiments with antibodies against key mediators of DNA damage response (DDR), which facilitate checkpoint activation and repair. In particular, we used a phosphorylated form of histone variant H2AX (γH2AX) (Shiloh (2003) Nat. Rev. Cancer, 3: 155-168) and p53 binding protein-1 (53BP1) (Aguilera and Gomez-Gonzalez (2008) Nat. Rev. Genet., 9: 204-217; Shiloh (2003) Nat. Rev. Cancer, 3: 155-168; Stewart (2009) Cell Cycle, 8: 1532-1538) to provide evidence of the presence of molecular characteristics of cells bearing DNA double-strand breaks (DSB) in both self-renewing and senescent hADSC cultures. As hADSCs approached senescence, both γH2AX and 53BP1 localized (FIG. 1C), forming characteristic foci, named persistent DNA damage foci (previously reported by Rodier et al. (2009) Nat. Cell Biol., 11: 973-979). Persistent γH2AX/53BP1 foci formation upon cellular senescence has been associated with the presence of unresolved DSB, as determined by colocalization with several DNA repair factors (d'Adda di Fagagna et al. (2003) Nature, 426: 194-198). Occurrences of γH2AX/53BP1 foci formations were very rare in self-renewing ADSCs, and their formation increased as cultured hADSCs approached senescence (SEN hADSCs) (FIG. 8).
Combined, these results reveal that senescence-associated effects in hADSCs are not restricted to senescent passages, but are continuously acquired from the onset of their ex-vivo expansion, similar to phenomena previously reported for human fibroblasts and human hematopoietic stem cells.
We also noticed that senescent hADSCs contained activated forms of the DNA damage checkpoint kinases CHK1 and CHK2, phosphorylated on S345 and T68, respectively (FIG. 9). The phosphorylation of these sites by ATM/ATR is required for full execution of DNA-damage-induced cell-cycle arrest in human somatic cells (d'Adda di Fagagna et al. (2003) Nature, 426: 194-198; Sedelnikova et al. (2008) Aging Cell 7: 89-100; Tanaka et al. (2006) Cell Prolif., 39: 313-323). To further delineate the state of these cells, we performed differential (SR vs. SEN hADSCs) transcriptional analysis of the 96 genes involved in multiple aspects of the cell cycle (Human Cell Cycle qPCR array), described in detail in Experimental Procedures. Genes that were statistically significantly downregulated with a p value <0.05 in SEN hADSCs are shown in Table. 1, and include genes involved in cell cycle regulation, DNA replication, and mitosis, suggesting that senescent hADSC cultures follow a DDR program directly or indirectly related to the formation of senescence-associated DNA-damage foci. The causal factors that might mediate this process in human adult stem cells are as yet unknown.
Genome-Wide ChIP-Seq Location Analysis of γH2AX in Senescent hADSCs.
To identify genomic loci directly engaged in DDR in SEN hADSCs, we performed genome-wide profiling of hADSC chromatin using chromatin immunoprecipitation with an antibody against the γH2AX modified histone, followed by next generation sequencing (ChIP-seq) on the ABI SOLiD platform (FIG. 2A). Asynchronous SR samples of hADSCs were used in similar experiments to establish an overall representation of γH2AX modified chromatin, if any, to define the locations of repairable DNA damage which does not affect the SR properties of the cells. ChIP-seq was performed on four replicates each of SR and SEN hADSC cultures. Genomic mapping of the resulting sequence tags (Table 2) was followed by outlier removal, noise reduction, and merging of the data from replicate experiments. Details of the algorithms developed and approaches used for ChIP-seq data analyses can be found in Supplemental Methods. The ChIP-seq experimental protocol and data merging we employed were supported by the highly consistent mapping results seen for the replicate experiments. Having mapped and processed the ChIP-seq tags, we evaluated tag counts with respect to an empirically-derived count threshold (Supplemental Methods) for approximately mono-nucleosomal-sized windows of 200 bp in order to identify individual γH2AX modified nucleosomes on the human genome. The resulting genomic distributions of γH2AX modified nucleosomes were compared for SR and SEN cells (FIG. 2B) in order to assess the positional and quantitative changes in γH2AX modified chromatin associated with these phenotypes.
Majority of γH2AX Modified Chromatin Maps to the Retrotransposal Portion of the Human Genome
Past studies reported that topological constraints on chromatin structures upon DNA replication and transcription pose the biggest danger to DNA integrity (reviewed in Shrivastav et al. (2008) Cell Res., 18: 134-147). Altogether, there are four potential sources of damage in aging adult stem cells: transient DSB generated by inducible gene transcription, DNA origin firing (Ju et al. (2006) Science, 312: 1798-1802; Ju and Rosenfeld (2006) Cell Cycle, 5: 2557-2560; Rampakakis and Zannis-Hadjopoulos (2009) Nucleic Acids Res., 37: 5714-5724), DNA damage resulting from impediments of replication forks due to collision of replication and transcription machineries, and difficulties arising from particularities in the replication of centromeric and telomeric regions of the genome (Dalal and Bui (2010) Curr. Opin. Cell Biol., 22: 392-402; Dotiwala et al. (2010) Curr. Biol., 20: 328-332; Morris and Moazed (2007) Cell, 128: 647-650; Schoeftner and Blasco (2009) EMBO J. 28: 2323-2336). Recent studies of γH2AX distribution in the genome of cycling Saccharomyces cerevisiae cells revealed “fragile” genomic locations (Szilard et al. (2010) Nat. Struct. Mol. Biol., 17: 299-305), suggesting that mapping sites of γH2AX enrichment could be fruitful to pinpoint at-risk genomic elements in other genomes. Since mechanistic aspects of “fragile sites” are linked to regulation of cell cycle checkpoints and DNA repair (Durkin and Glover (2007) Annu. Rev. Genet., 41: 169-192), one can expect that the gradual accumulation of unresolved DNA damage sites at these locations in asynchronously cycling cells can result in the triggering of cellular senescence.
Our mapping experiments revealed that γH2AX modified chromatin was distributed non-randomly and, unexpectedly, represented a small fraction of total human chromatin in asynchronously cycling SR hADSCs:  ̃1.4% (200,977 γH2AX modified nucleosomes), and it represented an even smaller portion in SEN hADSCs:  ̃1.2% of total chromatin (173,877 γH2AX modified nucleosomes). The majority of γH2AX modifications (SR: 65.4%, SEN: 65.2%) mapped to transposable elements (TEs), most of which were SINE/Alu, L1, or LTR retrotransposons (FIG. 10). In addition to the overall abundance of retrotransposons bearing γH2AX modified nucleosomes, this genomic element also showed the greatest difference in relative frequencies of γH2AX marks for SR versus SEN hADSCs (FIG. 2C). SINE/Alu elements were the most enriched for γH2AX modified nucleosomes in SR cells compared to SEN cells, whereas L1s had the greatest relative increase of γH2AX modified nucleosomes in SEN cells (FIG. 2C). Apart from genic sequences, the retrotransposal portion of the genome is known to impede the progression of replication machinery (Scheifele et al. (2009) Proc. Natl. Acad. Sci. USA, 106: 13927-13932; Voineagu et al. (2008) Proc. Natl. Acad. Sci. USA, 105: 9936-9941; de la Loza et al. (2009) DNA Repair (Amst), 8: 620-626), suggesting that a portion of γH2AX detected in asynchronously dividing cells (SR sample) might be caused by replication-fork pausing or collapse as described previously in yeast (Admire et al. (2006) Genes Dev., 20: 159-173; Cha and Kleckner (2002) Science, 297: 602-606; de la Loza et al. (2009) DNA Repair (Amst), 8: 620-626; Deshpande and Newlon (1996) Science, 272: 1030-1033).
Our data suggest a role for retrotransposons in DNA damage-mediated cellular response related to normal cell cycle progression (Curcio et al. (2007) Mol. Cell Biol., 27: 8874-8885; Scheifele et al. (2009) Proc. Natl. Acad. Sci. USA, 106: 13927-13932), and imply that these elements may contribute to the aging phenotype in hADSCs (Rudin and Thompson (2001) Genes Chromosomes Cancer, 30: 64-71; Wang et al. (1999) Mutat. Res., 433: 147-157). Our data further corroborate the observation that the retrotransposal portion of the genome is particularly sensitive to DSB, a critical finding considering that this type of DNA breakage has been recently linked directly to genomic rearrangements in cancer (Eickbush (2002) Nat. Genet., 31: 126-127; Gosselin et al. (2009) Cancer Res., 69: 7917-7925; Lin et al. (2009) Cell, 139: 1069-1083; Rudin and Thompson (2001) Genes Chromosomes Cancer, 30: 64-71; Weinstock et al. (2006) DNA Repair (Amst) 5: 1065-1074).
Size and Distribution of γH2AX Chromatin in SR and SEN hADSCs
Previous studies have indicated that γH2AX forms within minutes of DSB formation and can cover large (thousands to millions of base pairs) regions of chromatin (Redon et al. (2003) EMBO Rep., 4: 678-684; Rogakou et al. (1999) J. Cell Biol., 146: 905-916). Therefore, we investigated the continuity of γH2AX chromatin in both asynchronously dividing SR and SEN hADSCs.
We developed a maximum segment-based algorithm to identify contiguous genomic regions that have anomalously high densities of γH2AX modified sites (Supplemental Methods). Such γH2AX-dense regions were taken to represent individual γH2AX chromatin clusters, and the distributions of the clusters were evaluated for SR and SEN cells. There was a single dominant γH2AX cluster size peak in both SR and SEN cells, which corresponded to approximately mono-nucleosomal-sized fragments that probably marked single DSB sites (FIG. 11A). The total amount of γH2AX mono-nucleosomal chromatin did not change significantly upon cellular senescence of adult stromal cells (FIG. 11B): there was  ̃21 MB of mononucleosomal sized chromatin in each cell type and the difference between SR and SEN cells was only  ̃0.6%. Our computational analysis did not reveal a particular genomic category that is significantly enriched for mono-nucleosomal damage accumulation upon cell cycle arrest in SEN hADSCs (Table 3). However, a majority of the mono-nucleosomal γH2AX sites (67.3% in SR and 65.8% in SEN) were associated with the retrotransposal portion of the genome, with SINEs contributing 34.9% and 30.0% and LINEs accounting for 32.4% and 35.78% of these sites in SR and SEN hADSCs, respectively (Table 3).
The γH2AX cluster size distributions were largely coincident for the majority of the clusters, but began to diverge substantially in tails of the distributions, which were occupied by the largest clusters (FIG. 11A). This difference between the SR versus SEN γH2AX cluster size distributions was used to delineate mid-size and large clusters. Relative entropy values, calculated as a measurement of the difference between the two distributions (Supplemental Methods), were calculated over a range of cluster length threshold values. The cluster length threshold value that maximized the relative entropy between the SR versus SEN γH2AX cluster size distributions was taken as a cut-off between mid-size and large clusters (FIG. 11C).
In contrast to what was observed for mono-nucleosomal sites, there were substantial differences in the amount of, as well as the genomic features occupied by, large γH2AX clusters. The total amount of large γH2AX clusters dropped more than three-fold from the SR (37,467,892 bp) to the SEN (11,243,429 bp) samples (FIG. 11B). In SR cells, 47.9% of large cluster chromatin was associated with the genic portion of the genome including: (i) gene promoters, (ii) introns, (iii) exons and, (iv) intron-exon junctions (Table 3). In SEN non-replicating samples, large γH2AX clusters were shifted to intergenic regions, with a decrease in genic sequences to 35.2%.
We hypothesized that this γH2AX deposition might result from topological problems arising from replication fork collision with transcription machinery. Our data support such a hypothesis since the dynamic of large clusters was shifted to intergenic regions in non-replicating SEN samples, with drastic decreases in exonic and intronic sequences. It is tempting to speculate that large genic and intergenic clusters of γH2AX might represent hot spots not only for chromosome fragility at the G2-M transition (SEN samples), but also for sister chromatid entanglement during the S phase of SR hADSCs.
γH2AX Chromatin and Gene Density
Transcriptional activity may represent an obstacle for progressing replication forks, resulting in their collapse and the accumulation of DSB (reviewed in Zegerman and Diffley (2009) DNA Repair (Amst) 8: 1077-1088). Therefore, functional genic regions such as exons and promoters are possibly relatively enriched with γH2AX modified nucleosomes in SR hADSCs, in contrast to SEN cells that have ceased their replication activity. Also, one may expect that gene-dense human chromosomes would be prone to γH2AX accumulation, and if this damage were not resolved during checkpoint activation, perhaps due to wear-and-tear of the DNA repair machinery, this could trigger the senescent phenotype. If this were the case, one would expect to observe γH2AX enrichment at similar locations within the chromatin of SR and SEN cells, and the amounts of γH2AX chromatin would be positively correlated with gene density in both phenotypes.
To test these two hypotheses, we evaluated the overlap between γH2AX chromatin sites in SR and SEN cells and compared the gene density of human chromosomes to the density of γH2AX sequence tags in SR and SEN cells. The overlap of γH2AX chromatin sites between cell types was small:  ̃5%. Gene density and γH2AX tag density were significantly positively correlated in SR samples, and negatively correlated in SEN samples (FIG. 2D and Table 4).
This difference in correlations for SR versus SEN cells is consistent with the transcription versus replication complex conflict model explaining the excess of genic-region-specific damage in SR hADSCs, and points out that the majority of damage in SEN cells is not related to unrepaired damage accumulation caused by the collision of replication forks and transcriptional complexes.
Considering that about 50% of γH2AX modifications are associated with the retrotransposal portion of the genome (FIG. 10), and it has been previously reported that the two most abundant categories of the retrotransposons, such as SINES and LINE, are differentially enriched in gene-dense and gene-poor chromatin (intergenic), respectively, with a bias toward GC-rich DNA (Lander et al. (2001) Nature, 409: 860-921; Mouse Genome Sequencing Consortium, Waterston et al. (2002) Nature 420: 520-562), we next investigated the correlation between γH2AX accumulation and the GC-content of human chromosomes. GC-rich genomic areas are also prone to the accumulation of DNA damage (Cha and Kleckner (2002) Science, 297: 602-606), but there is no a priori reason to expect a difference in GC-related damage accumulation between SR and SEN cells. Indeed, when chromosomal GC content and γH2AX tag density were compared, as was done for gene density, we observed significantly positive correlations for both SR and SEN hADSCs (FIG. 2E and Table 4). The positive correlation of GC content and γH2AX tag density in SEN cells stood in contrast to the negative correlation between gene density and γH2AX tag density in the same cells. This result is unexpected, since it is known that GC content is positively correlated with gene density. To tease apart these two genomic features, we performed a partial correlation analysis between gene density and γH2AX tag density in SEN cells by controlling for GC content. When partial correlation was performed in this way, the correlation between gene density and γH2AX tag density in SEN cells became more highly negative and statistically significant (Table 4).
Our data indicate a redistribution of γH2AX modified nucleosomes from genic regions in SR to non-genic regions in SEN hADSCs. This is consistent with what is seen when the relative enrichments of γH2AX modified nucleosomes are compared for SR and SEN cells genome-wide (FIG. 2C). The redistribution of γH2AX during senescence may also explain the excess of SINE/Alu-associated γH2AX modified nucleosomes in SR cells, since SINE/Alus are enriched in and around genes, along with the excess of L1 γH2AX modified nucleosomes in SEN cells, as L1s are primarily intergenic (FIG. 2C).
Promoter Distribution of γH2AX
Previously, transcriptional activation has been reported to require the formation of transient DSB in the promoters of inducible genes (Ju et al. (2006) Science, 312: 1798-1802). Later it was suggested that transcriptional misregulation upon cellular aging might stem from the inability to successfully repair DSB (Tower (2006) Cell Metab., 4: 101-103). Under these conditions, the transient DSB in SR hADSCs would become persistent DNA damage sites in SEN cells.
We observed significant changes in γH2AX accumulation within proximal promoter regions of protein-coding genes between SR and SEN cells. Consistent with the overall genomic distribution of γH2AX, there was a twofold increase in γH2AX accumulation in SR cell promoters than seen for SEN cells;  ̃3.9% of SR cell promoter sequences were occupied by γH2AX compared to 2.0% for SEN cells (Table 5). Furthermore, there was relatively little overlap in genes with γH2AX-modified promoters between SR and SEN cells (FIG. 2G). For both SR and SEN hADSCs, γH2AX showed periodic enrichment at specific positions from −2 kb to +2 kb relative to transcription start sites (TSS) (FIG. 2F). These enrichment patterns are consistent with nucleosome phasing around TSS in both SR and SEN cells; however, the distributions were shifted, suggesting a repositioning of γH2AX modified nucleosomes relative to TSS in SEN samples.
We evaluated genes with γH2AX modified promoters in SEN hADSCs (Table 6) in order to assess their potential impact on cellular function related to the aging phenotype. There were a number of over-represented gene ontology (GO) functional categories among this set of genes, including the related cell cycle (GO:0007049) and cell proliferation (GO:0008283) categories (Table 7). Genes in these categories have demonstrated effects on cell cycle progression and cell division (Table 8). When we performed a similar analysis for the group of cell cycle regulator genes (Table 1 and data not shown), we did not observe any correlation between senescence-associated downregulation of transcriptional activity of the genes and γH2AX accumulation in their promoters.
Our data suggest that down-regulation of tested cell cycle regulators in SEN hADSCs at the transcriptional level is not related to accumulation of DSB as previously proposed (Tower (2006) Cell Metab., 4: 101-103).
Peri-Telomeric Distribution of γH2AX Chromatin and Human Adult Stem Cell Senescence
Previous studies have reported that DSBs causing senescence in somatic cells arise directly from dysfunctional telomeres (Celli and de Lange (2005) Nat. Cell Biol., 7: 712-718; d'Adda di Fagagna et al. (2003) Nature, 426: 194-198; Takai et al. (2003) Curr. Biol., 13: 1549-1556). It was speculated that telomeric erosion might provide the reservoir of persistent DNA damage signal resulting in sustained p53 activation and manifestation of the SEN phenotype.
To ascertain whether dysfunctional telomeres are directly engaged in DDR in SEN hADSCs, we investigated the chromosomal enrichment of γH2AX in the telomeric and peri-telomeric areas of the human genome.
Telomeric regions are highly repetitive and have not been extensively characterized at the sequence level; therefore, we could not unambiguously map telomeric γH2AX ChIP-seq tags to the human genome. To evaluate γH2AX accumulation at telomeres, we therefore analyzed the counts of ChIP-seq tags that bore a specific telomeric repeat sequence motif (TTAGGG/AATCCC, (SEQ ID NO:9)) among all sequencing tags (mapped uniquely and unmapped due to their repetitive nature). SR cell samples had an average of 1.66% of such telomeric tags while SEN cells had 1.63%. These percentages did not reveal an over-representation of telomeric tags, and the difference between the two cell types was not significant.
We defined peri-telomeres as 100 kb regions from the end of the sequenced human chromosomes, and evaluated 15 Mb of genomic DNA extending into the chromosome arms for SR and SEN hADSCs (FIG. 3A). Overall, SR samples showed an enrichment of γH2AX over the entire region, while SEN samples were depleted for γH2AX. However, for both asynchronously cycling adult stem cells and for their SEN counterparts there was a marked decrease in the γH2AX density approaching and into the peri-telomeric regions (FIG. 3A). In fact, the total fraction of γH2AX modified chromatin found in peri-telomeres was quite small (SR=0.07%, SEN=0.05%; FIG. 10), but this could be due to the relatively small portion of the genome made up of peri-telomeres as defined here. To evaluate this possibility, we computed the genomic enrichment of γH2AX modified chromatin in peri-telomeres within SR and SEN cells as the fraction of modified chromatin normalized by the fraction of the genome occupied by peri-telomeric sequences. When peri-telomeric regions were evaluated in this way, they showed 1.5-fold (SR) and 2.1-fold (SEN) depletions in γH2AX modified nucleosomes (FIG. 12).
Our data support the notion that, contrary to what has been observed in human somatic cells (d'Adda di Fagagna et al. (2003) Nature, 426: 194-198), the aging of human adult stem cells might not be directly triggered by extensive peri-telomeric damage due to shortening of telomeres. This observation further corroborates findings previously reported (Sedelnikova et al. (2004) Nat. Cell Biol., 6: 168-170). However, close inspection revealed a number of individual chromosomes with significant telomeric accumulations of γH2AX. The peri-telomeric γH2AX distribution on these chromosomes was markedly asymmetrical with respect to the chromosome arms (FIG. 3C). In SR cells, chromosomes 10, 12, 17, and 18 had γH2AX enriched telomeres, as did chromosomes 4, 7, and 18 in SEN hADSCs. Despite the differences in peri-telomeric γH2AX accumulation between SR and SEN cells, the chromosome arm asymmetry was always preserved. The relative levels of chromosome arm-specific γH2AX peri-telomeric accumulation were identical for SR and SEN cells (FIG. 3C).
Pericentric distribution of γH2AX
Compelling evidence indicates that pericentric chromatin is of critical importance for the regulation of kinetochore assembly, sister chromatid cohesion, spindle attachment, and chromosome segregation (Blower and Karpen (2001) Nat. Cell Biol., 3: 730-739; Bernard et al. (2001) Science 294: 2539-2542; Schueler and Sullivan (2006) Annu. Rev. Genomics Hum. Genet., 7: 301-313). Data in yeast also point to a causal relationship between fork stalling and chromosomal DSB in areas known as “replication slow zones” (RSZ) (Cha and Kleckner (2002) Science, 297: 602-606), many of which are associated with pericentric and centromeric chromatin. Also, spindle assembly checkpoint (SAC) acts semi-redundantly with the DNA damage checkpoint to enact long-term cell-cycle arrest in the presence of unresolved DSB, thereby preventing the segregation of the damaged chromatin (Dotiwala et al. (2010) Curr. Biol., 20: 328-332; Elledge (1996) Science, 274: 1664-1672; Harrison and Haber (2006) Annu. Rev. Genet., 40: 209-235; Lim et al. (2009) Trends Cell Biol., 19: 325-333). Therefore, we further investigated whether human pericentric chromatin shows signs of fragility upon aging of adult stem cells, and whether or not it is resistant to γH2AX accumulation in cycling cells.
In our data, the distributions of γH2AX modified chromatin at peri-centromeric regions were markedly different from those seen for peri-telomeres. While peri-centromeres also contained a small fraction of the overall γH2AX modified chromatin across the genome (SR=0.78%, SEN=0.82%; FIG. 10), there was substantially more γH2AX modified chromatin in peri-centromeres than expected based on the fraction of the genome they occupy (FIG. 3B). Similar to chromosome-specific damage in peri-telomers, there were a number of individual chromosomes with highly enriched levels of peri-centromeric γH2AX accumulation (FIG. 3D). Overall peri-centromeres exhibited 2.0- and 2.1-fold enrichments for SR and SEN cells respectively (FIG. 12), thus indicating intrinsic susceptibility (“fragility”) of these chromosomal regions. Peri-centromeres were not only enriched for γH2AX, but they were also one of the few genomic features that showed relatively greater γH2AX presence in SEN cells (FIG. 2B, FIG. 3D, 3E). Chromosomes 6, 14, 15, 16 and 21 demonstrated the most significant γH2AX enrichment in SEN cells (FIG. 3E).
Interestingly, our data did not reveal uniform and gradual accumulation of γH2AX in pericentric chromatin on the same chromosomes upon senescence, suggesting that damage accumulation in senescence is not necessarily a result of simply passing on a cell-cycle-associated defect in the DNA repair of these regions.
CENP-A and CREST Colocalization with Persistent DNA Damage Foci in Senescent ADSCs
An increase in pericentric γH2AX accumulation only on a subset of human chromosomes in SEN hADSCs could be indicative of: i) unresolved DSB (possibly due to a change in the characteristics of their pericentric heterochromatin and the molecular machinery responsible for its repair), and/or, ii) defects in the assembly of kenetochore that provokes spindle-generated DNA damage, resulting in SAC (Guerrero et al. (2010) Proc. Natl. Acad. Sci. USA, 107: 4159-4164). Both of these events, acting separately or synergistically, might trigger senescence of human adult stem cells.
To uncouple these two possible scenarios, we first evaluated colocalization of central to kinetochore assembly centromeric histone H3 variant CENP-A with persistent γH2AX/53BP1 foci in SEN hADSCs. SR and SEN hADSCs were immunolabeled with antibodies to the 53BP1 and CENP-A proteins and studied by confocal fluorescent microscopy. In nearly 75% of the cases, CENP-A colocalized with large 53BP1/γH2AX in SEN hADSCs (FIG. 4A, B, FIG. 13A, B). Similar overlapping immunostaining patterns were observed when human anti-kinetochore serum (α-CREST) and anti-53BP1 antibodies or α-CREST and anti-γH2AX antibodies were assessed by confocal microscopy (FIG. 13C, D). As expected, we did not observe (detectable by our method) colocalization of centromeric regions with either 53BP1 (FIG. 4A) or γH2AX (data not shown) in interphase of SR hADSCs, since no prolonged cell cycle arrest was triggered in these cells (FIG. 1B, FIG. 7B) nor γH2AX/53BP1 foci were formed (FIG. 1C). Our data indicate that centromeric regions are embedded within persistent γH2AX/53BP1 DNA damage foci, formed upon senescence of hADSCs, and yet no defects in centromeric chromatin CENP-A incorporation/inner kinetochore assembly are observed. CENP-A is required to recruit many other centromere and kinetochore proteins (McClelland et al. (2007) EMBO J. 26: 5033-5047), with the exception of proteins located in adjacent heterochromatin domains, such as heterochromatic protein 1 (HP1) (Blower and Karpen (2001) Nat. Cell Biol., 3: 730-739). We cannot rule out a possibility that defects in the other kinetochore/spindle components acting downstream of CENP-A affect the assembly of mitotic spindle, and that such deficiencies result in increased levels of γH2AX in peri-centromeric regions due to inappropriate chromosome segregation and cell cycle arrest (Dalton et al. (2007) Cancer Res., 67: 11487-11492; Guerrero et al. (2010) Proc. Natl. Acad. Sci. USA, 107: 4159-4164; Quignon et al. (2007) Oncogene, 26: 165-172). In fact, we observed significant transcriptional downregulation upon hADSCs senescence of KNTC1 (hROD), a component of tension-sensitive kinetochore complex, known as the RZZ (Table 1) (Chan et al. (2000) Nat. Cell Biol., 2: 944-947).
Senescence-Associated γH2AX Foci are Sites of Active Pol-III Transcription
There is a body of literature suggesting that pericentric chromatin is essential for the establishment and function of centromeres (Bernard et al. (1994) Exp. Cell Res., 214: 373-380; Dalal and Bui (2010) Curr. Opin. Cell Biol., 22: 392-402). In fission yeast, native pericentric chromosomal regions are permissive to RNA Pol-III (Chen et al. (2008) Nature, 451: 734-737; Scott et al. (2007) PLoS One 2: e1099), indicating a complex relationship between heterochromatic assembly, maturation of centromeric chromatin, and transcription. Mammalian chromatin modifying activities associated with cellular aging, such as sirtuins, have been previously reported to contribute not only to the formation of facultative heterochromatin (Vaquero et al. (2007) Nature 450, 440-444), but to also be involved in the mediation of repression at constitutive heterochromatic regions such as pericentromeric chromatin. Importantly, the generation of DSB by oxidative stress leads to increased transcription of pericentric satellite repeat DNA in a model of mammalian embryonic stem cells (Oberdoerffer et al. (2008) Cell, 135: 907-918).
With this in mind, we investigated the possibility of transcriptional activity associated with persistent γH2AX/53BP1 foci in SEN hADSCs. We combined the transcriptional assay of 5-fluorouridine (5-FUr) incorporation with detection of the 53BP1 and promyelocytic leukemia (PML) proteins upon senescence of hADSCs (described in Experimental Procedures and Casafont et al. (2006) Neuroscience, 140: 453-462). Nuclear incorporation of a halogenated nucleotide analog into nascent RNA was visualized in the whole nucleus by immunocytochemistry with an anti-BrdU antibody (Boisvert et al. (2000) J. Cell Biol., 148: 283-292). We chose a specific nuclear compartment, PML body, since it has been suggested to play a role in aspects of transcriptional regulation and/or protein segregation (Carbone et al. (2002) Oncogene 21: 1633-1640; Dellaire et al. (2006) J. Cell Biol., 175: 55-66; Dellaire et al. (2009) Cell Cycle, 8: 3750-3769). We observed a close juxtapositioning of PML nuclear bodies and senescence-associated persistent 53BP1 foci (FIG. 4C). Nascent RNA transcripts visibly decorated the periphery of the PML nuclear body and showed clear colocalization with the 53BP1 signal (FIG. 4C). These observations are consistent with previous reports indicating a possible involvement of RNA in 53BP1 foci formation after IR-induced damage in NIH3T3 and Hela cells (Pryde et al. (2005) J. Cell Sci., 118: 2043-2055).
It has been proposed that transcriptional control through retroelements may facilitate pericentric transcription (Ugarkovic (2005) EMBO Rep., 6: 1035-1039). The large clusters of γH2AX chromatin in our experiments colocalized with pericentric regions known to be enriched for SINE/Alu retrotransposal repeats (FIGS. 3B, 3D, 3E, Table 3, and Schueler and Sullivan (2006) Annu. Rev. Genomics Hum. Genet., 7: 301-313). Considering that transcriptional activity of SINE/Alu repeats was largely upregulated in SEN hADSCs (FIG. 14) and driven by the Pol-III transcriptional complex (Deininger (1989) Mobile DNA (Washington, D.C., American Society for Microbiology)), we examined the dynamic formation of the persistent 53BP1/γH2AX foci and FUr-incorporation in response to the inhibition of Pol-III transcription by the drug tagetin (Allen et al. (2004) Nat. Struct. Mol. Biol., 11: 816-821; Wang et al. (2003) Mol. Biol. Cell, 14: 2425-2435).
Senescent hADSCs were either cultured in the presence of 10 tM inhibitor of Pol III transcriptional activity, tagetin, for 2 hrs at 37° C. (+tagetin) or in the absence of the inhibitor treatment (-tagetin). Nuclear RNA was labeled by addition of 2 mM FUr to the culture for 10 min at 37° C. After fixation, cells were immunolabelled with anti-BrdU antibody (red) to detect FUr incorporation sites in combination with anti-53BP1. Double labeling experiment revealed FUr incorporation sites exclusively localized with persistent DNA damage sites throughout entire depth of z-stack images. Tagetin inhibition of Pol III dependent transcription resulted in complete disappearance of FUr incorporation, and loss of compaction of the DNA damage sites as detected by more defuse 53BP1 staining.
Our data revealed that SEN hADSCs treated with the inhibitor of Pol-III tagetin showed impaired 53BP1 focus-forming capacity. Further, comparative strand-specific RT-PCR analysis of a number of SINE/Alu sequences associated with DNA damage in pericentric areas of chromosomes 10 (designated MIR and Alu) and 21 (designated AluSx and AluJb) revealed that upregulation of Alu transcriptional activity correlates with the presence of persistent DNA damage in SEN samples, as shown for the Alu repeat in the vicinity of the 7.6 kb γH2AX cluster. Our data demonstrate that, despite the fact that the transcriptional activity of the chosen repeats (AluJb, AluSx, and Alu) is recorded regardless of the state of the hADSC. Alu transcription is upregulated on chromosome 10 where damage is observed only upon cellular senescence. No transcriptional activity from the MIR element was recorded in either the SR or SEN state of hADSCs. These data also support our observation that γH2AX/53PB1 foci in SEN hADSC are centers of Pol-III dependent transcriptional activity (FIG. 4C). To our surprise, by using a conventional ChIP assay, we observed an increase in the recruitment of a Pol-III transcriptional complex (designated TFIIIC) to the MIR repeat during senescence even in the absence of the MIR-recorded transcription (FIG. 5). This could be due to an ability of TFIIIC to not only be essential for loading PoI-III machinery (Young et al. (1991) Science, 252: 542-546; Kundu et al. (1999) Mol. Cell. Biol., 19: 1605-1615; Noma et al. (2006) Cell, 125: 859-872), but also for its ability to participate in the structural organization of chromatin cohesion and condensation within the eukaryotic nucleus as recently demonstrated in yeast (Iwasaki et al. (2010) Mol. Biol. Cell, 21: 254-265).
The presence of large tracts of satellite DNA limits the selection of strand-specific primers for reverse transcription due to a higher degree of sequence complementation, thus preventing further meticulous assessment of multiple, densely-packed SINE/Alu repeats within and near these genomic loci. Nevertheless, our data, together with multiple reports from yeast and mammals (Lunyak et al. (2007) Science, 317: 248-251; Noma et al. (2006) Cell, 125: 859-872; Oki and Kamakaka (2005) Mol. Cell, 19: 707-716; Scott et al. (2006) Curr. Biol., 16: 119-129; Willoughby et al. (2000) J. Biol. Chem., 275: 759-768), suggest a general role for the Pol-III transcription in genome organization, and support the hypothesis that, similar to centromeric tRNA genes in yeast (Iwasaki et al. (2010) Mol. Biol. Cell, 21: 254-265), pericentric SINE/Alu retrotransposons in humans may be integral to centromere functioning in self-renewing hADSCs, and upon senescence of hADSCs.
Impaired Cohesin and Condensin I Association with Pericentric γH2AX Foci in Senescent hADSCs
Multiple lines of evidence indicate that the processes of chromosome repair and segregation are directly linked through cohesin, an evolutionarily-conserved protein complex (Heidinger-Pauli et al. (2008) Mol. Cell, 31: 47-56; Kim et al. (2002) Proc. Natl. Acad. Sci. USA, 99: 1241-1246; Sjogren and Nasmyth (2001) Curr. Biol., 11: 991-995; Strom et al. (2004) Mol. Cell, 16: 1003-1015; Unal et al. (2004) Mol. Cell, 16: 991-1002). Specific association of cohesin with SINE/Alu repeats was also previously reported (Hakimi et al. (2002) Nature, 418L 994-998). The Scc1/Rad21/Mcd1 subunit of this complex is central to cohesin function and has been shown to be necessary for sister chromatid cohesion and kinetochore function in vertebrate cells (Sonoda et al. (2001) Dev. Cell, 1: 759-770), as well as G1 and G2-M DNA damage checkpoints (Jessberger (2009) EMBO J., 28: 2491-2493). Reportedly, the cohesin complex becomes enriched at DSB sites and facilitates DNA repair by homologous recombination (HR) (Bekker-Jensen et al. (2006) J. Cell Biol., 173: 195-206; Potts et al. (2006) EMBO J., 25: 3377-3388). Inefficient postreplicative repair of DSB can arise from a deficiency in either cohesin loading or the conversion of cohesin to its cohesive state by Eco1 (ctf7) (Strom et al. (2004) Mol. Cell, 16: 1003-1015; Unal et al. (2004) Mol. Cell, 16: 991-1002; Unal et al. (2007) Science, 317: 245-248). In addition, one cannot overlook the importance of the condensin-related pathway in these events. Condensin loads cohesin onto the chromosome and is known to form two different complexes: condensin I and II (Samoshkin et al. (2009) PLoS One, 4: e6831). Importantly, the condensin I complex has been implicated in the function of pericentric heterochromatin. The depletion of the Cap-H subunit of the condensin I complex results in defects associated with alterations in the structural integrity of centromere-proximal heterochromatin in Drosophila (Oliveira et al. (2005) Mol. Cell. Biol., 25: 8971-8984). It has been demonstrated that Cap-H depletion does not affect CENP-A incorporation into the centric chromatin or kinetochore assembly, but does result in severe depletion of cohesin Scc1/Rad21/Mcd1, triggering cell cycle defects.
We hypothesized that SINE/Alu transcription in pericentric chromatin can affect condensin and cohesin loading, thus blocking DNA repair by HR. This inability to repair DNA damage will directly or indirectly result in the cessation of the SR capacity of hADSCs by triggering cellular senescence. Therefore, we investigated the recruitment components of the cohesin complex Scc1/Rad21/Mcd1, condensin I complex (Cap-H), and histone acetylase Eco1 to the SINE/Alu repeats in the vicinity of a persistent pericentric γH2AX cluster on chromosome 10. Data obtained by conventional ChIP analysis demonstrated a statistically significant loss in the recruitment of Scc1/Rad21/Mcd1 and Cap-H to the MIR and Alu repeats during senescence of hADSCs (SEN red bars in FIG. 5C) when compared to the status of the same genomic locations in SR cells (SR blue bar in FIG. 5C). Surprisingly, the recruitment of histone acetylase Eco1 was largely correlated with the formation of persistent γH2AX clusters in senescence, and appeared to be Scc1/Rad21/Mcd1-independent. This suggests another, not yet identified, role for the Eco1 protein in DDR, different from its previously reported participation in the modulation of cohesiveness of Scc1/Rad21/Mcd1 (Hakimi et al. (2002) Nature, 418L 994-998; Strom et al. (2004) Mol. Cell, 16: 1003-1015; Unal et al. (2004) Mol. Cell, 16: 991-1002; Unal et al. (2007) Science, 317: 245-248).
Together these data further support the correlation between an increase in SINE/Alu transcription, and, (i) DNA damage accumulation, (ii) defects in pericentric recruitments of condensin I (Cap-H) and cohesin (Scc1/Rad21/Mcd1), and (iii) senescence of hADSCs.
Functional Significance of the SINE/Alu Transcripts in Senescence of hADSCs
SINE/Alu elements occupy 6% of the human genome, significantly outnumbering other families of pseudogenes generated by retrotransposition (Weiner et al., 1986). The human SINE/Alu retrotransposon is a tandem repeat of two B1 elements connected by an A-rich linker (FIG. 6A), and the secondary structure of its RNA was previously reported (Sinnett et al. (1991) J. Biol. Chem., 266: 8675-8678). In order to evaluate the functional significance of SINE/Alu transcription in establishing and/or mediating cellular senescence upon ex-vivo aging, SEN hADSCs were genetically manipulated to stably express shRNA, targeting the generic SINE/Alu transcript. We took advantage of the common sequence feature of SINE/Alu retrotransposons to generate a number of lenti-virus-delivered shRNA constructs carrying GFP for assessment of transduction efficiency (FIG. 6B; Experimental Procedures).
Knockdown of the generic SINE/Alu transcript in transduced hADSCs stably expressing shRNA against different portions of SINE/Alu was demonstrated by Northern Blot hybridization (FIG. 6C) with a transduction efficiency of nearly 99% (FIG. 6B). hADSCs transduced with lentiGFP expressing sh-132Alu RNA demonstrated a near complete knockdown of the generic SINE/Alu transcript. hADSCs transduced with lentiGFP (control) or lentiGFP sh-193Alu exhibited little or no change at the SINE/Alu transcription level. Surprisingly, SEN hADSCs lines stably expressing sh-132Alu RNA had a dramatically altered morphology and exhibited an increase in proliferation as detected in 3[H] thymidine uptake DNA synthesis experiments (FIG. 6D). In contrast, SEN hADSCs transduced with lentiGFP (control), or treated with polybrene alone (PB), did not show any significant changes in their proliferation rate when compared with wild type (wt) SEN hADSCs (FIG. 6D). Faithful re-establishment of DNA synthesis upon knockdown of the SINE/Alu transcript was further recorded 96 hours after transduction. Unexpectedly, when compared to wt SEN hADSCs, prior SEN hADSCs lines stably expressing sh-132Alu RNA demonstrated statistically significant upregulation of pluripotency factors Nanog and Oct4 as detected by qPCR analysis (FIG. 6E). The levels of Nanog and Oct4 in wt SEN hADSCs were below the threshold of the qPCR method.
In summary, this finding raises the possibility that SINE/Alu RNA serves as an unexpected but key component of the machinery that controls stem cell pluripotency. Consistently, we observed that prior SEN hADSC with stable knockdown for generic SINE/Alu RNA were viable, and after 7 days in culture had formed cell aggregates (embryoid-like structures) which tested positive for alkaline phosphatase activity, a marker of induced pluripotency (FIG. 6F).
Discussion
Repetitive elements are implicated in changing/controlling properties of the chromatin in many systems, but few studies have addressed their functional significance in the context of cellular aging, in particular, in human adult stem cells. Several observations have linked persistent DDR to the manifestation of cellular senescence phenotype and ultimately to cell aging. Although cellular senescence is thought to be naturally irreversible cell-cycle arrest induced by DDR signaling, it is still unclear what determines the inability of cells with activated DDR to promptly and properly fix DNA lesions and restore their proliferative capacity. However, what has become clear from the cumulative studies of different models of cellular senescence is that these determinants can vary among cell types and in intensity, duration and nature of the DNA damage.
This raises the possibility that specific genomic regions/elements exist within cells where DNA damage is less easily repaired, possibly in a manner which is affected by age-dependent changes in chromatin structure and/or chromosomal organization. These functional elements have not been previously identified. In this study we have addressed this question.
Central to this working hypothesis was our initial effort to perform a location analysis of γH2AX histone modification, which appears on the nucleosomes adjacent to the sites of DNA lesions. By using ChIP-seq analysis with single nucleosome resolution we were able to map locations of repairable DNA damage, which did not affect the proliferative capacity of hADSCs, and perform a comparative analysis of the dynamic of this damage in the onset of cellular senescence. Based on the distribution of γH2AX modifications, our data allowed us to conclude that neither repairable nor persistent DNA damage occurs randomly in SR or SEN hADSCs, and both types of damage are biased toward the transposable portion of the genome. Increased transcriptional activity of SINE/Alu retrotransposons in the aging human adult stem cells significantly correlates with the formation of persistent DNA damage foci, indicating a role in interfering with DNA repair. We provide a mechanistic explanation for retrotransposons' transcriptional interference with DDR: defective recruitment of the cohesin and condensin I complexes to pericentric chromatin upon their activation. Both of these complexes are critical to chromatin organization and damage repair of these genomic regions. We propose a novel model whereby SINE/Alu transposons act by altering chromatin structure in the vicinity of DNA lesions, thus blocking their repair (FIG. 6G). This event is integral to establishing/mediating persistent DDR upon ex-vivo aging of human adult stem cells.
Although it is still an open question what regulates a transcriptional activity of SINE/Alu retrotransposons, it is tempting to speculate that this might be due to the loss of epigenetic control for DNA methylation with ex-vivo aging of hADSCs. However, we cannot exclude the possibility that upregulation of the SINE/Alu transcripts is not directly related to an increase in their transcriptional activity, but rather results from senescence-associated deficiencies in their posttranscriptional processing, turn over or even degradation. Whatever their nature, our finding represents the first example of SINE/Alu transcription's role in the altering the properties of pericentric chromatin in conjunction with persistent DDR and cellular senescence of human adult stem cells.
Importantly, our results demonstrate a functional significance of SINE/Alu transcripts in the mechanism of ex-vivo aging of hADSC, and challenge the dogma that cellular senescence, and ultimately cellular aging, is an irreversible process. Since a depletion of generic SINE/Alu transcripts alone is sufficient to reinstate the proliferative capacity of hADSCs, and to increase the plasticity of previously niche-restricted human adult stem cells, it is plausible to suggest that SINE/Alu retrotransposons are playing an important role in mesenchymal niche specification through yet unknown, but interconnected with adult stem cell aging, pathways.
Experimental Procedures
Antibodies
Primary antibodies used were: b-actin (Abcam #ab62760), 53BP1 (Bethyl Laboratories #A300-273A), BrdU (BD, 7580), CAP-H (Bethyl Laboratories #A300-603A), CD31 (Invitrogen #HMCD3101), CD44 (Invitrogen #HMCD4401), CD45 (Invitrogen #HMCD4501), CD105 (Invitrogen #HMCD10520), Phospho-cdc2 (Tyr15) (Cell Signaling Technologies #9111), Phospho-Chk1 (Ser345) (CST #2341), Phospho-Chk2 (Thr68) (CST #2661), CENP-A (Abcam #ab13939), human anti-centromeric autoantibody, a-CREST (Antibodies Incorporated #15-235), Eco1 (Bethyl Laboratories #A300-312A), γH2AX (Millipore #05-636), PML (Santa Cruz Biotechnology #sc-966), and Rad21 (Abcam #ab992). Secondary antibodies were AlexaFlour® conjugated donkey antibodies (Invitrogen).
Human ADSC Isolation and Expansion
Human adipose derived stem cells were isolated from human subcutaneous white adipose tissue collected during liposuction procedures. The lipoaspirate was suspended in Hank s Buffered Salt Solution (HBSS), 3.5% Bovine Serum Albumin (BSA), 1% Collagenase, type II (Sigma) in 1:3 w/v ratio and shaken at 37° C. for 50 min The cells were filtered through a 70 μm mesh cell strainer (BD Falcon #352350), treated with Red Blood Cell Lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3), and expanded ex-vivo in DMEM/F12 complete medium (DMEM/F12, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin B; Invitrogen) in 10% CO2 at 37° C. and passaged at 80% confluency, changing medium every 72-96 hours. Cumulative population doublings were calculated by summing the population doublings (PD=log(N/N0)×3.33, where N0 is the number of cells plated in the flask and N is the number of cells harvested at this passage) across multiple passages as a function of the number of days it was grown in culture.
Surface Marker Characterization
5×105 cells each were incubated for 30 min on ice in the dark with fluorochrome-conjugated antibodies (CD31, CD44, CD45 and CD105; Invitrogen) in PBS with 1% BSA (Sigma), washed and analyzed in a Guava EasyCyte Mini System (Guava Technologies, Millipore). Data analysis was done with FlowJo software (Tree Star, Ashland, Oreg.).
Assessment of Cellular Senescence and Pluripotency Phenotype
Senescence-associated J3-galactosidase activity assay was done as described in manufacturer's kit (BioVision). Cellular phenotype towards induced pluripotent cells was assayed with alkaline phosphatase staining kit (Stemgent).
Immunofluorescence
1-3×104 cells/well in 4-well slides were fixed with 4% paraformaldehyde and permeabilized with PBS, 0.5% Triton X-100. The blocking and antibody incubations were carried out in 4% normal donkey serum (NDS; Jackson Immunochemicals) in PBS. The nuclei were counter-stained with 100 ng/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma), and the slides were mounted in ProLong® Gold antifade aqueous mounting medium (Invitrogen).
Epifluorescence images were acquired on an Olympus BX60 fluorescence microscope with Spotfire 3.2.4 software (Diagnostics Instruments). Confocal images (z-series slice thickness 0.39 μm) were acquired on Zeiss LSM 510 NLO with 488 nm Argon, 543 nm HeNe, and Coherent Chameleon 2-photon lasers using a 63× planapo objective and 0.08×0.08×0.39 μm voxel dimensions. Image stacks were deconvolved using Huygens Professional 3.4.0 (Scientific Volume Imaging, Netherlands) and visualized in Bitplane Imaris 6.3.1 (Bitplane Inc., Saint Paul, Minn.).
5-Fluorouridine Labeling for RNA Transcript Detection In Situ
Senescent hADSCs, 1×104 cells/well in 4-well slides, were treated with 2 mM 5-Fluorouridine (FUr; Sigma) for 10 minutes. The nuclei were exposed with ice-cold CSK buffer (100 mM KCl, 300 mM sucrose, 10 mM Pipes pH 6.8, 3 mM MgCl2, 1 mM EGTA, 1.2 mM phenylmethylsulfonyl fluoride) with 0.5% Triton X-100 and fixed with 4% paraformaldehyde. Immunostaining was performed as described above for immunoflourescence.
Expression Profiling
Total RNAs from  ̃1.5×106 SR and SEN ADSCs were prepared by TRIZOL® method (Invitrogen). Human Cell Cycle qRCR Array (SABiosciences #PAHS-020C-2) was used for the profiling (7500 Fast PCR System, Applied Biosystems). Three RNA samples from SR hADSCs were matched to RNA samples for their replicatively SEN counterparts. Statistical significance of the transcriptional differences was calculated by T-test as described in SABiosciences manual and “www.sabiosciences.com/RTPCR”. The model-based expression values were calculated and filtered for the genes with higher or lower than two-fold differences in expression between SEN and SR samples. Categories of the genes were annotated using the PANTHER (protein analysis through evolutionary relationships) classification system.
Preparation of Nucleosomal Lysates
3×105 SR and SEN hADSCs were washed with PBS, and crosslinked in 1% formaldehyde. The cells were scraped from the plate in CSK buffer with 0.5% Triton X-100, washed, and sonicated in microccocal nuclease buffer (50 mM Tris-HCl, pH 7.9, 5 mM CaCl; NEB) with a Covaris S2 Sonicator (frequency sweep at 1% duty cycle, 5 intensity and 50 cycles per burst for 12 cycles of 10 sec each at 4° C.). Sonicates were cenrtrifuged to get rid of the cellular debris, and supernatants were incubated with 25U/tl of microccocal nuclease (NEB).
ChIP-Seq: Nucleosomal ChIP and SOLiD Library Preparation
Nucleosomal lysates were adjusted to antibody Binding buffer (25 mM Tris-HCl pH 7.9, 0.15% SDS, 1% Triton X-100, 150 mM NaCl), and immunoprecipitated with 10 μg γH2AX antibody overnight at 4° C. Protein A Sepharose-beads were blocked with 10 μg/ml glycogen, 10 μg/ml lysozyme, 5 μg/mltRNA and 20 μg/ml BSA for 1 hour at 4° C. before binding to the immuno-complexes for 1 hour at 4° C. Beads were washed with Binding buffer and again with the same buffer containing 0.5M NaCl and finally in TE (10 mM Tris-HCl pH 7.6, 1 mM EDTA). Immuno-complexes bound to beads were re-suspended in TE buffer, de-crosslinked, and purified by Proteinase K treatment, phenol:chloroform extraction, and isopropanol precipitation. DNA fragments were prepared for adapter ligation by filling-in ends by DNA polymerase I (Klenow fragment) and phosphorylating 5′ ends of PCR primers by Polynucleotide kinase (NEB), and ligated to 30-fold molar excess of SOLIDTM System 2.0 (Applied Biosystems) library adapters according to manufacturer's protocol. DNA libraries were amplified by PCR using cloned Pfu DNA Polymerase (Stratagene, Agilent Technologies. Mononucleosomal size DNA fragments were size-selected in a 2% agarose gel, cut around 200 bp size, and purified with QIAQUICK® Gel Extraction Kit (Qiagen). Emulsion PCR (ePCR) for sequencing bead preparations were done with 0.05 and 0.1 pg/μl of library DNA for each sample. Samples were sequenced on SOLiDTM System 2.0 (Applied Biosystems) according to manufacturer's protocol.
RT PCR Analysis of Retrotransposal Transcription and qPCR
Genomic coordinates of the SINE/Alu elements tested in RT-PCR were from the March 2006 Assembly (NCBI36/hgl8) of the Human Genome Browser at UCSC (genome.ucsc.edu); MIR: chr10:41922815-41922906, Alu: chr10:41928992-41929118, AluJb: chr21:10141132-10141429 and AluSx: chr21:10145344-10145644. 100 ng of total RNA was used with the RT2 First Strand Kit (SABiosciences) per reaction. The primers for first strand synthesis are at locations outside of the SINE/Alu element sequences (external or reverse primers, Table 9) and forward primers within the SINE/Alu element sequence (internal forward primers, Table 9). RPL13A, GAPDH was used as a positive control.
For real-time quantitative PCR, 1 mg of each total RNA was used for first strand cDNA synthesis with SUPERSCRIPT® III First-Strand Synthesis System for RT-PCR (Invitrogen) with random priming. qPCR was performed using RT2 SYBR Green qPCR MasterMix (SABiosciences) and run in LIGHTCYCLER® 480 II (Roche). qPCR primers are listed in Table 9. Data analysis of relative gene expression was done by 2-DCt method.
Lentiviral shRNA Constructs
Lentiviral shRNA constructs to knockdown genetic SINE/Alu transcript were designed as follows: oligonucleotides Lenti sh-132 Alu RNA: 5′-GAT CCC CCC ACC ACG CCC GGC TAA TTT TCA AGA GAA ATT AGC CGG GCG TGG TGG TTT TTG GAA A-3′ (SEQ ID NO:10) and Lenti sh-193 Alu RNA 5′-GAT CCC CCC CGG GTT CAA GCG ATT CTT TCA AGA GAA GAA TCG CTT GAA CCC GGG TTT TTG GAA A-3′ (SEQ ID NO:11), were annealed with equal amounts of their complementary strands, creating restriction site specific overhangs for cloning, and ligated into HindIII and BglII digested, gel purified pENTR/pTER+vector (Campeau et al., 2009). The constructs were confirmed by sequencing (sense strand sequence is shown above). Equal amounts of each constructs was mixed with pLenti-CMV-GFP DEST vector (Campeau et al., 2009) in LR Clonase reaction to recombine cloned shRNA production elements into a destination vector according to manufacturer's instructions (Invitrogen). The produced lentiviral plasmid was transformed into E. coli Stb13 cells (Invitrogen) for amplification.
Lentiviral Production and Transduction
293T cells were grown in DMEM complete medium (DMEM, high glucose, 10% FBS, 0.1M nonessential amino acid, 6 mM L-glutamine, 1 mM pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin) for lentiviral production, and transfected for 12 hours with pLenti sh-132Alu or pLenti sh-193Alu and pCgpV, pRSV-Rev, and pCMV-VSV-G helper plasmids (Allele Biotechnology) in 2:1:1:1 molar ratio using Lipofectamine 2000 (Invitrogen) according to standard protocol (Campeau et al., 2009). Medium was collected at 48 and 72 hours and filtered (0.45 μm). Virus was precipitated with PEG and frozen in aliquots (−80° C.). Lentiviral transductions were done in complete medium with 5 μg/ml Polybrene (Santa Cruz Biotechnology) for 12 hours. Viral titers were determined by comparing GFP positive cells counts to total population.
Proliferation Index
For each condition in 3[H]-thymidine uptake assay, 10,000 cells were treated with 1 μCi 3[H]-thymidine (Perkin-Elmer, Boston, Mass.) in DMEM/F12 complete for 24 hours. DNA was isolated from harvested cells and quantified with NanoDrop (ND-1000; NanoDrop Technologies Inc). 3[H]-thymidine uptake into cellular DNA was measured with liquid scintillation counter (LS 6500; Beckman Instruments). Lentiviral transduction was done as above, and untreated and Polybrene treated controls were carried out in parallel.
Northern Hybridization
RNA was isolated from SR and SEN hADSCs (with or without lentiviral transduction) with mirVANA (Ambion, Invitrogen) kit, and 2 mg of total RNA/lane was run on a 7M Urea, 6% polyacrylamide, TBE. Gel was stained with ethidium bromide, photographed and electroblotted onto HybondTM-N+(Amersham, GE Healthcare). Hybridizations were performed in 6×SSC, 4×Denhardts′, 0.1% SDS at 37° C. Oligonucleotide probes were labeled with Biotin-16-dUTP (Roche) by terminal transferase (NEB). Northern was visualized with streptavidin-HRP (Invitrogen) using ECL Plus Western Blotting Detection Reagent (Amersham, GE Healthcare) and Amersham Hyperfilm (GE Healthcare). Oligonucleotide used as probes were: SINE/Alu 132:5′-CCA CCA CGC CCG GCT AAT TT-3′ (SEQ ID NO:12) and SINE/Alu 90: 5′-CGC GCG CCA CCA CGC CCG GCT AAT TTT TGT ATT TTT AGT AGA GAC GGG GTT TCA CCA TGT TGG CC-3′ (SEQ ID NO:13).
Example 2
Supplemental Methods
Outlier Removal.
γH2AX ChIP-seq tags were mapped to the March 2006 human genome reference sequence (NCBI build 36.1, UCSC hg18) using the SOLiDTM System Analysis Pipeline Tool (Corona Lite). After tag-to-genome mapping of the ChIP-Seq data for each biological replicate experiment, tag counts were compared between replicates for identical genomic positions across all chromosomes. To do this, tag counts for each individual nucleotide position in each replicate experiment were determined. Then nucleotide positions that have a tag count of 0 in either replicate were eliminated from consideration. This is because if a position has a tag count of 0 in both data sets, it will not be considered in the subsequent analysis since there is no signal there. In addition, if a position has a 0 tag count in one replicate and a nonzero tag count in the other replicate, then it is considered to be an unreliable position. For nucleotide positions that have non-zero tag counts in both replicates, linear regression was used to compare the tag counts over all nucleotide positions. Based on the regression line and the data distribution around the line, 95% confidence bounds are determined using the Working-Hotelling formula as follows: For each value of x, an expected value of y−E(y)—is estimated based on the linear regression model E(y)=mx+b. Then for each estimated value of E(y), a standard deviation−s(y)—is computed based on the data distribution around E(y) for the corresponding value of x. For each value of E(y), the upper and lower confidence values are computed as E(y)±w·s(y) where the constant w is determined based on the F distribution at the confidence level α with 2 and n−2 degrees of freedom: w=√(2·F[(1−α),2,n−2]). This regression analysis and confidence bound inference is done for each individual chromosome. Nucleotide positions that fall outside of the confidence bounds determined in this way are removed from further consideration. Replicate experiments were highly consistent.
Noise Reduction and Data Merging.
After removal of outliers, noise reduction was performed in order to empirically determine a tag count threshold value th above which an individual nucleosome sized genomic position (i.e. mono-nucleosome) is considered to be modified. A window size of 200 bp was chosen for this analysis based on 147 bp of DNA in a single nucleosome plus linker sequence. The average tag count in 200 bp windows—λ200—is computed as the total number of tags in the genome n divided by the genome length l times the window size of 200: λ200=(n/l)·200. The Poisson distribution is parameterized by the 200 value in order to model the background noise and determine the tag count signal threshold th for modified nucleosome positions:




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where τ=the number of tags in a window and P is a Bonferroni corrected P-value threshold. To do this, corresponding position-specific tag count values were summed between replicate experiments and compared to a Poisson distribution parameterized by the sum of the 200-values computed for each replicate. This approach was taken because the replicates are independent biological replicates, each of which is modeled by a separate Poisson distribution. Therefore, the summation of position-specific tag counts across the genome can also be modeled by a Poisson distribution with a parameter equal to the sum of the individual 200-values from each replicate. This procedure resulted in a classification of all nucleosome size genomic positions as modified or unmodified based on data that has been merged between replicates and purged of outliers.

Maximal-Segment Based Clustering Algorithm for Continuous Modified Nucleosome Positions.
In addition to the analysis of mono-nucleosome size fragments of modified genomic DNA described previously, we also investigated clusters of adjacent co-located modified nucleosomes. Clusters were operationally defined as contiguous genomic regions where the number of modified mono-nucleosome size fragments is significantly greater than the average genomic background level of modified positions. To identify and demarcate such clusters, we applied the Maximal Segment algorithm. To do this, we first devised a binary scoring scheme that characterizes mono-nucleosome size fragments (200 bp) as either modified or unmodified. This procedure is used to define a binary genome-wide map of nucleosome scores. Then the Maximal Segment algorithm was applied to the genomic map of binary nucleosome scores to define clusters. The details of the Maximal Segment algorithm are presented elsewhere (Ruzzo and Tompa (1999) Proc. Int. Conf Intell. Syst. Mol. Biol., 234-241). Below, we describe our scoring scheme for individual nucleosome positions.
Binary Nucleosome Scoring Scheme
A binary scoring scheme is implemented in such a way as to assign the log likelihood that an individual mono-nucleosome size fragment either modified or unmodified. The scores are assigned as: s=ln(q/p) where p is the density of modified nucleosome positions over the whole genome computed as the number of modified positions divided by the total number of positions and q is the density of modified nucleosome positions in real clusters. There is no way to directly calculate q since the clusters are unknown a priori. In order to estimate q, a Poisson distribution is parameterized with p, the density of modified nucleosome positions over the whole genome. Then, given a confidence level P, a threshold value of q can be chosen using the Poisson distribution as shown:




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                  !
                
              
              ⁢
              
                e
                
                  -
                  λ
                
              
            
          
        
        ≤
        P
      
      }
    
  




where τ=number of modified nucleosome positions.

This scoring scheme results in an assignment of a single positive score to all modified nucleosome positions and a single negative score to all unmodified positions. This approach is taken based on the proof that log likelihood ratios of this kind are optimal scores for the identification of contiguous genomic segments (Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA, 87: 2264-2268). In addition, the value of q chosen for the scoring scheme allows for control over cluster selection in the sense that the density of modified nucleosomes per cluster will be greater than or equal to the value of q.
Relative Entropy to Define Large Clusters.
After getting the size distributions of contiguous modified nucleosome regions (i.e. clusters), we classified the clusters into 3 groups: 1) mononucleosomal clusters, 2) mid-sized clusters and 3) large clusters. In order to set a size threshold between mid-sized and large clusters, we calculated the relative entropies for different thresholds between the size distributions in self-renewing and in senescent cells (FIG. 11A). The threshold is set as the one which gives the maximal relative entropy between the two distributions (FIG. 11C). Thus, the difference between fractions of mid-sized and large clusters in the two cell types is maximized.
Comparison with Genomic Features.
The tag-to-genome mapping and the quality control procedures described above (see Outlier removal and Noise reduction and data merging sections) yielded maps of nucleosome positions in the human genome that can be confidently considered to be modified by ãH2AX for self-renewing (SR) and senescent (SEN) hADSCs.
These maps were used to compare the locations of ãH2AX modified nucleosomes to various genomic features of interest within and between SR and SEN cells. For instance, we compared the locations of modified nucleosomes in the genome to the locations of transcription start sites, exons, introns, repetitive sequence elements, pericentric regions and peri-telomeric regions. These comparisons were done using the UCSC Table Browser tool (Karolchik et al. (2004) Nucleic Acids Res., 32: D493-D496), which is a part of the UCSC Genome Browser suite of tools and genome annotations (Karolchik et al. (2003) Nucleic Acids Res., 31: 51-54). In addition, custom Perl scripts were used to modify UCSC Genome Browser tracks, such as the gene annotation tracks, to pull out specific features of interest including transcription start site locations and exon/intron boundaries for further analysis.











TABLE 1








Fold up-/





Gene
down-


Biological process
Gene name
Symbol
regulation
P value



















cell cycle/cell
cyclin B1
CCNB1
−2.66
0.0006


cycle control
cyclin B2
CCNB2
−3.10
0.0005



Cyclin D2
CCND2
2.55
0.0001



cyclin F
CCNF
−2.40
0.008



cell division cycle 2, G1
CDC2
−4.51
0.0001



to S and G2 to M



cell division cycle 20
CDC20
−3.15
0.0001



homolog (S. cerevisiae)



cyclin-dependent kinase 2
CDK2
−2.75
0.0012



cyclin-dependent kinase
CDKN3
−3.28
0.0029



inhibitor 3 (CDK2-



associated dual



specificity phosphatase)



CHK1 checkpoint
CHEK1
−3.00
0.008



homolog (S. pombe)



retinoblastoma-like 1
RBL1
−2.56
0.0012



(p107)


DNA replication
MCM2
MCM2
−3.29
0.0001



minichromosome



mantenacne deficient 2,



mitotin (S. cerevisiae)



MCM3
MCM3
−2.33
0.0184



minichromosome



maintenance deficient 3



(S. cerevisiae)



MCM4
MCM4
−3.44
0.0022



minichromosome



maintenance deficient 4



(S. cerevisiae)



MCM5
MCM5
−3.57
0.0001



minichromosome



maintenance deficient 5,



cell division cycle 46 (S. cerevisiae)



replication protein A3,
RPA3
−2.52
0.0014



14 kDa



proliferating cell nuclear
PCNA
−2.53
0.0009



antigen


Chromosome
MAD2 mitotic arrest
MAD2L1
−4.37
0.0021


segregation
deficient-like 1 (yeast)



kinetochore associated 1
KNTC1
−3.22
0.0049


DNA repair
breast cancer 1, early
BRCA1
−3.83
0.0032



onset



breast cancer 2, early
BRCA2
−3.32
0.0011



onset



RAD51 homolog (RecA
RAD51
−3.75
0.0005



homolog, E. coli) (S. cerevisiae)


mRNA
transcription factor Dp-1
TFDP1
−2.21
0.0097


transcription


regulation


inhibition of
baculoviral IAP repeat
BIRC5
−3.70
0.002


apoptosis
containing 5 (surviving)


stress response
G-2 and S-phase
GTSE1
−4.51
0.0017



expressed 1


Biological process
CDC28 protein kinase
CKS1B
−2.35
0.0382


unclassified
regulatory subunit 1B



CDC28 protein kinase
CKS2
−2.42
0.001



regulatory subunit 2



antigen identified by
MKI67
−4.04
0.0053



monoclonal antibody Ki-



67
















TABLE 2







γH2AX ChIP-seq tag and mapping data.












Experiment
Total tags
Mapped tags
% Mapped tags







SR1
25650863
6427476
25.058%



SR2
22864646
6740098
29.478%



SR3
23172888
7153067
30.868%



SR4
20554101
6610682
32.162%



SEN1
32114970
7915256
24.647%



SEN2
28469416
7569634
26.589%



SEN3
26694829
4911085
18.397%



SEN4
23942531
4780551
19.967%

















TABLE 3







Fractions of γH2AX clusters occupied by genomic features.











Mono-





nucleosomal
Mid-size
Large













Features
SR
SEN
SR
SEN
SR
SEN










Genic Regions













All genic
41.5%
41.0%
42.9%
39.7%
47.9%
35.2%


regions








Promoter
0.75%
0.63%
0.83%
0.61%
0.94%
0.56%


Exon
2.86%
2.43%
3.20%
2.14%
4.02%
1.78%


Intron
37.9%
38.0%
38.9%
36.9%
42.9%
32.9%


Exon-intron
0.19%
0.16%
0.21%
0.14%
0.28%
0.11%


Junction













Intergenic Regions













All Intergenic
59.6%
59.9%
57.8%
61.4%
52.5%
66.8%


regions













All Transposable Elements (TEs)













All TEs
72.7%
70.6%
69.6%
70.2%
64.4%
65.6%







Retrotransposons













All
67.3%
65.8%
66.0%
65.8%
63.5%
61.4%


Retro-








transposons













SINE Elements













All SINEs
34.9%
30.0%
36.3%
30.9%
37.5%
28.6%


Alu
23.4%
17.8%
22.6%
18.8%
20.8%
16.9%


MIR
10.4%
11.2%
12.7%
11.2%
15.8%
11.0%


SINE-other
1.06%
0.97%
1.03%
0.88%
0.91%
0.71%







LINE Elements













All LINEs
32.4%
35.8%
29.7%
34.9%
26.0%
32.8%


L1
20.3%
23.6%
17.2%
22.8%
12.9%
21.9%


L2
10.7%
10.5%
10.9%
10.4%
11.5%
9.19%


LINE-other
1.42%
1.68%
1.55%
1.65%
1.64%
1.75%







Other Transposable Elements













LTR
20.8%
17.7%
18.6%
17.1%
14.9%
15.9%


DNA
8.50%
9.15%
8.75%
9.35%
8.70%
8.57%
















TABLE 4







γH2AX tag density compared to chromosome gene density


and GC content








SR
SEN












Slope
Correlation
P-value
Slope
Correlation
P-value










Correlation: γH2AX tag density × chromosome gene


density [RγH2AX|gene density]












    0.48
0.64
5.2E−04
−0.19
−0.22
0.16







Correlation: γH2AX tag density × chromosome


GC content [RγH2AX|GC]












2366
0.67
2.6E−04
2429
0.54
3.9E−03







Partial correlation: γH2AX tag density × chromosome


gene density controlling for GC content [rγH2AX|gene density.GC]












N/A
0.49
0.01
N/A
−0.65
1.1E−04
















TABLE 5







Length fractions of genomic features intersected with clusters of γH2AX











Mono-nucleosomal
Mid-size
Large













Features
SR
SEN
SR
SEN
SR
SEN





TSS
0.51%
0.43%
2.29%
1.36%
1.12%
0.20%


Exon
0.64%
0.55%
2.90%
1.56%
1.58%
0.21%


Intron
0.46%
0.46%
1.91%
1.45%
0.91%
0.21%


Junction
0.63%
0.53%
2.81%
1.48%
1.60%
0.19%


Intergenic
0.67%
0.68%
2.65%
2.26%
1.04%
0.40%


peri-
0.09%
0.09%
0.39%
0.40%
0.73%
0.52%


centromere








peri-
0.31%
0.21%
1.42%
0.75%
0.29%
0.26%


telomere








TE
0.75%
0.73%
2.90%
2.35%
1.16%
0.36%


intergenic
0.76%
0.74%
2.88%
2.43%
1.04%
0.39%


TE








genic TE
0.73%
0.71%
2.94%
2.21%
1.36%
0.31%


Alu
0.67%
0.51%
2.61%
1.74%
1.04%
0.25%


MIR
0.69%
0.75%
3.42%
2.43%
1.84%
0.39%


SINE-other
0.77%
0.71%
3.02%
2.08%
1.16%
0.27%


L1
0.49%
0.57%
1.68%
1.78%
0.54%
0.28%


L2
0.79%
0.78%
3.26%
2.50%
1.49%
0.36%


LINE-other
0.65%
0.77%
2.87%
2.45%
1.31%
0.42%


LTR
0.81%
0.70%
2.93%
2.17%
1.02%
0.33%


DNA
0.65%
0.70%
2.69%
2.31%
1.16%
0.34%
















TABLE 6





List of genes with promoter γH2AX accumulation in SR 


or SEN cells.


















NM_001001933
LHX8
NM_152418
WDR21C


NM_139240
C1orf105
NM_052832
SLC26A7


NM_015658
NOC2L
NM_015435
RNF19A


NM_198317
KLHL17
NM_030788
TM7SF4


NM_148902
TNFRSF18
NM_139166
ABRA


NM_080875
MIB2
NM_177531
PKHD1L1


NM_033467
MMEL1
NM_021021
SNTB1


NM_152492
CCDC27
NM_078480
PUF60


NM_207370
GPR153
NM_201384
PLEC1


NM_181864
ACOT7
NM_024531
GPR172A


NM_006786
UTS2
NM_003923
FOXH1


NM_005026
PIK3CD
NM_001029976
ZNF16


NM_198544
APITD1
NR_023392
ZNF252


NM_001127325
MAD2L2
NM_021240
DMRT3


NM_001009611
PRAMEF4
NM_005511
MLANA


NM_001018001
KIAA1026
NM_000170
GLDC


NM_207348
SLC25A34
NM_144966
FREM1


NM_001089591
UQCRHL
NM_017645
FAM29A


NM_153213
ARHGEF19
NM_002172
IFNA14


NM_017940
NBPF1
NM_078487
CDKN2B


NM_012387
PADI4
NM_016410
CHMP5


NM_014589
PLA2G2E
NM_007343
PRSS3


NM_207334
FAM43B
NM_015297
KIAA1045


NM_001113347
ECE1
NM_002732
PRKACG


NM_002167
ID3
NM_001007470
TRPM3


NM_054016
FUSIP1
NM_021154
PSAT1


NM_178122
C1orf201
NM_030940
ISCA1


NM_015627
LDLRAP1
NM_005226
S1PR3


NM_198137
CATSPER4
NM_145006
SUSD3


NM_145345
UBXN11
NM_177995
PTPDC1


NM_001039775
AIM1L
NM_003837
FBP2


NM_152365
C1orf172
NM_000507
FBP1


NM_005281
GPR3
NM_173200
NR4A3


NM_001990
EYA3
NM_207299
PRG-3


NM_001269
RCC1
NM_000035
ALDOB


NM_004437
EPB41
NM_018112
TMEM38B


NM_002379
MATN1
NM_006687
ACTL7A


NM_004814
SNRNP40
NM_173521
C9orf84


NM_023009
MARCKSL1
NM_033051
SLC46A2


NM_005268
GJB5
NM_138424
KIF12


NM_017629
EIF2C4
NM_017418
1-Dec


NM_172313
CSF3R
NM_198188
ASTN2


NM_017850
C1orf109
NM_001011649
CDK5RAP2


NM_001113482
MANEAL
NM_012164
FBXW2


NR_003929
LOC728448
NM_001735
C5


NM_152373
ZNF684
NM_007018
CEP110


NM_144990
SLFNL1
NM_001004450
OR1B1


NM_007102
GUCA2B
NR_003071
SNORD90


NM_144626
TMEM125
NM_030978
ARPC5L


NM_182517
C1orf210
NM_005833
RABEPK


NM_001005417
B4GALT2
NM_006195
PBX3


NR_000024
SNORD46
NM_001011703
FAM125B


NM_153274
BEST4
NM_203305
FAM102A


NM_005897
IPP
NM_032728
PPAPDC3


NM_004474
FOXD2
NR_003050
SNORD62A


NM_032110
DMRTA2
NM_018956
C9orf9


NM_004153
ORC1L
NM_152572
C9orf98


NM_001010978
LDLRAD1
NM_002003
FCN1


NM_152607
C1orf177
NM_178469
LCN8


NM_015888
HOOK1
NM_016219
MAN1B1


NM_001083592
ROR1
NM_080877
SLC34A3


NM_152489
UBE2U
NM_052817
MID2


NM_001924
GADD45A
NM_003662
PIR


NM_003838
FPGT
NM_007220
CA5B


NM_001112808
TNNI3K
NM_001080975
REPS2


NM_001002912
C1orf173
NM_006089
SCML2


NM_012152
LPAR3
NM_001037343
CDKL5


NM_016620
ZNF644
NR_023358
SCARNA9L


NM_001127215
GFI1
NM_001415
EIF2S3


NM_007358
MTF2
NM_177404
MAGEB1


NM_001114106
SLC44A3
NM_004229
MED14


NM_080630
COL11A1
NM_001356
DDX3X


NM_001122961
C1orf194
NM_017776
ZNF673


NM_172212
CSF1
NR_015378
LOC401588


NM_032414
PROK1
NM_001039891
ZNF674


NM_001025197
CHI3L2
NM_153280
UBA1


NM_024102
WDR77
NM_007130
ZNF41


NM_000677
ADORA3
NM_001114123
ELK1


NM_033020
TRIM33
NM_007137
ZNF81


NM_001002810
PDE4DIP
NM_001037735
ZNF630


NM_144697
C1orf51
NM_203475
PORCN


NM_182578
THEM5
NM_000377
WAS


NM_016190
CRNN
NM_002049
GATA1


NM_178354
LCE1F
NM_017602
OTUD5


NM_001128600
LCE6A
NM_007075
WDR45


NM_001024209
SPRR2E
NM_015698
GPKOW


NM_000427
LOR
NM_020717
SHROOM4


NM_005979
S100A13
NM_005448
BMP15


NM_004515
ILF2
NM_001010862
SPIN3


NM_173852
KRTCAP2
NM_138737
HEPH


NM_001039517
C1orf104
NM_014725
STARD8


NM_006912
RIT1
NM_207320
OTUD6A


NM_006365
C1orf61
NM_002565
P2RY4


NM_021948
BCAN
NM_004312
ARR3


NM_001004469
OR10J5
NM_007363
NONO


NM_001013661
VSIG8
NR_002226
INGX


NM_012337
CCDC19
NM_001013627
NHSL2


NM_001001734
ATP1A4
NM_017669
ERCC6L


NM_052931
SLAMF6
NR_003255
TSIX


NM_003037
SLAMF1
NM_015975
TAF9B


NM_017625
ITLN1
NM_153252
BRWD3


NM_030916
PVRL4
NM_000307
POU3F4


NM_053282
SH2D1B
NM_033048
CPXCR1


NM_178550
C1orf110
NM_006729
DIAPH2


NM_018417
ADCY10
NM_022144
TNMD


NM_001460
FMO2
NM_021637
TMEM35


NM_001531
MR1
NM_022053
NXF2


NM_005562
LAMC2
NM_182541
TMEM31


NM_002597
PDC
NM_017416
IL1RAPL2


NM_005298
GPR25
NM_138382
RIPPLY1


NM_003281
TNNI1
NM_018301
RBM41


NM_001048230
ADORA1
NM_173494
CXorf41


NM_018208
ETNK2
NM_002764
PRPS1


NM_030952
NUAK2
NM_001522
GUCY2F


NM_181644
MFSD4
NM_001004308
ZCCHC16


NM_134325
SLC26A9
NM_001031855
LONRF3


NR_004389
SNORA16B
NM_001152
SLC25A5


NM_206594
ESRRG
NM_181777
UBE2A


NR_001587
AURKAPS1
NM_017544
NKRF


NM_024709
C1orf115
NM_004541
NDUFA1


NM_014184
CNIH4
NM_006978
RNF113A


NM_033445
HIST3H2A
NM_006777
ZBTB33


NM_004578
RAB4A
NM_003588
CUL4B


NM_152379
C1orf131
NM_001008222
ZDHHC9


NM_022051
EGLN1
NM_001077188
HS6ST2


NM_032435
KIAA1804
NM_004484
GPC3


NM_021186
ZP4
NM_021796
PLAC1


NM_024804
ZNF669
NM_001013403
LOC347487


NM_175911
OR2L13
NM_004065
CDR1


NM_019044
CCDC93
NM_173078
SLITRK4


NM_152670
C2orf51
NM_001011544
MAGEA11


NM_001040649
ACP1
NM_005342
HMGB3


NM_015677
SH3YL1
NM_004224
GPR50


NM_175722
TPO
NM_153478
CSAG1


NM_138799
MBOAT2
NM_005367
MAGEA12


NM_033090
GREB1
NM_007150
ZNF185


NM_012344
NTSR2
NM_001001344
ATP2B3


NM_182828
GDF7
NM_001025243
IRAK1


NM_181713
UBXN2A
NR_000011
SNORA70


NM_001040710
C2orf84
NM_004728
DDX21


NM_153759
DNMT3A
NM_020397
CAMK1D


NM_017877
C2orf18
NM_022365
DNAJC1


NM_007046
EMILIN1
NM_020824
ARHGAP21


NM_032434
ZNF512
NM_014915
ANKRD26


NM_020744
MTA3
NM_052997
ANKRD30A


NM_022436
ABCG5
NM_003421
ZNF37A


NM_001003937
TSPYL6
NM_145312
ZNF485


NM_014562
OTX1
NM_014696
GPRIN2


NM_006759
UGP2
NM_001098512
PRKG1


NM_152792
ASPRV1
NM_022079
HERC4


NM_001692
ATP6V1B1
NM_030625
TET1


NM_020459
PAIP2B
NM_014767
SPOCK2


NM_144579
SFXN5
NM_001114133
SYNPO2L


NM_003960
NAT8
NR_002724
MBL1P1


NM_001615
ACTG2
NM_032372
DYDC2


NM_021196
SLC4A5
NM_138812
DYDC1


NM_016170
TLX2
NM_006829
C10orf116


NM_021103
TMSB10
NM_148978
PANK1


NM_031283
TCF7L1
NM_006413
RPP30


NM_017952
PTCD3
NM_004523
KIF11


NR_003503
GGT8P
NM_015385
SORBS1


NM_002371
MAL
NM_020349
ANKRD2


NM_013434
KCNIP3
NM_138413
C10orf65


NM_001037228
LOC285033
NM_001009997
C10orf62


NM_001079
ZAP70
NM_031212
SLC25A28


NM_138798
MITD1
NM_003988
PAX2


NM_001025108
AFF3
NM_013274
POLL


NM_173343
IL1R2
NM_002779
PSD


NM_173205
IL1F7
NM_002925
RGS10


NM_173161
IL1F10
NM_024834
C10orf119


NM_173842
IL1RN
NR_003570
FLJ46361


NM_012184
FOXD4L1
NM_001105574
HMX3


NM_002830
PTPN4
NM_001007793
BUB3


NM_032740
SFT2D3
NM_153442
GPR26


NM_138770
CCDC74A
NM_000375
UROS


NM_144586
LYPD1
NM_018180
DHX32


NM_019845
RPRM
NM_001380
DOCK1


NM_001009959
ERMN
NM_006426
DPYSL4


NM_002349
LY75
NM_003577
UTF1


NM_173162
KCNH7
NM_001098483
C10orf125


NM_000523
HOXD13
NM_145651
SCGB1C1


NM_012086
GTF3C3
NM_001039490
CD151


NM_199440
HSPD1
NM_138567
SYT8


NM_001228
CASP8
NM_001013254
LSP1


NM_001102659
LOC200726
NM_199292
TH


NM_005210
CRYGB
NM_014555
TRPM5


NM_000634
IL8RA
NM_001039165
MRGPRE


NM_007127
VIL1
NM_003141
TRIM21


NM_001105537
ZNF142
NM_001004137
OR52M1


NM_003936
CDK5R2
NM_001004757
OR51Q1


NM_001005176
SP140
NR_002777
TRIMP1


NM_030926
ITM2C
NM_176875
CCKBR


NM_031313
ALPPL2
NM_012192
FXC1


NM_005199
CHRNG
NM_016229
CYB5R2


NM_007120
UGT1A4
NM_001003745
OR10A3


NM_018410
HJURP
NM_005418
ST5


NM_001005853
OR6B2
NM_001031853
INSC


NM_001080835
LOC643905
NM_017508
SOX6


NM_005301
GPR35
NM_000525
KCNJ11


NM_015148
PASK
NM_002478
MYOD1


NM_002712
PPP1R7
NM_194285
SPTY2D1


NM_175727
IL5RA
NM_003986
BBOX1


NM_020873
LRRN1
NM_001031854
ACCSL


NM_033337
CAV3
NM_001031730
TSPAN18


NM_014850
SRGAP3
NM_000256
MYBPC3


NM_033084
FANCD2
NM_001004727
OR4X2


NM_003178
SYN2
NM_001005201
OR8H3


NM_005037
PPARG
NM_001004064
OR8J3


NM_025265
TSEN2
NM_001004746
OR5T2


NM_012298
CAND2
NM_001005210
LRRC55


NM_001007073
RPL32
NM_000062
SERPING1


NM_024827
HDAC11
NM_145008
YPEL4


NM_001080423
GRIP2
NM_005838
GLYAT


NM_020839
WDR48
NM_001004706
OR4D11


NM_001337
CX3CR1
NM_152716
PATL1


NM_005201
CCR8
NM_178031
TMEM132A


NM_001098414
ZNF621
NM_152718
VWCE


NM_020707
HHATL
NM_021727
FADS3


NM_173658
ZNF660
NM_020238
INCENP


NM_145044
ZNF501
NM_006473
TAF6L


NM_013270
TSP50
NM_199337
TMEM179B


NM_182702
TESSP2
NM_198897
FIBP


NM_001008269
TMEM89
NM_178864
NPAS4


NM_000884
IMPDH2
NM_003793
CTSF


NM_001640
APEH
NM_014578
RHOD


NM_007024
TMEM115
NM_017857
SSH3


NM_152397
IQCF1
NM_000695
ALDH3B2


NM_144641
PPM1M
NM_002335
LRP5


NM_020163
SEMA3G
NM_005247
FGF3


NM_002218
ITIH4
NM_000803
FOLR2


NM_205853
MUSTN1
NM_033388
ATG16L2


NM_001126128
PROK2
NM_004154
P2RY6


NM_001105580
GABRR3
NM_021200
PLEKHB1


NM_001042459
FILIP1L
NM_004041
ARRB1


NM_032787
GPR128
NM_015516
TSKU


NM_014981
MYH15
NM_182833
GDPD4


NM_001008273
TAGLN3
NM_153696
PSMAL


NM_001025073
C3orf17
NM_001105522
LOC729384


NM_152538
IGSF11
NM_033135
PDGFD


NM_005191
CD80
NM_002906
RDX


NM_175924
ILDR1
NM_015191
SIK2


NM_182628
CCDC37
NM_001101389
LOC644672


NM_013336
SEC61A1
NM_152315
FAM55A


NM_153330
DNAJB8
NM_001077263
TMPRSS13


NM_020187
C3orf37
NM_001467
SLC37A4


NM_001042384
CEP63
NM_024791
PDZD3


NM_004189
SOX14
NM_004205
USP2


NM_001002026
CLDN18
NM_001005197
OR8D4


NM_016161
A4GNT
NM_001002918
OR8D2


NM_004766
COPB2
NM_017425
SPA17


NM_080862
SPSB4
NM_025004
CCDC15


NM_139209
GRK7
NM_022112
P53AIP1


NM_013308
GPR171
NM_138342
GLB1L2


NM_014575
SCHIP1
NM_003044
SLC6A12


NM_000882
IL12A
NM_001039029
LRTM2


NM_173084
TRIM59
NM_020996
FGF6


NM_004122
GHSR
NM_001769
CD9


NM_130770
HTR3C
NM_020400
LPAR5


NM_001100120
ECE2
NM_002075
GNB3


NM_001102416
KNG1
NM_002543
OLR1


NM_130834
OPA1
NM_020853
KIAA1467


NM_152531
C3orf21
NM_005504
BCAT1


NM_138461
TM4SF19
NM_001098531
RAPGEF3


NM_182524
ZNF595
NM_014554
SENP1


NM_001039127
ZNF718
NM_012272
PRPF40B


NM_017733
PIGG
NM_001971
ELA1


NM_022042
SLC26A1
NM_175053
KRT74


NM_003023
SH3BP2
NM_153633
HOXC4


NM_198229
RGS12
NM_006163
NFE2


NM_001113361
TBC1D14
NM_020370
GPR84


NM_001001290
SLC2A9
NM_006741
PPP1R1A


NM_004787
SLIT2
NM_021191
NEUROD4


NM_147183
KCNIP4
NM_002206
ITGA7


NM_015187
KIAA0746
NM_002429
MMP19


NM_018302
C4orf19
NM_022465
IKZF4


NM_207406
BEND4
NM_012064
MIP


NM_198353
KCTD8
NR_003046
SNORD59B


NM_000809
GABRA4
NM_148897
SDR9C7


NM_032622
LNX1
NM_024779
PIP4K2C


NM_032495
HOPX
NM_001478
B4GALNT1


NM_020368
UTP3
NM_000075
CDK4


NM_000584
IL8
NM_015026
MON2


NM_033214
GK2
NM_022496
ACTR6


NM_001003810
HNRNPD
NM_016053
CCDC53


NM_001098540
HPSE
NM_006825
CKAP4


NM_032717
AGPAT9
NM_002920
RFX4


NM_138980
MAPK10
NM_203436
ASCL4


NM_178135
HSD17B13
NM_057169
GIT2


NM_198281
GPRIN3
NM_002710
PPP1CC


NM_183049
TMSL3
NM_001034025
ERP29


NM_000671
ADH5
NM_181578
RFC5


NM_000670
ADH4
NM_002859
PXN


NM_000667
ADH1A
NM_015918
POP5


NM_000673
ADH7
NM_004276
CABP1


NM_033430
PDE5A
NM_000545
HNF1A


NM_152778
MFSD8
NM_016237
ANAPC5


NM_000910
NPY2R
NM_207437
DNAH10


NM_005651
TDO2
NM_144669
GLT1D1


NM_005038
PPID
NM_183044
RNF6


NM_017923
1-Mar
NM_017826
SOHLH2


NM_006792
MORF4
NM_001009814
KIAA0564


NR_003612
FAM92A3
NM_002498
NEK3


NM_207352
CYP4V2
NM_001011724
HNRNPA1L2


NM_000128
F11
NM_022843
PCDH20


NM_013232
PDCD6
NM_006346
PIBF1


NM_024786
ZDHHC11
NM_014305
TGDS


NM_001044
SLC6A3
NM_019616
F7


NM_020227
PRDM9
NM_000504
F10


NM_018356
C5orf22
NM_198235
RNASE1


NM_005983
SKP2
NM_201535
NDRG2


NM_001007527
LMBRD2
NM_001012264
RNASE13


NM_018034
WDR70
NM_007192
SUPT16H


NM_152403
EGFLAM
NM_001344
DAD1


NM_000163
GHR
NM_000359
TGM1


NM_022483
C5orf28
NM_138452
DHRS1


NM_022132
MCCC2
NM_032594
INSM2


NM_004291
CARTPT
NM_032352
BRMS1L


NM_153217
TMEM174
NM_003317
NKX2-1


NM_001004441
ANKRD34B
NM_001202
BMP4


NM_173061
CAST
NM_015589
SAMD4A


NM_001962
EFNA5
NM_014992
DAAM1


NM_022828
YTHDC2
NM_004857
AKAP5


NM_014350
TNFAIP8
NM_001039465
SFRS5


NM_153223
CEP120
NM_015351
TTC9


NM_001017974
P4HA2
NM_018228
C14orf115


NM_021982
SEC24A
NM_002632
PGF


NM_145282
LOC153328
NM_004452
ESRRB


NM_015564
LRRTM2
NM_024496
C14orf4


NM_001033112
PAIP2
NM_033426
KIAA1737


NM_016459
MGC29506
NM_145870
GSTZ1


NM_001077693
ECSCR
NM_015859
GTF2A1


NM_032289
PSD2
NM_001275
CHGA


NM_018911
PCDHA8
NM_022151
MOAP1


NM_031857
PCDHA9
NM_006215
SERPINA4


NM_031883
PCDHAC2
NR_003234
SNORD113-6


NM_018936
PCDHB2
NR_003196
SNORD114-4


NM_031947
SLC25A2
NR_003316
SNORD116-1


NM_004576
PPP2R2B
NR_003324
SNORD116-3


NM_001040174
HTR4
NR_003330
SNORD116-15


NM_024577
SH3TC2
NR_003311
SNORD115-17


NM_133263
PPARGC1B
NR_003313
SNORD115-20


NM_001804
CDX1
NR_003496
HBII-52-27


NM_130899
FAM71B
NR_003350
SNORD115-35


NM_001037333
CYFIP2
NR_003353
SNORD115-38


NM_007017
SOX30
NR_003355
SNORD115-40


NM_000816
GABRG2
NM_017762
MTMR10


NM_199246
CCNG1
NM_001103184
FMN1


NM_145266
NUDCD2
NM_005135
SLC12A6


NM_001102609
LOC133874
NM_170675
MEIS2


NM_001029886
PFN3
NM_152453
TMCO5A


NM_019057
FLJ10404
NM_033286
C15orf23


NM_001024649
CANX
NM_133639
RHOV


NM_201627
TRIM41
NM_020857
VPS18


NR_002592
SNORD96A
NM_016642
SPTBN5


NM_138296
PTCRA
NM_022473
ZFP106


NM_018303
EXOC2
NM_000119
EPB42


NM_004568
SERPINB6
NM_004245
TGM5


NM_205864
CAGE1
NM_014985
CEP152


NM_153005
RIOK1
NM_016194
GNB5


NR_004855
HULC
NM_130810
DYX1C1


NM_153003
OFCC1
NM_012182
FOXB1


NM_003220
TFAP2A
NM_004663
RAB11A


NM_016462
TMEM14C
NM_016166
PIAS1


NM_207582
HERV-FRD
NM_022369
STRA6


NM_138574
HDGFL1
NM_020447
C15orf17


NM_014722
FAM65B
NM_173469
UBE2Q2


NM_170610
HIST1H2BA
NM_003978
PSTPIP1


NM_006355
TRIM38
NM_022566
MESDC1


NM_003523
HIST1H2BE
NM_201651
SLC28A1


NM_003532
HIST1H3E
NM_000326
RLBP1


NM_001732
BTN1A1
NM_198925
SEMA4B


NM_003511
HIST1H2AL
NM_002569
FURIN


NM_005322
HIST1H1B
NM_018668
VPS33B


NM_003447
ZNF165
NM_006011
ST8SIA2


NM_021253
TRIM39
NM_001102612
LOC145814


NM_005803
FLOT1
NM_016310
POLR3K


NR_003948
HCG22
NM_016541
GNG13


NM_000595
LTA
NM_207419
C1QTNF8


NR_002971
SNORA38
NM_024164
TPSB2


NM_025258
C6orf27
NM_023076
UNKL


NM_006929
SKIV2L
NM_004970
IGFALS


NM_033554
HLA-DPA1
NR_002327
SNORA10


NM_005453
ZBTB22
NM_174903
RNF151


NM_018679
TCP11
NM_005262
GFER


NM_153487
MDGA1
NM_022372
GBL


NM_031460
KCNK17
NM_182563
C16orf79


NM_024807
TREML2
NM_001374
DNASE1L2


NM_018643
TREM1
NM_001919
DCI


NM_002630
PGC
NM_001089
ABCA3


NM_000322
PRPH2
NM_007108
TCEB2


NM_003192
TBCC
NM_172229
KREMEN2


NM_023932
DLK2
NR_002169
OR1F2


NM_021572
ENPP5
NM_138440
VASN


NM_005588
MEP1A
NM_000833
GRIN2A


NM_000847
GSTA3
NM_170664
OTOA


NM_004282
BAG2
NM_173806
C16orf65


NM_183227
RAB23
NM_006043
HS3ST2


NM_078485
COL9A1
NM_032038
SPNS1


NM_014989
RIMS1
NM_006319
CDIPT


NM_002395
ME1
NM_152458
ZNF785


NM_016230
CYB5R4
NM_001080417
ZNF629


NM_000865
HTR1E
NM_000887
ITGAX


NM_001042493
C6orf162
NM_016633
ERAF


NM_020320
RARS2
NM_145186
ABCC11


NM_002042
GABRR1
NM_017839
LPCAT2


NM_015891
CDC40
NM_005949
MT1F


NM_001013734
RFPL4B
NM_005950
MT1G


NM_173560
RFX6
NM_153837
GPR114


NM_001029858
SLC35F1
NM_170776
GPR97


NM_020755
SERINC1
NM_001297
CNGB1


NM_033515
ARHGAP18
NM_001080492
KLKBL4


NM_022121
PERP
NM_020465
NDRG4


NM_020340
KIAA1244
NM_001796
CDH8


NM_015439
CCDC28A
NM_173815
CES8


NM_033071
SYNE1
NM_014329
EDC4


NM_014892
RBM16
NM_002801
PSMB10


NM_207118
GTF2H5
NM_012320
PLA2G15


NM_138810
TAGAP
NM_032178
SLC7A6OS


NM_016098
BRP44L
NM_018667
SMPD3


NR_002787
LOC154449
NM_138612
HAS3


NM_001098201
GPER
NM_030579
CYB5B


NM_007353
GNA12
NM_006927
ST3GAL2


NR_015343
LOC389458
NM_138994
CNTNAP4


NM_014038
BZW2
NM_014861
ATP2C2


NR_022006
KIAA0087
NM_031476
CRISPLD2


NM_000823
GHRHR
NM_152287
ZNF276


NM_006658
C7orf16
NM_145068
TRPV3


NM_207173
NPSR1
NM_144611
CYB5D2


NM_181791
GPR141
NM_001140
ALOX15


NM_001105282
FAM183B
NM_003562
SLC25A11


NM_021223
MYL7
NM_020795
NLGN2


NM_013389
NPC1L1
NM_001251
CD68


NR_002990
SNORA5B
NM_153007
ODF4


NM_021116
ADCY1
NM_001010855
PIK3R6


NM_000790
DDC
NM_004822
NTN1


NM_182546
VSTM2A
NM_139162
SMCR7


NM_016220
ZNF107
NM_144775
SMCR8


NM_015852
ZNF117
NM_004618
TOP3A


NM_031468
CALN1
NM_007148
RNF112


NM_002835
PTPN12
NM_001080837
SEBOX


NM_012395
PFTK1
NM_003593
FOXN1


NM_019004
ANKIB 1
NM_031934
RAB34


NM_004912
KRIT1
NR_000014
SNORD42A


NM_006528
TFPI2
NM_020791
TAOK1


NM_018842
BAIAP2L1
NM_015986
CRLF3


NM_003496
TRRAP
NM_015544
TMEM98


NM_057096
CYP3A43
NM_001094
ACCN1


NM_001185
AZGP1
NM_145272
C17orf50


NM_006833
COPS6
NM_024864
MRM1


NM_003227
TFR2
NM_000458
HNF1B


NM_002291
LAMB1
NM_003673
TCAP


NM_182597
C7orf53
NM_021724
NR1D1


NM_003391
WNT2
NM_019010
KRT20


NM_016087
WNT16
NM_031961
KRTAP9-2


NM_002851
PTPRZ1
NM_001524
HCRT


NM_001024613
FEZF1
NM_001991
EZH1


NM_003117
SPAM1
NM_080863
ASB16


NM_006193
PAX4
NM_145663
DBF4B


NR_002187
LOC286016
NM_005892
FMNL1


NM_001869
CPA2
NM_199282
ARHGAP27


NM_001105543
PLXNA4
NM_175882
IMP5


NM_002825
PTN
NM_001007532
STH


NM_001085429
TMEM213
NM_024320
ATAD4


NM_032295
SLC37A3
NM_152244
SNX11


NM_176817
TAS2R38
NM_000023
SGCA


NM_019841
TRPV5
NM_001267
CHAD


NM_001031690
FAM131B
NM_004655
AXIN2


NM_175571
GIMAP8
NM_000346
SOX9


NM_014020
TMEM176B
NM_000835
GRIN2C


NM_144727
CRYGN
NM_015353
KCTD2


NM_001040633
PRKAG2
NM_001005849
SUMO2


NM_033225
CSMD1
NM_001005619
ITGB4


NM_002052
GATA4
NM_180990
ZACN


NM_000237
LPL
NM_006640
9-Sep


NM_001114137
EPB49
NM_001042573
FLJ21865


NM_144962
PEBP4
NM_178543
ENPP7


NM_007257
PNMA2
NM_024110
CARD14


NM_171982
TRIM35
NM_181483
C18orf1


NM_013959
NRG1
NM_003799
RNMT


NM_152413
GOT1L1
NM_020805
KLHL14


NR_003129
RNF5P1
NM_032980
DTNA


NR_001569
tMDC
NM_018170
RPRD1A


NM_014420
DKK4
NM_001008239
C18orf25


NM_002027
FNTA
NM_001101654
CXXC1


NM_006269
RP1
NM_018696
ELAC1


NM_138969
SDR16C5
NM_012397
SERPINB13


NM_001007070
SDCBP
NM_001037331
C18orf62


NM_032466
ASPH
NM_001025101
MBP


NM_000370
TTPA
NM_014913
ADNP2


NM_002604
PDE7A
NM_198969
AES


NM_144650
ADHFE1
NM_001017392
SFRS14


NM_013257
SGK3
NM_182577
ODF3L2


NM_025170
PREX2
NM_005860
FSTL3


NM_006540
NCOA2
NM_002777
PRTN3


NM_007332
TRPA1
NM_001001975
ATP5D


NM_004770
KCNB2
NM_005883
APC2


NM_024721
ZFHX4
NM_003021
SGTA


NM_198584
CA13
NM_021231
C19orf29


NM_020209
SHD
NM_001103176
CNPY1


NM_001013706
LSDP5
NM_014671
UBE3C


NM_005817
M6PRBP1
NM_001040032
C8orf74


NM_015318
ARHGEF18
NM_031439
SOX7


NM_020533
MCOLN1
NM_001127505
ASAH1


NM_006949
STXBP2
NM_003155
STC1


NM_001042462
TRAPPC5
NM_015254
KIF13B


NM_002002
FCER2
NM_021631
FKSG2


NM_001004456
OR1M1
NR_003671
LOC728024


NM_001005193
OR7G2
NM_003012
SFRP1


NM_003451
ZNF177
NM_001556
IKBKB


NM_003259
ICAM5
NM_000749
CHRNB3


NM_001611
ACP5
NM_001005365
POTEA


NM_006397
RNASEH2A
NM_144651
PXDNL


NM_006563
KLF1
NM_005098
MSC


NM_004907
IER2
NM_000971
RPL7


NM_003765
STX10
NM_172037
RDH10


NM_024323
C19orf57
NM_024699
ZFAND1


NM_001007525
NWD1
NM_152284
CHMP4C


NM_015260
SIN3B
NM_001100393
RALYL


NM_000923
PDE4C
NM_001083588
E2F5


NM_004750
CRLF1
NM_019098
CNGB3


NM_004720
LPAR2
NM_002380
MATN2


NM_021047
ZNF253
NM_001029860
FBXO43


NM_033468
ZNF257
NM_152888
COL22A1


NM_001098626
ZNF98
NM_207414
FLJ43860


NM_021175
HAMP
NM_177458
LYNX1


NM_015302
KIAA0841
NM_201379
PLEC1


NM_001863
COX6B1
NM_001129888
CYHR1


NM_052948
SNX26
NM_025251
KIAA1688


NM_004646
NPHS1
NM_021951
DMRT1


NM_020951
ZNF529
NM_005059
RLN2


NM_152605
ZNF781
NM_001099223
IFT74


NM_001042507
LGALS7B
NM_145005
C9orf72


NM_020862
LRFN1
NM_004512
IL11RA


NM_016941
DLL3
NM_020944
GBA2


NM_182704
SELV
NM_016042
EXOSC3


NM_152361
EID2B
NM_153237
C9orf71


NM_033543
CEACAM21
NM_032171
CEP78


NM_001817
CEACAM4
NM_004938
DAPK1


NM_198850
PHLDB3
NM_001912
CTSL1


NM_182498
ZNF428
NM_001023564
CTSL3


NM_198478
NKPD1
NM_033014
OGN


NM_033258
GNG8
NM_000136
FANCC


NM_019855
CABP5
NM_014930
ZNF510


NM_014959
CARD8
NM_002486
NCBP1


NM_031485
GRWD1
NM_014788
TRIM14


NM_177973
SULT2B1
NM_197977
ZNF189


NM_000979
RPL18
NM_001017998
GNG10


NM_145807
NTN5
NM_032303
HSDL2


NM_006179
NTF4
NM_021218
C9orf80


NM_031886
KCNA7
NM_005118
TNFSF15


NM_001040031
CD37
NM_001244
TNFSF8


NM_020309
SLC17A7
NM_002077
GOLGA1


NM_017432
PTOV1
NM_001100877
PHYHD1


NM_152899
IL4I1
NM_014581
OBP2B


NM_052884
SIGLEC11
NM_015837
FCN2


NM_001030049
KLK3
NM_001080849
DNLZ


NM_004917
KLK4
NM_024718
C9orf86


NM_001012964
KLK6
NM_004479
FUT7


NM_016543
SIGLEC7
NM_000718
CACNA1B


NM_053003
SIGLEC12
NM_001727
BRS3


NM_001462
FPR2
NM_005044
PRKX


NM_173857
VN1R4
NM_001042480
GEMIN8


NR_003699
ZNF525
NM_005089
ZRSR2


NM_001110514
EBF4
NM_004057
S100G


NM_024958
NRSN2
NM_001024666
SH3KBP1


NM_001040007
RSPO4
NM_152577
ZNF645


NM_198994
TGM6
NM_031442
TMEM47


NM_014731
ProSAPiP1
NM_152856
RBM10


NM_025220
ADAM33
NM_019056
NDUFB11


NM_024960
PANK2
NM_175723
SSX5


NM_012409
PRND
NM_003173
SUV39H1


NM_170774
RASSF2
NM_013271
PCSK1N


NM_144773
PROKR2
NM_000084
CLCN5


NM_003434
ZNF133
NM_031407
HUWE1


NM_016652
CRNKL1
NM_152424
FAM123B


NM_153675
FOXA2
NM_207422
FLJ44635


NM_001052
SSTR4
NM_152694
ZCCHC5


NM_005492
CST8
NM_003022
SH3BGRL


NM_032609
COX4I2
NM_001105243
PCDH19


NM_001011718
XKR7
NM_001031834
RAB40AL


NM_182658
C20orf185
NM_001113490
AMOT


NM_021215
RPRD1B
NM_007231
SLC6A14


NR_002911
SNORA71A
NM_145800
6-Sep


NM_003064
SLPI
NM_012084
GLUD2


NM_181502
SPINLW1
NM_001042751
STAG2


NM_147198
WFDC9
NM_001114937
SH2D1A


NM_172131
WFDC10B
NM_001078172
FAM127B


NM_052951
DNTTIP1
NM_006359
SLC9A6


NM_080752
ZSWIM3
NM_000074
CD40LG


NM_080608
C20orf165
NM_032512
PDZD4


NM_172113
EYA2
NM_024332
BRCC3


NM_005985
SNAI1
NM_001818
AKR1C4


NM_199129
TMEM189
NM_153256
C10orf47


NM_199203
TMEM189-
NM_153244
C10orf111



UBE2V1




NM_018431
DOK5
NR_023388
PRINS


NM_080617
CBLN4
NM_017433
MYO3A


NM_017495
RBM38
NM_173576
MKX


NR_003505
PPP4R1L
NM_021738
SVIL


NM_207034
EDN3
NM_003591
CUL2


NM_152757
C20orf200
NM_153368
GJD4


NM_017896
C20orf11
NM_001004297
OR13A1


NM_172109
KCNQ2
NM_020945
WDFY4


NM_005975
PTK6
NM_001079516
ASAH2B


NM_033405
PRIC285
NM_194298
SLC16A9


NM_012384
GMEB2
NM_030759
NRBF2


NM_182482
BAGE2
NM_201262
DNAJC12


NM_182484
BAGE5
NM_024045
DDX50


NM_001187
BAGE
NM_015634
KIAA1279


NR_003087
ABCC13
NM_018649
H2AFY2


NM_022136
SAMSN1
NM_020799
STAMBPL1


NM_001338
CXADR
NR_002779
NUDT9P1


NM_181600
KRTAP13-4
NM_032373
PCGF5


NM_181615
KRTAP20-1
NM_173497
HECTD2


NM_175858
KRTAP11-1
NM_003061
SLIT1


NM_005806
OLIG2
NM_018425
PI4K2A


NM_058182
FAM165B
NM_182639
HPS1


NM_130436
DYRK1A
NM_001278
CHUK


NM_033173
B3GALT5
NM_005029
PITX3


NM_182832
PLAC4
NM_015916
CALHM2


NM_002462
MX1
NM_001129742
CALHM3


NM_207629
ABCG1
NM_000681
ADRA2A


NM_003226
TFF3
NM_002313
ABLIM1


NM_018961
UBASH3A
NR_003251
INPP5F


NM_018964
SLC37A1
NM_014468
VENTX


NM_003307
TRPM2
NM_001109
ADAM8


NM_198699
KRTAP10-12
NM_000773
CYP2E1


NM_003343
UBE2G2
NM_025092
ATHL1


NM_032261
C21orf56
NM_001012416
KRTAP5-6


NM_006272
S100B
NM_002555
SLC22A18


NM_053006
TSSK2
NM_001005161
OR52B4


NM_003776
MRPL40
NM_001004753
OR51F2


NM_003325
HIRA
NM_001005567
OR51B5


NM_007310
COMT
NM_001005288
OR51I1


NM_002650
PI4KA
NM_130389
TRIM34


NM_004173
SLC7A4
NM_198185
OVCH2


NM_007128
VPREB1
NM_001004461
OR10A6


NM_002073
GNAZ
NM_001025389
AMPD3


NM_001007468
SMARCB1
NM_012250
RRAS2


NR_001283
TOP1P2
NM_016451
COPB1


NM_001008566
TPST2
NM_000922
PDE3B


NM_001886
CRYBA4
NM_001741
CALCA


NM_012399
PITPNB
NM_000728
CALCB


NM_013387
UCRC
NM_006292
TSG101


NM_004861
GAL3ST1
NM_032781
PTPN5


NM_014227
SLC5A4
NM_213599
ANO5


NM_001098535
RFPL3
NM_177553
GAS2


NR_001450
RFPL3S
NM_001018080
FSHB


NM_182486
C1QTNF6
NM_001127612
PAX6


NM_032561
C22orf23
NM_001752
CAT


NM_181773
APOBEC3H
NM_002391
MDK


NM_153497
MAP3K7IP1
NM_016223
PACSIN3


NM_005297
MCHR1
NM_024783
AGBL2


NM_003932
ST13
NM_001005272
OR4A5


NM_152513
MEI1
NM_001003750
OR8I2


NM_145733
3-Sep
NM_198183
UBE2L6


NM_007326
CYB5R3
NM_001085468
CTNND1


NM_001012986
LOC388910
NM_001005212
OR9Q1


NM_001123225
C22orf41
NM_001079807
PGA3


NM_152228
TAS1R3
NM_004111
FEN1


NM_001003808
C1orf222
NM_022830
TUT1


NM_207306
KIAA0495
NM_012200
B3GAT3


NM_181866
ACOT7
NM_198334
GANAB


NM_001042665
PLEKHG5
NR_002560
SNORD31


NM_004401
DFFA
NR_002561
SNORD30


NM_001103170
AADACL3
NR_002559
SNORD29


NM_032341
DDI2
NM_017490
MARK2


NM_178840
C1orf64
NM_014067
MACROD1


NM_004070
CLCNKA
NM_024650
C11orf80


NM_152376
UBXN10
NM_020441
CORO1B


NM_001077195
ZNF436
NM_145200
CABP4


NM_018202
TMEM57
NM_004055
CAPN5


NM_203401
STMN1
NM_021827
CCDC81


NM_024869
GRRP1
NM_001101321
UBTFL2


NR_003066
SNORD85
NM_004621
TRPC6


NM_001856
COL16A1
NM_004347
CASP5


NM_001042771
LCK
NM_052889
CARD16


NM_206837
C1orf102
NM_152434
CWF19L2


NM_022756
C1orf149
NM_207430
C11orf88


NM_003819
PABPC4
NM_001082970
C11orf57


NM_148960
CLDN19
NM_012459
TIMM8B


NM_173484
KLF17
NM_198971
HINFP


NM_020365
EIF2B3
NM_012101
TRIM29


NM_000778
CYP4A11
NR_003125
LOC85391


NM_057176
BSND
NM_001005188
OR6X1


NM_152377
C1orf87
NM_001004474
OR10S1


NM_032852
ATG4C
NM_001005468
OR8B2


NM_203464
AK3L1
NM_001005196
OR8B4


NR_003042
SNORD45C
NM_001005195
OR8B12


NM_005263
GFI1
NM_014987
IGSF9B


NM_005665
EVI5
NM_002234
KCNA5


NM_002061
GCLM
NM_001039916
ZNF384


NM_139156
AMPD2
NM_007273
PHB2


NM_147148
GSTM4
NM_000319
PEX5


NM_139053
EPS8L3
NM_015509
NECAP1


NM_004696
SLC16A4
NM_020661
AICDA


NM_004185
WNT2B
NM_016523
KLRF1


NM_006699
MAN1A2
NM_205852
CLEC12B


NM_006099
PIAS3
NM_030817
APOLD1


NM_005399
PRKAB2
NM_001013698
C12orf69


NM_212551
LYSMD1
NM_001175
ARHGDIB


NM_178431
LCE3A
NM_032918
RERG


NM_207308
NUP210L
NM_004570
PIK3C2G


NM_170782
KCNN3
NM_018638
ETNK1


NM_000157
GBA
NM_001004329
DBX2


NM_032323
TMEM79
NM_015401
HDAC7


NM_001004476
OR10K2
NM_015086
DDN


NM_001639
APCS
NM_021044
DHH


NM_015331
NCSTN
NM_006337
MCRS1


NM_005149
TBX19
NM_000424
KRT5


NM_018186
C1orf112
NR_003716
HOTAIR


NM_033418
C1orf156
NM_014212
HOXC11


NM_152663
RALGPS2
NM_014620
HOXC4


NM_173533
TDRD5
NR_003084
HOXC5


NM_052966
FAM129A
NM_153693
HOXC6


NM_006469
IVNS1ABP
NM_005538
INHBC


NM_002113
CFHR1
NM_014770
AGAP2


NM_001994
F13B
NM_002076
GNS


NM_001024594
C1orf53
NM_020525
IL22


NM_004767
GPR37L1
NM_032735
BEST3


NM_002832
PTPN7
NM_005447
RASSF9


NM_201348
PRELP
NM_001037671
C12orf74


NM_020439
CAMK1G
NM_018838
NDUFA12


NM_052843
OBSCN
NM_001093
ACACB


NM_005999
TSNAX
NM_024072
DDX54


NM_019090
KIAA1383
NM_194286
KIAA1853


NM_080738
EDARADD
NM_001014336
IL31


NM_152666
PLD5
NM_019887
DIABLO


NM_006642
SDCCAG8
NM_178314
RILPL1


NM_014812
CEP170
NR_002979
SNORA49


NM_031844
HNRNPU
NM_175066
DDX51


NM_001004492
OR2B11
NM_001007537
RP11-45B20.2


NM_001005487
OR13G1
NM_002097
GTF3A


NM_001005286
OR6F1
NM_145657
GSX1


NM_001005522
OR2T8
NM_178007
STARD13


NM_001004695
OR2T33
NM_005584
MAB21L1


NM_001004693
OR2T10
NM_001004127
ALG11


NM_001042521
MGC13057
NM_001005918
ATP7B


NM_013388
PREB
NM_001005501
OR4K2


NM_007266
GPN1
NM_001004480
OR11H6


NM_017910
TRMT61B
NM_138376
TTC5


NM_005102
FEZ2
NM_017807
OSGEP


NM_002759
EIF2AK2
NM_019852
METTL3


NM_148962
OXER1
NM_016609
SLC22A17


NM_001101330
LOC728819
NM_138460
CMTM5


NM_001042385
PREPL
NM_006177
NRL


NM_178313
SPTBN1
NM_006263
PSME1


NM_001039348
EFEMP1
NM_014169
CHMP4A


NM_144709
PUS10
NM_203355
CTAGES


NM_138458
WDR92
NM_020937
FANCM


NM_006507
REG1B
NM_018139
C14orf104


NM_017750
RETSAT
NM_006832
FERMT2


NM_018271
THNSL2
NM_022571
GPR135


NM_020151
STARD7
NM_018373
SYNJ2BP


NM_012214
MGAT4A
NM_014982
PCNX


NM_201557
FHL2
NM_001024674
LIN52


NM_032411
C2orf40
NM_004755
RPS6KA5


NM_032528
ST6GAL2
NM_000624
SERPINA5


NM_176825
SULT1C2
NM_152592
C14orf49


NM_000575
IL1A
NR_003237
SNORD113-9


NM_025181
SLC35F5
NM_001311
CRIP1


NM_005270
GLI2
NM_144599
NIPA1


NM_004805
POLR2D
NR_003317
SNORD116-2


NM_032357
CCDC115
NR_003298
SNORD115-6


NM_018328
MBD5
NR_003311
SNORD115-17


NM_004522
KIF5C
NR_003314
SNORD115-22


NM_052917
GALNT13
NR_003495
HBII-52-24


NM_006593
TBR1
NR_003347
SNORD115-32


NM_004490
GRB14
NR_003498
HBII-52-45


NM_021193
HOXD12
NM_001042495
SLC12A6


NM_001077197
PDE11A
NM_175741
C15orf55


NM_145739
OSBPL6
NM_207445
C15orf54


NM_000885
ITGA4
NM_001080791
C15orf57


NM_002500
NEUROD1
NM_015540
RPAP1


NM_005019
PDE1A
NM_014994
MAPKBP1


NM_000090
COL3A1
NM_173088
CAPN3


NM_014585
SLC40A1
NM_016396
CTDSPL2


NM_144708
ANKAR
NM_003758
EIF3J


NM_022353
OSGEPL1
NM_152448
C15orf43


NM_016192
TMEFF2
NM_207581
DUOXA2


NM_138395
MARS2
NM_000338
SLC12A1


NM_014670
BZW1
NM_014701
SECISBP2L


NM_006139
CD28
NM_153374
LYSMD2


NM_014929
FASTKD2
NM_001104554
PAQR5


NM_020989
CRYGC
NM_201526
ISLR


NM_000599
IGFBP5
NM_015162
ACSBG1


NM_001557
IL8RB
NM_003847
PEX11A


NM_005381
NCL
NM_020211
RGMA


NM_002601
PDE6D
NM_152449
LYSMD4


NM_001631
ALPI
NM_006453
TBL3


NM_017974
ATG16L1
NM_004209
SYNGR3


NR_003006
SCARNA6
NM_001103175
CCDC64B


NM_021027
UGT1A9
NM_004220
ZNF213


NR_004428
EGO
NM_016292
TRAP1


NM_015869
PPARG
NM_004380
CREBBP


NM_015199
ANKRD28
NM_001127448
ABAT


NM_054110
GALNTL2
NM_002761
PRM1


NM_017897
OXSM
NM_014153
ZC3H7A


NM_001025068
ARPP-21
NM_015161
ARL6IP1


NM_175888
ZNF620
NM_001002911
GPR139


NM_016305
SS18L2
NM_003460
ZP2


NM_001295
CCR1
NM_013302
EEF2K


NM_003965
CCRL2
NM_181078
IL21R


NM_138615
DHX30
NM_024516
C16orf53


NM_005051
QARS
NM_001114380
ITGAL


NM_080865
GPR62
NR_002966
SNORA30


NM_018313
PBRM1
NM_033226
ABCC12


NM_015224
C3orf63
NM_001006610
SIAH1


NM_002841
PTPRG
NM_144602
C16orf78


NM_182920
ADAMTS9
NM_031885
BBS2


NM_015123
FRMD4B
NM_002990
CCL22


NM_001080393
GLT8D4
NR_002978
SNORA46


NM_001080448
EPHA6
NR_003079
SNORD111


NM_014648
DZIP3
NM_020995
HPR


NM_005944
CD200
NM_001105663
NUDT7


NM_001085357
BTLA
NM_021197
WFDC1


NM_173799
TIGIT
NM_024735
FBXO31


NM_005694
COX17
NM_006822
RAB40B


NM_016372
GPR175
NM_182705
FAM101B


NM_007283
MGLL
NM_022463
NXN


NM_007354
C3orf27
NR_003073
SNORD91B


NM_004164
RBP2
NM_018553
C17orf85


NM_152616
TRIM42
NM_198501
SMTNL2


NM_023915
GPR87
NM_001100812
CXCL16


NM_000902
MME
NM_001024937
MINK1


NM_207015
NAALADL2
NM_019013
FAM64A


NM_014693
ECE2
NM_003985
TNK1


NM_005787
ALG3
NM_152766
C17orf61


NM_004366
CLCN2
NM_032356
LSMD1


NM_006232
POLR2H
NM_004217
AURKB


NM_004443
EPHB3
NM_201432
GAS7


NM_018138
TBCCD1
NM_001372
DNAH9


NM_181573
RFC4
NM_016113
TRPV2


NM_001114980
TP63
NM_016078
FAM18B


NM_178335
CCDC50
NM_016231
NLK


NM_015274
MAN2B2
NM_015584
POLDIP2


NM_001105662
USP17
NM_144683
DHRS13


NM_031911
C1QTNF7
NM_005408
CCL13


NM_001775
CD38
NM_017559
FNDC8


NM_182592
YIPF7
NM_001104588
SLFN11


NM_173536
GABRG1
NM_032875
FBXL20


NM_006587
CORIN
NM_199334
THRA


NM_018475
TMEM165
NM_033187
KRTAP4-3


NM_002703
PPAT
NM_033060
KRTAP4-1


NM_001012763
GNRHR
NM_021013
KRT34


NM_016323
HERC5
NM_032387
WNK4


NM_145244
DDIT4L
NM_001466
FZD2


NM_002006
FGF2
NM_152343
C17orf46


NM_007080
LSM6
NM_005486
TOM1L1


NM_183375
ESSPL
NM_138363
CCDC45


NM_004564
PET112L
NM_014960
ARSG


NM_001013415
FBXW7
NM_139177
SLC39A11


NM_000824
GLRB
NM_022036
GPRCSC


NM_201592
GPM6A
NM_178233
OTOP3


NM_138464
LOC116349
NR_004397
SNORD1C


NM_004174
SLC9A3
NM_199167
CLUL1


NM_018140
CEP72
NM_005433
YES1


NM_007030
TPPP
NM_173211
TGIF1


NM_182632
SLC6A18
NM_173464
L3MBTL4


NM_001001702
FLJ33360
NM_003826
NAPG


NM_001369
DNAH5
NM_172241
CTAGE1


NR_003921
PMCHL1
NM_002647
PIK3C3


NM_178140
PDZD2
NR_002970
SNORA37


NM_001042625
CAPSL
NM_005024
SERPINB10


NM_173489
HEATR7B2
NM_152721
DOK6


NM_001005473
PLCXD3
NM_006566
CD226


NM_175921
C5orf51
NM_130760
MADCAM1


NM_153361
MGC42105
NM_006830
UQCR


NM_021147
CCNO
NM_198532
C19orf35


NM_181523
PIK3R1
NM_024333
FSD1


NR_003014
SNORA47
NM_153359
MGC24975


NM_005779
LHFPL2
NM_174918
C19orf59


NM_005654
NR2F1
NR_002931
CLEC4GP1


NM_001002796
MCTP1
NM_012335
MYO1F


NM_031952
SPATA9
NM_001004699
OR2Z1


NM_004772
C5orf13
NM_004461
FARSA


NM_001077654
TNFAIP8
NM_080864
RLN3


NM_001048252
CTXN3
NM_032207
C19orf44


NM_002154
HSPA4
NM_145046
CALR3


NM_017415
KLHL3
NM_002248
KCNN1


NM_013983
NRG2
NM_025249
KIAA1683


NM_018931
PCDHB11
NM_001080409
ZNF99


NM_018929
PCDHGC5
NM_001105570
NUDT19


NM_001079812
DIAPH1
NM_019849
SLC7A10


NM_181677
PPP2R2B
NM_021902
FXYD1


NM_054023
SCGB3A2
NM_207392
KRTDAP


NM_133371
MYOZ3
NM_014364
GAPDHS


NM_052860
ZNF300
NM_021232
PRODH2


NR_002168
PPP1R2P3
NM_144689
ZNF420


NM_021911
GABRB2
NM_003890
FCGBP


NM_001129891
LOC-
NM_198540
B3GNT8



100131897




NM_144769
FOXI1
NM_004363
CEACAM5


NM_001008220
CPLX2
NM_002780
PSG4


NM_016290
UIMC1
NM_001005376
PLAUR


NM_197975
BTNL3
NM_001033719
ZNF404


NM_001085401
C6orf201
NM_001081563
DMPK


NM_145649
GCNT2
NM_004943
DMWD


NM_003529
HIST1H3A
NM_001425
EMP3


NR_003504
MGC22265
NM_182575
IZUMO1


NM_003535
HIST1H3J
NM_014475
DHDH


NM_001509
GPX5
NM_001459
FLT3LG


NM_006510
TRIM27
NM_001079907
ZNF331


NM_021904
GABBR1
NM_001037731
DEFB116


NM_001077511
TCF19
NM_153269
C20orf96


NM_001105564
CCHCR1
NM_080725
SRXN1


NM_005514
HLA-B
NM_001083910
SIRPB1


NM_002904
RDBP
NM_001039508
SIRPG


NM_022107
GPSM3
NR_003684
SNORD119


NM_001077516
SLC39A7
NM_000490
AVP


NM_002931
RING1
NM_198798
ANKRD5


NM_002263
KIFC1
NM_130811
SNAP25


NM_007104
RPL10A
NM_001195
BFSP1


NM_032115
KCNK16
NM_002509
NKX2-2


NM_014623
MEA1
NM_178312
GGTLC1


NM_152882
PTK7
NM_024893
C20orf39


NM_170609
CRISP1
NR_004846
LOC10013486


NM_014464
TINAG
NM_002110
HCK


NM_019036
HMGCLL1
NM_031483
ITCH


NM_183050
BCKDHB
NM_006404
PROCR


NR_003038
SNHG5
NM_080748
ROMO1


NM_018064
AKIRIN2
NM_213632
C20orf132


NM_002043
GABRR2
NR_003018
SNORA71D


NM_015323
KIAA0776
NR_003239
SNHG11


NM_005190
CCNC
NM_000022
ADA


NM_014028
OSTM1
NM_198139
SEMG1


NM_006016
CD164
NM_001048226
DBNDD2


NM_153048
FYN
NM_021035
ZNFX1


NM_002269
KPNA5
NM_080829
FAM65C


NM_024581
FAM184A
NM_173485
TSHZ2


NM_152730
C6orf170
NM_177980
CDH26


NM_001446
FABP7
NM_080606
BHLHE23


NM_030963
RNF146
NM_080823
SRMS


NM_014702
KIAA0408
NM_017806
LIME1


NM_001079823
LAMA2
NM_003906
MCM3AP


NM_005021
ENPP3
NM_058181
C21orf57


NM_053278
TAAR8
NM_021219
JAM2


NM_078488
VNN2
NM_181614
KRTAP19-7


NM_052831
C6orf192
NM_000454
SOD1


NM_005923
MAP3K5
NM_170737
KCNJ15


NM_001100166
PHACTR2
NM_004915
ABCG1


NM_001080951
PLAGL1
NM_198693
KRTAP10-2


NM_138785
C6orf72
NM_198690
KRTAP10-9


NM_017909
RMND1
NM_198692
KRTAP10-11


NM_001007466
TULP4
NM_197966
BID


NM_020823
TMEM181
NM_002688
5-Sep


NM_003057
SLC22A1
NR_003714
DKFZp434P21


NM_021977
SLC22A3
NM_002430
MN1


NM_024492
LPAL2
NM_030641
APOL6


NM_000301
PLG
NM_013356
SLC16A8


NM_031409
CCR6
NM_004810
GRAP2


NM_018974
UNC93A
NM_024053
CENPM


NM_144781
PDCD2
NM_058238
WNT7B


NM_020144
PAPOLB
NM_032257
ZMYND12


NM_032172
USP42
NM_002440
MSH4


NM_019005
MIOS
NM_031936
GPR61


NM_019029
CPVL
NM_001042552
TATDN3


NM_175887
PRR15
NM_007357
COG2


NM_032639
PLEKHA8
NM_001004698
OR2W5


NM_006774
INMT
NM_175735
LYG2


NM_016489
NT5C3
NM_001106
ACVR2B


NM_015283
DPY19L1
NM_007246
KLHL2


NM_002192
INHBA
NM_016604
JMJD1B


NM_015052
HECW1
NM_005565
LCP2


NM_001013398
IGFBP3
NM_014068
PSORS1C1


NR_003595
MGC26484
NM_004289
NFE2L3


NM_198570
VWC2
NM_018697
LANCL2


NM_030798
WBSCR16
NR_015374
LOC401463


NM_000927
ABCB1
NM_014637
MTFR1


NM_006980
MTERF
NM_172060
EYA1


NM_001742
CALCR
NM_199160
LHX6


NM_001099400
SGCE
NM_012364
OR1Q1


NM_015395
TECPR1
NM_000216
KAL1


NM_015545
PTCD1
NM_001099857
IKBKG


NM_000777
CYP3A5
NM_001002295
GATA3


NM_018275
C7orf43
NM_018294
CWF19L1


NM_013439
PILRA
NM_003054
SLC18A2


NM_145030
C7orf47
NM_001128202
C10orf122


NM_022574
GIGYF1
NM_030754
SAA2


NM_021930
RINT1
NM_012194
C11orf41


NM_007356
LAMB4
NM_199418
PRCP


NM_015723
PNPLA8
NM_145018
C11orf32


NM_182529
THAP5
NM_001009562
LST-3TM12


NM_001662
ARF5
NM_152590
IFLTD1


NM_012133
COPG2
NM_020782
KLHDC5


NM_012450
SLC13A4
NM_015319
TENC1


NM_003852
TRIM24
NM_201550
LRRC10


NM_130840
ATP6V0A4
NR_002801
DKFZp686A1627


NM_016019
LUC7L2
NM_016350
NIN


NM_022750
PARP12
NM_001756
SERPINA6


NM_001080392
KIAA1147
NM_004918
TCL1B


NM_018980
TAS2R5
NM_005926
MFAP1


NM_004668
MGAM
NM_152450
FAM81A


NR_002140
OR6W1P
NM_002558
P2RX1


NM_001004685
OR2F2
NM_000442
PECAM1


NM_001001659
OR2A14
NM_001040185
ZNF765


NM_002889
RARRES2




NM_198285
WDR86



















Supplemental Table 7. Gene Ontology (GO) analysis of genes


with promoter γH2AX accumulation in SEN cells.









GO term
Name
P-value





GO: 0043170
Biopolymer metabolic process
2.83E−26


GO: 0006139
Nucleobase, nucleoside, nucleotide and
1.43E−21



nucleic acid metabolic process


GO: 0007165
Signal transduction
4.91E−19


GO: 0019538
Protein metabolic process
7.67E−17


GO: 0019222
Regulation of metabolic process
1.25E−16


GO: 0031323
Regulation of cellular metabolic process
3.47E−16


GO: 0016070
RNA metabolic process
1.35E−14


GO: 0010468
Regulation of gene expression
1.35E−14


GO: 0044267
Cellular protein metabolic process
1.11E−13


GO: 0044260
Cellular macromolecule metabolic process
1.14E−13


GO: 0019219
Regulation of nucleobase, nucleoside,
3.05E−13



nucleotide and nucleic acid metabolic



process


GO: 0006350
transcription
3.16E−13


GO: 0045449
Regulation of transcription
1.15E−12


GO: 0048522
Positive regulation of cellular process
1.78E−11


GO: 0048518
Positive regulation of biological process
3.02E−11


GO: 0032774
RNA biosynthetic process
5.03E−11


GO: 0006351
Transcription DNA dependent
5.85E−11


GO: 0007275
Multicellular organismal development
6.55E−11


GO: 0051234
Establishment of localization
1.16E−10


GO: 0048519
Negative regulation of biological process
3.29E−10


GO: 0006355
Regulation of transcription, DNA dependent
3.35E−10


GO: 0006810
transport
3.75E−10


GO: 0048731
System development
5.44E−10


GO: 0051252
Regulation of RNA metabolic process
6.32E−10


GO: 0048523
Negative regulation of cellular process
9.86E−10


GO: 0048856
Anatomical structure development
1.25E−09


GO: 0051641
Cellular localization
4.91E−09


GO: 0035556
Intracellular signaling cascade
1.35E−08


GO: 0022607
Cellular component assembly
2.00E−08


GO: 0051649
Establishment of cellular localization
2.15E−08


GO: 0006366
Transcription from RNA polymerase II
2.38E−08



promoter


GO: 0007166
Cell surface receptor linked signal
2.81E−08



transduction


GO: 0009966
Regulation of signal transduction
4.65E−08


GO: 0006464
Protein modification process
5.35E−08


GO: 0006996
Organelle organization and biogenesis
7.78E−08


GO: 0065003
Macromolecular complex assembly
9.38E−08


GO: 0007049
Cell cycle
1.23E−07


GO: 0009058
Biosynthetic process
3.04E−07


GO: 0046907
Intracellular transport
4.31E−07


GO: 0048468
Cell development
5.91E−07


GO:
Macromolecule localization
6.70E−07


GO 0008283
Cell proliferation
8.91E−07


GO: 0045934
Negative regulation of nucleobase,
1.07E−06



nucleoside, nucleotide and nucleic acid



metabolic process


GO: 0009892
Negative regulation of metabolic process
1.99E−06


GO: 0031324
Negative regulation of cellular metabolic
2.53E−06



process


GO: 0006950
Response to stress
2.98E−06


GO: 0006357
Regulation of transcription from RNA
3.77E−06



polymerase II promoter


GO: 0008104
Protein localization
4.31E−06


GO: 0016481
Negative regulation of transcription
7.94E−06
















TABLE 8







Cell Cycle (GO: 0007049) and Cell Proliferation (GO: 0008283) genes with


promoter γH2AX accumulation in SEN cells.











Gene




GO term
symbol
Gene name
Function*





GO: 0007049
INHBA
inhibin, beta A
The inhibin beta A subunit joins the alpha




(activin A, activin
subunit to form a pituitary FSH secretion




AB alpha
inhibitor. Inhibin has been shown to regulate




polypeptide)
gonadal stromal cell proliferation negatively





and to have tumor-suppressor activity.


GO: 0007049
GFI1
growth factor
This gene encodes a nuclear zinc finger




independent 1
protein that functions as a transcriptional





repressor. This protein plays a role in diverse





developmental contexts, including





hematopoiesis and oncogenesis. It functions





as part of a complex along with other





cofactors to control histone modifications





that lead to silencing of the target gene





promoters.


GO: 0007049
CD28
CD28 molecule
CD28 costimulation is essential for CD4


GO: 0008283


(MIM 186940)-positive T-cell proliferation,





survival, interleukin-2 (IL2; MIM147680)





production, and T-helper type-2 (Th2)





development.


GO: 0007049
STMN1
stathmin
This gene belongs to the stathmin family of




1/oncoprotein 18
genes. It encodes a ubiquitous cytosolic





phosphoprotein proposed to function as an





intracellular relay integrating regulatory





signals of the cellular environment. The





encoded protein is involved in the regulation





of the microtubule filament system by





destabilizing microtubules.


GO: 0007049
CUL2
cullin 2
Core component of multiple cullin-RING-


GO: 0008283


based ECS (ElonginB/C-CUL2/5-SOCS-box





protein) E3 ubiquitin-protein ligase





complexes, which mediate the ubiquitination





of target proteins. May serve as a rigid





scaffold in the complex and may contribute





to catalysis through positioning of the





substrate and the ubiquitin-conjugating





enzyme.


GO: 0007049
GAS7
growth arrest-
Growth arrest-specific 7 is expressed




specific 7
primarily in terminally differentiated brain





cells and predominantly in mature cerebellar





Purkinje neurons. GAS7 plays a putative





role in neuronal development.


GO: 0007049
HERC5
hect domain and
This gene is a member of the HERC family




RLD 5
of ubiquitin ligases and encodes a protein





with a HECT domain and five RCC1





repeats. Pro-inflammatory cytokines





upregulate expression of this gene in





endothelial cells. The protein localizes to the





cytoplasm and perinuclear region and





functions as an interferon-induced E3





protein ligase that mediates ISGylation of





protein targets.


GO: 0008283
GLI2
GLI-Kruppel
This gene encodes a protein which belongs




family member
to the C2H2-type zinc finger protein




GLI2
subclass of the Gli family. Gli family zinc





finger proteins are mediators of Sonic





hedgehog (Shh) signaling and they are





implicated as potent oncogenes in the





embryonal carcinoma cell. The protein





encoded by this gene localizes to the





cytoplasm and activates patched Drosophila





homolog (PTCH) gene expression. It is also





thought to play a role during embryogenesis.





The encoded protein is associated with





several phenotypes - Greig





cephalopolysyndactyly syndrome, Pallister-





Hall syndrome, preaxial polydactyly type





IV, postaxial polydactyly types A1 and B.


GO: 0008283
TCF19
transcription factor
Potential trans-activating factor that could




19 (SC1)
play an important role in the transcription of





genes required for the later stages of cell





cycle progression.


GO: 0008283
CD40LG
CD40 ligand (TNF
The protein encoded by this gene is




superfamily,
expressed on the surface of T cells. It




member 5, hyper-
regulates B cell function by engaging CD40




IgM syndrome)
on the B cell surface. A defect in this gene





results in an inability to undergo





immunoglobulin class switch and is





associated with hyper-IgM syndrome.


GO: 0008283
CALCA
calcitonin/calcitonin-
This gene encodes the peptide hormones




related
calcitonin, calcitonin gene-related peptide




polypeptide, alpha
and katacalcin by tissue-specific alternative





RNA splicing of the gene transcripts and





cleavage of inactive precursor proteins.





Calcitonin is involved in calcium regulation





and acts to regulate phosphorus metabolism.


GO: 0008283
TNFSF15
tumor necrosis
The protein encoded by this gene is a




factor (ligand)
cytokine that belongs to the tumor necrosis




superfamily,
factor (TNF) ligand family. This cytokine




member 15
is a ligand for receptor TNFRSF25 and





decoy receptor TNFRSF21/DR6. It can





activate NF-kappaB and MAP kinases, and





acts as an autocrine factor to induce





apoptosis in endothelial cells. This cytokine





is also found to inhibit endothelial cell





proliferation, and thus may function as an





angiogenesis inhibitor.


GO: 0008283
ADRA2A
adrenergic, alpha-
Alpha-2-adrenergic receptors are members




2A-, receptor
of the G protein-coupled receptor





superfamily. These receptors have a critical





role in regulating neurotransmitter release





from sympathetic nerves and from





adrenergic neurons in the central nervous





system.


GO: 0008283
IL8RB
interleukin 8
The protein encoded by this gene is a




receptor, beta
member of the G-protein-coupled receptor





family. This protein is a receptor for





interleukin 8 (IL8). It binds to IL8 with high





affinity, and transduces the signal through a





G-protein activated second messenger





system. This receptor also binds to





chemokine (C—X—C motif) ligand 1





(CXCL1/MGSA), a protein with melanoma





growth stimulating activity, and has been





shown to be a major component required for





serum-dependent melanoma cell growth.





This receptor mediates neutrophil migration





to sites of inflammation.


GO: 0008283
TNFSF8
tumor necrosis
The protein encoded by this gene is a




factor (ligand)
cytokine that belongs to the tumor necrosis




superfamily,
factor (TNF) ligand family. This cytokine is




member 8
a ligand for TNFRSF8/CD30, which is a cell





surface antigen and a marker for Hodgkin





lymphoma and related hematologic





malignancies. This cytokine was shown to





enhance cell proliferation of some





lymphoma cell lines, while to induce cell





death and reduce cell proliferation of other





lymphoma cell lines.


GO: 0008283
FLT3LG
fms-related
Stimulates the proliferation of early




tyrosine kinase 3
hematopoietic cells. Synergizes well with a




ligand
number of other colony stimulating factors





and interleukins.


GO: 0008283
CD164
CD164 molecule,
CD164 is a type I integral transmembrane




sialomucin
sialomucin that functions as an adhesion





receptor.


GO: 0008283
PLG
plasminogen
The protein encoded by this gene is a





secreted blood zymogen that is activated by





proteolysis and converted to plasmin and





angiostatin.


GO: 0008283
FABP7
fatty acid binding
The protein encoded by this gene is a brain




protein 7, brain
fatty acid binding protein.


GO: 0008283
EVI5
ecotropic viral
Functions as a regulator of cell cycle




integration site 5
progression by stabilizing the FBXO5





protein and promoting cyclin-A





accumulation during interphase. May play a





role in cytokinesis.


GO: 0008283
IL1A
interleukin 1,
The protein encoded by this gene is a




alpha
member of the interleukin 1 cytokine family.





This cytokine is a pleiotropic cytokine





involved in various immune responses,





inflammatory processes, and hematopoiesis.


GO: 0008283
RERG
RAS-like,
RERG, a member of the RAS superfamily of




estrogen-regulated,
GTPases, inhibits cell proliferation and




growth inhibitor
tumor formation.


GO: 0008283
EIF2AK2
eukaryotic
Following activation by double-stranded




translation
RNA in the presence of ATP, the kinase




initiation factor 2-
becomes autophosphorylated and can




alpha kinase 2
catalyze the phosphorylation of the





translation initiation factor EIF2S1, which





leads to an inhibition of the initiation of





protein synthesis.


GO: 0008283
EMP3
epithelial
Probably involved in cell proliferation and




membrane protein 3
cell-cell interactions.


GO: 0008283
CRIP1
cysteine-rich
Cysteine-rich intestinal protein (CRIP)




protein 1
belongs to the LIM/double zinc finger




(intestinal)
protein family. CRIP may be involved in





intestinal zinc transport.


GO: 0008283
REG1B
regenerating islet-
This gene encodes a protein secreted by the




derived 1 beta
exocrine pancreas that is highly similar to




(pancreatic stone
the REG1A protein.




protein, pancreatic




thread protein)
















SUPPLEMENTAL TABLE 9







RT-PCR and qPCR primers











SEQ 


Primer
Sequence
ID NO





MIRm
Forward: 5′-AGGATTTAAAGCTCTCTCTGCAGG-3′
14



Reverse: 5′-ATGACTGAACTCTAAGATAAAGATCACAGC-3′
15


 


Alu
Forward: 5′-AGACAATCCTGGCCAACTTGG-3′
16



Reverse: 5′-GCATTCCTGGACTGTGATGTGG-3′
17


 


AluJb
Forward: 5′-TGTGTGCCTGTAGTCCTAGCTACTAGG-3′
18



Reverse: 5′-TTCAGGTTAGAGCTCTGAAGTCACG-3′
19


 


AluSx
Forward: 5′-TCTGCTCGGGAGGCTGAGG-3′
20



Reverse: 5′-CCACCCACGAAGAATACATTTGC-3′
21


 


RPL13A
Forward: 5′-AAGGTGTTTGACGGCATCC-3′
22



Reverse: 5′-GTTCTTCTCGGCCTGTTTCC-3′
23


 


Oct4
Forward: 5′-CTTCGCAAGCCCTCATTTC-3′
24



Reverse: 5′-GAGAAGGCGAAATCCGAAG-3′
25


 


Nanog
Forward: 5′-CCCCAGCCTTTACTCTTCCT-3′
26



Reverse: 5′-CTGGTTGCTCCAGGTTGAAT-3′
27


 


b-actin 
Forward: 5′-CAACTCCATCATGAAGTGTGAC-3′
28



Reverse: 5′-GCCATGCCAATCTCATCTTG-3′
29









Example 3
Protein Complex Assembled on piRNA Derived from Alu Retrotransposal Transcript Indicates Putative Participation of retroRNA in the Cell Cycle, DNA Repair and Chromatin Assembly
Recent genome wide analysis indicates that thousands of short Interspersed elements (SINEs) are present in constrained non-exonic elements (Lowe and Haussler, PNAS 2007) suggesting that these repeated retrotransposal elements might play a unique and yet to be discovered role in shaping and/or specializing the genomic landscape as well as gene regulation during mammalian evolution. Here we report LC-MS/MS RNA-affinity complex isolation using piRNA derived from Alu retrotransposal RNA. Our data indicate the role of piAlu RNAs in DNA repair, cell cycle, and chromatin regulation.
Recently, we have demonstrated that a majority of the repairable DNA damage sites in self-renewing human adult stem cells is associated with the retrotransposal portion of the genome, in particular, with Alu retrotransposons (Wang et al. (2011) Cell Cycle 10: 3016-3030). Our group has shown that the up-regulation of transcriptional activity from Alu can be triggered by genotoxic stress-induced damage and can be recorded in human retinal pigment epithelium (RPE) and human adult stem cells (Id.) upon in-vivo and ex-vivo aging, respectively. Evidence indicates that increased accumulation of Alu RNA within cells is directly linked to Dicer-1 deficiency (Kaneko et al. (2011) Nature 471: 325-330), suggesting that Alu transcript accumulation might not necessarily be due to increased transcriptional activity from Alu retrotransposons, but rather may result from the absence of concomitant Alu transcript processing, leading to cytotoxicity. Our recent data indicates that the accumulation of unprocessed Alu transcripts triggers chromatin deterioration, loss of DNA repair in pericentromeric areas eliciting the persistent DNA damage response and, ultimately, cellular senescence. Our data also indicate that human adult stem cells stably expressing an shRNA against Alu transcripts override cellular senescence and reinstate their DNA repair capacity (Wang et al. (2011) Cell Cycle 10: 3016-3030), suggesting that RNA interference (RNAi) machinery is involved in these events. This data also suggests two equally possible mechanisms through which an shRNA against Alu might mediate the observed function: 1) through the PTGS Dicer-dependent pathway via the cytoplasmic degradation of unprocessed Alu RNA or 2) through facilitating transcriptional silencing by recruiting either the Dicer-dependent or Piwi-dependent arms of the RNAi pathway to act directly on the chromatin as shown in FIG. 21. Both of these pathways are plausible and either could depend on the assembly of single or multiple protein complexes that are capable of cross-talk with DNA-damage-sensing/repair and chromatin and/or centromeric maintenance pathways. Interestingly, a recent study indicated that transcripts generated from telomere-repeat-encoded RNA (TERRA) interact with heterochromatin protein 1 (HP1), trimethlyated histone H3 Lysine 9 (H3K9me3), core components of the Shelterin complex as well as members of the DNA-damage-sensing pathway (Deng et al. (2009) Mol. Cell, 35: 403-413).
Similarly we believe that the portion of Alu RNA, which is functionally relevant to overriding the senescent phenotype of hADSCs (Wang et al. (2011) Cell Cycle 10: 3016-3030), can mediate a broad array of downstream effects associated with retrotransposal transcription. Since the cytoplasmic role of Alu RNA in the assembly of signal recognition particles has been previously reported (He et al. (1994) J. Cell Sci., 107(Pt 4): 903-912), we have focused our efforts specifically on the elucidation of nuclear complexes assembled on Alu RNA. Using an unbiased RNA affinity assay coupled with mass spectrometry, we provide evidence for the composition of molecular complexes assembled on processed Alu RNA transcripts. Our data suggests the pathways and molecular processes through which processed intermediates of Alu RNA may participate in a multitude of nuclear regulations within human adult stem cells.
Results
Alu RNA and its Homology to Human PIWI-Interacting RNA (piRNA)
Previously it was reported that Alu RNA participates in the cytoplasmic assembly of the signal recognition particle (SRP) in mammalian cells (He et al. (1994) J. Cell Sci., 107(Pt 4): 903-912). The mammalian SRP is composed of a single RNA, the 7SL RNA, and six proteins (Walter and Blobel (1981) J. Cell Biol., 91: 557-561). SRP9 and SRP14 bind to the 5′ end of the RNA (Alu-domain), which functions in translational arrest (Siegel and Walter (1988) Trends Biochem. Sci., 13: 314-316; Siegel and Walter (1986) Nature 320:81-84). However, this functional shRNA reported in Wang et al. (2011) Cell Cycle 10: 3016-3030 has no complementarity to 7SL RNA. In addition, the region of Alu that corresponds to the shRNA is more highly conserved between repeats than the rest of the element (FIG. 22B). This conservation is consistent with the ability of the shRNA to effectively knock down transcription from multiple repeated Alu copies, including members of distinct subfamilies. Taken together, these data illuminate the functional relevance of the Alu shRNA sequence analyzed here, and indicate that this sequence may represent a processed Alu RNA species that functions in the nucleus. We queried the Alu shRNA sequence analyzed here against previously reported PIWI interacting RNA (piRNA) sequences previously reported in Girard et al. (2006) Nature 442: 199-202.
The exact sequence of the shRNA was identified as a PIWI-interacting RNA, pir-52207 and pir-57460 (Id.) (NCBI accession #DQ585095 (tgcctgtaat cccagctact caggaggctg, (SEQ ID NO:30)), and is referred to herein as piALU. This suggests that Alu RNA is subjected to processing by an analog of the human PIWI complex, the molecular function of which is unknown, but nevertheless indicates that this piAlu transcript, derived from processed Alu transcripts, might function in the nucleus.
Alu RNA Interaction with Nuclear Factors
We attempted to obtain a comprehensive list of proteins that interact, both directly and indirectly, with piALU. For this purpose, we engaged an unbiased RNA-affinity assay using synthetic biotinylated RNA oligos encompassing Alu nucleotides 140-183, shown in FIG. 22B, with nuclear extracts from self-renewing human adult stem cells. This portion of Alu is conserved among different types of Alu retrotransposons: AluSx, AluJb, AluJo, and AluY (FIG. 22B).
Our piALU RNA affinity assay was combined with a comprehensive LC-MS/MS (Liquid Chromatography-Electrospray Tandem Mass Spectrometry) analysis of interacting proteins. Since a nuclear RNA surveillance pathway exists that protects from transcriptional noise, we added a control hexanucleotide repeat control RNA “bait”, described in Materials and Methods, to avoid non-sequence-specific interactions. The experimental procedure was previously reported by Deng et al. (2009) Mol. Cell, 35: 403-413 and is outlined in FIG. 22B. Our comparative analysis of RNA-protein binding by PAGE depicted the presence of multiple piALU probe-specific bands (FIG. 22C) that were excised for LC-MS/MS identification. An exhaustive list of the proteins identified is shown in Supplementary Table 1.
Alu RNA Interactions with Major Chromatin Modifiers, DNA Repair Complexes, Centromeric Chromatin/Kinetochore Assembly Factors and Transcriptional Factors
Our mass spectrometry analysis of unbiased piALU RNA-affinity precipitants revealed the presence four major protein categories such as chromatin modifiers, DNA repair complexes, centromeric chromatin/kinetochore assembly and transcriptional factors (FIG. 23). The abundance of each component is represented as a color gradient fading from true hues to black for singly represented peptides. Similar to protein interactions with telomeric RNA repeats (TERRA) (Id.), piALU binding proteins include DNA damage response proteins DNA-PKC, PARP and hnRNPM, suggesting that piALU RNA may be integral to DNA repair complexes.
To assess the relevance of piALU RNA to biological processes, we applied a Gene Ontology (GO) (The Gene Ontology Consortium (2010) Nucleic Acids Res., 38(suppl 1): D331-D335; Barrell et al. (2009) Nucleic Acids Res 37:D396-D403) analysis to the list of interacting proteins. All together, 19 cellular processes were significantly (3.12×10−13≦P≦1.5×10−3) enriched among Alu-interacting proteins including: DNA repair, organelle organization, cell cycle, chromatin remodeling and transcription (FIG. 24A). These broader categories encompass nucleotide excision repair, DSB repair via homologous recombination, DNA recombination, V(D)J recombination, response to stress, chromosome segregation, cell cycle check-points, regulation of cell cycle checkpoint arrest, interphase of mitotic cycle and mitosis (FIG. 24A). Complexes related to DNA duplex unwinding, regulation of transcription and transcriptional elongation from RNA polymerase II promoters were also significantly enriched.
Several nuclear proteins were associated with multiple GO terms (n=12) designated to cellular components including the centrosome, nucleoplasm, kinetochore, spindle, microtubule cytoskeleton, centromeric and telomeric regions of chromosomes, as well as the cohesin and DNA-directed RNA polymerase complexes (FIG. 24A).
These data suggest a possible role for Alu RNA in the regulation of chromatin organization in centromeric/pericentromeric regions as well as DNA repair processes and the transcriptional regulation of genes. All of these cellular processes have been proven to be indispensable to the regulation of cell cycle progression.
Protein-Protein Interaction Network of the piALU RNA-Affinity Complex
Understanding the cellular function(s) of an isolated protein complex(es) is facilitated by the ability to correctly uncover and annotate all functional interactions among LC-MS/MS identified proteins. To build a protein-protein interaction network among piALU RNA-affinity isolated proteins, we used the online database Search Tool for the Retrieval of Interacting Genes (STRING) version 9.0. This tool provides uniquely comprehensive coverage and ease of access to both experimental and predicted interaction information (Szklarczyk et al. (2011) Nucleic Acids Res., 39: D561-D568). The STRING model of our LC-MS/MS data of nuclear piALU RNA-protein interactions is shown in FIG. 24B. Only high confidence level (experimentally reported) evidence of interactions/associations in curated databases in conjunction with the Markov clustering algorithm (MCL) were used to build the network and assign proteins into families (stronger associations are represented by thicker lines in FIG. 24B and FIG. 25) (Enright et al. (2002) Nucleic Acids Res., 30: 1575-1584). Strikingly, 9 out of the 64 Alu RNA interacting partners are involved in chromosome segregation and include SGOL1, SGOL2, CENPE, CENPF, MRE11A, CDCA5 with 4 more (PBRM1, CENPL, CDC27 and MAP3K8) directly linked to mitosis, while the addition of PIN4 and KIF15 depicts a complex protein network regulating spindle assembly. This observation suggests that piALU transcripts might form a molecular connection between centromere cohesion and microtubule dynamics at the kinetochore. The previously reported loss of outer kinetochore proteins CENP-E and CENP-F upon knockdown of Sgo (Salic et al. (2004) Cell 118: 567-578), together with our recent observation that the accumulation of unprocessed Alu RNA blocks the recruitment the cohesin complex to centromeres and triggers cellular senescence in adult stem cells (Wang et al. (2011) Cell Cycle 10: 3016-3030), suggests that Alu RNA in its processed form may be an RNA adaptor in spindle microtubule dynamics.
Data shown in FIG. 25 also indicate that 34 out of the 64 analyzed piALU RNA interacting partners are somehow involved in the regulation of the cell cycle. The presence of centrosomal proteins CEP192, CEP110, CEP152 and CEP135 points to the intriguing possibility that Alu RNA intermediates play a role in this cellular organelle and together with CUL4A, GMNN and CTD1 (FIG. 25), may participate in regulating interphase and the G/M phase transition.
Excitingly, piALU RNA also pulled down components of the nucleotide excision repair (ERCC3, DDB1, MRE11A), non-homologous end joining (LIG4, XRCC6) and homologous recombination (PALB2, RAD50) DNA repair pathways as well as the BAZ1B protein, suggesting a putative role for piALU RNA in double-strand break repair. The assembly of the DNA damage repair complexes described herein may be a critical event in adult stem cell aging (Kaneko et al. (2011) Nature 471: 325-330; Wang et al. (2011) Cell Cycle 10: 3016-3030). It is tempting to speculate that a lack of proper assembly/function of these complexes together or separately with chromatin remodeling components (CHD6, SMARCA2, BAZ1A, ARID1A, 1N080, ASXL1 and KDM2A) may explain the gradual loss of DNA damage repair triggering the persistent DNA damage response and cellular senescence (Rodier et al. (2009) Nat. Cell Biol., 11: 973-979; Rodier et al. (2011) J. Cell Sci., 124: 68-81).
Discussion
A recent genome-wide ChIP-RNA sequencing project revealed that over a third of all known large conserved ncRNAs can be pulled down with just four different chromatin modifying proteins (Khalil et al. (2009) Proc. Natl. Acad. Sci. USA, 106: 11667-11672), suggesting that most long ncRNAs associate with chromatin modifiers. The piALU RNA interactome presented herein also includes a variety of chromatin modifiers, possibly elucidating another level of complexity in ncRNA/chromatin modifier protein complexes that depend on the presence of a shorter, processed form of the Alu transcript. The full-length sequence of Alu RNA can form four distinct stem-loop structures, which may act as individual nucleating centers for the formation of distinct RNA-protein complexes (FIG. 22). Only one of the four stem-loops was used as a synthetic RNA “bait”, since it encompasses the previously reported human PIWI-interacting RNA (Girard et al. (2006) Nature 442: 199-202) and is capable of nucleating protein complex(es) that are directly or indirectly responsible for reinstating self-renewal to senescent adult stem cells (Wang et al. (2011) Cell Cycle 10: 3016-3030).
Indeed, in accord with our previous data, the observed interactions supports the relevance of these nuclear piALU RNA-protein complex(es) to cell cycle control, DNA repair, chromatin maintenance, and the function of centromeres/pericentromeres in human adult stem cells. This data is currently lacking functional assessment, but nevertheless may hold the key to understanding how retrotransposal transcription/processing regulates human adult stem cell aging and senescence ex-vivo.
The data reported in this Example points to interesting possible involvements of short RNA species derived from Alu retrotransposons in the regulation of cell cycle control, RNA-pol II transcription and the maintenance of chromatin architecture. It appears that this participation is intimately linked to DNA repair processes.
Material and Methods
Human ADSC Isolation and Expansion
Human adipose derived stem cells (hADSCs) were isolated from human subcutaneous white adipose tissue collected during liposuction procedures. The lipoaspirate was suspended in Hank's Buffered Salt Solution (HBSS), 3.5% Bovine Serum Albumin (BSA), 1% Collagenase, type II (Sigma) in 1:3 w/v ratio and shaken at 37° C. for 50 min. The cells were filtered through a 70 μm mesh cell strainer (BD Falcon #352350), treated with Red Blood Cell Lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3), and expanded ex-vivo in DMEM/F12 complete medium (DMEM/F12, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin B; Invitrogen) in 10% CO2 at 37° C. and passaged at 80% confluency, changing medium every 72-96 hours.
Nuclear Chromatin Extraction
Nuclear and cytoplasmic proteins were isolated from self-renewing hADSCs using the ProteoJETTM Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas #K0311), according to the manufacturer's protocol.
RNA Secondary Structure Folding
5′ biotinylated single stranded synthetic RNA probes corresponding to the 140-183 nucleotide region of full-length Alu, (5′-/Bio/-GCC GGG CG UG AUG GUG GGC GCC UGU AGU CCC AGC UAC UCG GGA GGC-3′, SEQ ID NO:31) as well as a synthetic random octamer repeat 5′-/Bio/-(CACUGA)8 (SEQ ID NO:32) as a linear control, were purchased from Integrated DNA Technologies (IDT). These oligos were purified via RNase-Free HPLC. 8 μg of each oligo were heated in a PCR tube with 15 ml of 2× Folding Buffer (40 mM HEPES pH 7.9, 0.4 mM EDTA, 2 M NaCl) and 9 ml DEPC water with 50 U/ml SUPERase-In RNAse inhibitor (Ambion, #AM2694). The RNA oligonucleotides were heated to 78° C. to induce a linear conformation, then the reaction temperature was reduced to and held at 54° C. to accommodate and retain desired secondary structure formation (mimicking the secondary stem-loop structure of the full length RNA), based on calculations by UNAFold software (IDT). The control repeat RNA 5′-/Bio/-(CACUGA)8 (SEQ ID NO:32) is not capable of forming a secondary structure.
Protein Pulldown (Unbiased RNA Affinity Assay)
100 μl of streptavidin-coated Dynabeads (Dynabeads M-270 Streptavidin, #65306) per reaction were washed three times in 1×D150 buffer (20 mM HEPES pH 7.9, 20% glycerol, 0.2 mM EDTA, 150 mM NaCl, 0.05% NP-40, 1 mM PMSF, 10 mM β-mercaptoethanol) before a final high-salt wash with 1× Folding Buffer (containing 1 M NaCl). 30 μl of folded RNA solution was added to each reaction tube and incubated at RT for 15 min, then washed twice with 100 μl of low-salt Folding Buffer (containing 150 mM NaCl). Bead-bound RNA probes were incubated for 30 min at RT with nuclear extracts from self-renewing hADSCs, along with 1 μl of 0.01M PMSF and 3 μl SUPERase-In. After incubation, the Dynabeads were washed five times in 1×D150 buffer, leaving only directly and indirectly interacting partners attached to the beads. As a negative control, nuclear extracts were also incubated with Dynabeads alone to control for any interaction between nuclear molecules and the beads themselves.
Denaturing PAGE Analysis of Affinity Purified Protein Complexes.
XT Sample buffer 12 (BioRad #161-0791) and XT Reducing Agent (BioRad #161-0792) were added to 10 μg of protein. After denaturation for 5 min at 100° C., protein samples were loaded into a precast 4-12% NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen, #NP0321BOX), and run out at 150 V for two hours. Gels were stained with ProteoSilver Plus Silver Stain Kit (Sigma, #Prot-SIL1). A SeeBlue Plus2 Pre-Stained Standard (Invitrogen, cat #LC5925) was used for visualization and approximation of molecular weights of protein samples. Bands unique to the Alu probe lane were excised and the analyzed by LC-MS/MS for protein content.
Sample Preparation for MS Analysis
The gel samples were first rinsed with acetonitrile, then reduced using DTT followed by alkylation using iodoacetamide. We rinsed gel samples with three alternating washes of 50 mM ammonium bicarbonate and acetonitrile, then cooled and resuspended each gel slice in trypsin (5.5 μg/mL in 50 mM ammonium bicarbonate/10% acetonitrile) and incubated at 37° C. for 24 hours for digestion of proteins. We extracted peptides with one rinse of 50 mM ammonium bicarbonate/10% acetonitrile followed by one rinse of 50% acetonitrile/0.1% formic acid, and prepared samples for mass spectrometry by lyophilization and rehydration in 20 μL 5% acetonitrile/0.2% formic acid.
Identification of Protein Partners by Mass Spectrometry High Resolution LC-MS/MS
High resolution LC-MS/MS analysis was carried out on an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) as previously described ENREF 45 (Lopez et al. (2011) J. Proteome Res., 10: 133-142). Briefly, we loaded excised and digested samples into 96-well plates for mass spectrometry analysis on a LTQ-Orbitrap XL (Thermo Fisher Scientific) instrument as previously described (Lopez et al. (2011) J. Proteome Res., 10: 133-142). We injected 10 μL of each re-constituted sample using a Thermo Scientific EASY-nLC Autosampler. Reverse phase chromatographic separations were carried out using Hypersil GOLDTM C18TM 3 μm media packed into a fused silica 75 μm inner diameter, 20 cm long column running at 250 nL/min. A gradient was produced using 5-40% acetonitrile, 0.2% formic acid over 150 minutes. The LTQ-Orbitrap was run in a top 8 configuration at 60K resolution for a full scan, with monoisotopic precursor selection enabled, and +1, and unassigned charge state rejected. The analysis on the LTQ-Orbitrap instrument was carried out with CID fragmentation.
LC-MS/MS Data Analysis and Protein Identification
LC-MS/MS data analysis, protein identification and peaklist generation were performed using Proteome Discoverer (Thermo Fisher Scientific) algorithm incorporating the SEQUEST® search engine and PercolatorTM (Kall et al. (2007) Nat, Methods, 4: 923-925) as previously described (Lopez et al. (2011) J. Proteome Res., 10: 133-142). MS/MS data were searched using 10 ppm mass accuracy on precursor m/z and a 0.5 Da window on fragment ions. Fully enzymatic tryptic searches with up to three missed cleavage sites were allowed. Oxidized methionines were searched as a variable modification and alkylated cysteines were searched as a fixed modification. Human databases were downloaded from NCBI and supplemented with common contaminants. We filtered peptides for each charge state to a false discovery rate (FDR) of 1%, and then grouped peptides into proteins using Occam's razor logic.
Protein Interaction Network Analysis
All highly represented members of the RNA-protein complex which were isolated were analyzed using STRING 9.0 software (Szklarczyk et al. (2011) Nucleic Acids Res., 39: D561-D568). The obtained interaction network was subsequently analyzed with BINGO2 plug-in to determine statistical enrichments for 14 Gene Ontology (GO) categories (The Gene Ontology Consortium (2010) Nucleic Acids Res., 38(suppl 1) D331-D335; Barrell et al. (2009) Nucleic Acids Res 37:D396-D403).
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

## Claims

1. A method of inducing or restoring or maintaining proliferative capacity in a mammalian mesenchymal stem cell in vitro, said method comprising:
reducing the level or activity of SINE/Alu retrotransposon transcripts in said mammalian mesenchymal stem cell in an amount sufficient to induce or restore or maintain proliferative capacity to said mammalian mesenchymal stem cell, wherein said reducing the level or activity of SINE/Alu transcripts comprises introducing into said mammalian mesenchymal stem cell a construct that comprises or encodes a small interfering RNA (siRNA) molecule or a piRNA molecule that targets a 7SL conserved region of a SINE/Alu retrotransposon transcript.

2. The method of claim 1, wherein said method comprises:
restoring proliferative capacity to said mammalian mesenchymal stem cell when said mesenchymal mammalian stem cell has reduced or lost proliferative capacity; or
inducing proliferative capacity to said mammalian stem cell when said mesenchymal mammalian stem cell lacks such capacity; or
maintaining proliferative capacity in said mammalian mesenchymal stem cell.

3. The method of claim 1, wherein said mammalian mesenchymal stem cell shows one or more indicators of senescence.

4. The method of claim 1, wherein said small interfering RNA comprises a molecule selected from the group consisting of a single strand RNA, a paired double strand RNA (dsRNA), and a small hairpin RNA (shRNA).

5. The method of claim 1, wherein said construct comprises a nucleic acid comprising the sequence 5′-GAT CCC CCC ACC ACG CCC GGC TAA TTT TCA AGA GAA ATT AGC CGG GCG TGG TGG TTT TTG GAA A-3′ (SEQ ID NO:10).

6. The method of claim 1, wherein said construct produces a stable down regulation of SINE/Alu transcripts.

7. The method of claim 1, wherein said construct comprises a vector.

8. The method of claim 7, wherein the vector is a plasmid vector; or a viral vector.

9. The method of claim 8, wherein the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated vector.

10. The method of claim 1, wherein said introducing is achieved via transformation, transduction, transfection, or infection.

11. The method of claim 1, wherein said introducing is achieved via a lipid or liposome.

12. The method of claim 1, wherein said method does not comprise targeted upregulation of a Oct3/4, and/or Sox2, and/or Klf4, and/or c-myc, or providing one or more heterologous constructs in said cell encoding Oct3/4, and/or Sox2, and/or Klf4, and/or c-myc.

13. The method of claim 1, wherein said construct comprises or encodes a PIWI RNA (piRNA).