bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation
Authors: Chen Ching-Yi , Gherzi Roberto , Andersen Jens S. , Gaietta Guido , Jürchott Karsten , Royer Hans-Dieter , Mann Matthias , Karin Michael
Published: November 2021

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5′ untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.
(CA/GT)n microsatellites affect homologous recombination during yeast meiosis
Authors: Gendrel Christiane-Gabrielle , Boulet Annick , Dutreix Marie
Published: November 2021

One of the most common microsatellites in eukaryotes consists of tandem arrays of the dinucleotide GT. Although the study of the instability of such repetitive DNA has been extremely fruitful over the last decade, no biological function has been demonstrated for these sequences. We investigated the genetic behavior of a region of the yeast Saccharomyces cerevisiae genome containing a 39-CA/GT dinucleotide repeat sequence. When the microsatellite sequence was present at the ARG4 locus on homologous chromosomes, diploid cells undergoing meiosis generated an excess of tetrads containing a conversion of the region restricted to the region of the microsatellite close to the recombination-initiation double-strand break. Moreover, whereas the repetitive sequence had no effect on the frequency of single crossover, its presence strongly stimulated the formation of multiple crossovers. The combined data strongly suggest that numerous recombination events are restricted to the initiation side of the microsatellite as though progression of the strand exchange initiated at the ARG4 promoter locus was impaired by the repetitive sequence. This observation corroborates in vitro experiments that demonstrated that RecA-promoted strand exchange is inhibited by CA/GT dinucleotide tracts. Surprisingly, meiotic instability of the microsatellite was very high (>0.1 alterations per tetrad) in all the spores with parental and recombinant chromosomes.
PAT1, a new member of the GRAS family, is involved in phytochrome A signal transduction
Authors: Bolle Cordelia , Koncz Csaba , Chua Nam-Hai
Published: November 2021

Light signaling via the phytochrome A (phyA) photoreceptor controls basic plant developmental processes including de-etiolation and hypocotyl elongation. We have identified a new Arabidopsismutant, pat (phytochrome Asignal transduction)1-1, which shows strongly reduced responses in continuous far-red light. Physiological and molecular data indicate that this mutant is disrupted at an early step of phyA signal transduction. The PAT1 gene encodes a cytoplasmic protein of 490 amino acids with sequence homologies to the plant-specific GRAS regulatory protein family. In the pat1-1mutant, a T-DNA insertion introduces a premature stop codon, which likely results in the production of a truncated PAT1 protein of 341 amino acids. The semidominant phenotype of this mutant can be recapitulated by overexpression of an appropriately truncatedPAT1 gene in the wild type. The results indicate that the truncated PAT1 protein acts in a dominant-negative fashion to inhibit phyA signaling.
γ-Catenin is regulated by the APC tumor suppressor and its oncogenic activity is distinct from that of β-catenin
Authors: Kolligs Frank T. , Kolligs Barbara , Hajra Karen M. , Hu Gang , Tani Masachika , Cho Kathleen R. , Fearon Eric R.
Published: November 2021

β-Catenin and γ-catenin (plakoglobin), vertebrate homologs of Drosophila armadillo, function in cell adhesion and the Wnt signaling pathway. In colon and other cancers, mutations in the APC tumor suppressor protein orβ-catenin's amino terminus stabilizeβ-catenin, enhancing its ability to activate transcription of Tcf/Lef target genes. Thoughβ- and γ-catenin have analogous structures and functions and like binding to APC, evidence that γ-catenin has an important role in cancer has been lacking. We report here that APC regulates bothβ- and γ-catenin andγ-catenin functions as an oncogene. In contrast to β-catenin, for which only amino-terminal mutated forms transform RK3E epithelial cells, wild-type and several amino-terminal mutated forms of γ-catenin had similar transforming activity. γ-Catenin's transforming activity, like β-catenin's, was dependent on Tcf/Lef function. However, in contrast toβ-catenin, γ-catenin strongly activated c-Myc expression and c-Myc function was crucial for γ-catenin transformation. Our findings suggest APC mutations alter regulation of bothβ- and γ-catenin, perhaps explaining why the frequency of APC mutations in colon cancer far exceeds that of β-catenin mutations. Elevated c-Myc expression in cancers with APC defects may be due to altered regulation of both β- andγ-catenin. Furthermore, the data implyβ- and γ-catenin may have distinct roles in Wnt signaling and cancer via differential effects on downstream target genes.
E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis
Authors: Komada Masayuki , McLean Derek J. , Griswold Michael D. , Russell Lonnie D. , Soriano Philippe
Published: November 2021

Cell type-specific microtubules, such as the Sertoli cell microtubules and the manchette and flagellum microtubules of the spermatids, play essential roles in spermatogenesis. We identified the gene encoding E-MAP-115 (epithelial microtubule-associated protein of 115 kD) as a retinoic acid-inducible gene using gene trap mutagenesis in mouse embryonic stem cells. The gene trap insertion led to a null allele of the E-MAP-115 gene and, in agreement with its high expression in the testis, male mice homozygous for the mutation were sterile because of deformation of spermatid nuclei and subsequent gradual loss of germ cells. Consistent with a possible role for E-MAP-115 in stabilizing microtubules, microtubule associations in the mutant were morphologically abnormal in the manchette of spermatids and in Sertoli cells. We hypothesize that the abnormal microtubules in these two cell types are responsible for deformation of spermatid nuclei and germ cell loss, respectively, and indicate an essential role for E-MAP-115 in microtubule functions required for spermatogenesis.
Two lineage boundaries coordinate vertebrate apical ectodermal ridge formation
Authors: Kimmel Robin A. , Turnbull Daniel H. , Blanquet Veronique , Wurst Wolfgang , Loomis Cynthia A. , Joyner Alexandra L.
Published: November 2021

Proximal–distal outgrowth of the vertebrate limb bud is regulated by the apical ectodermal ridge (AER), which forms at an invariant position along the dorsal–ventral (D/V) axis of the embryo. We have studied the genetic and cellular events that regulate AER formation in the mouse. In contrast to implications from previous studies in chick, we identified two distinct lineage boundaries in mouse ectoderm prior to limb bud outgrowth using a Cre/loxP-based fate-mapping approach and a novel retroviral cell-labeling technique. One border is transient and at the limit of expression of the ventral gene En1, which corresponds to the D/V midline of the AER, and the second border corresponds to the dorsal AER margin. Labeling of AER precursors using an inducible Cre showed that not all cells that initially express AER genes form the AER, indicating that signaling is required to maintain an AER phenotype. Misexpression of En1 at moderate levels specifically in the dorsal AER of transgenic mice was found to produce dorsally shifted AER fragments, whereas high levels ofEn1 abolished AER formation. In both cases, the dorsal geneWnt7a was repressed in cells adjacent to theEn1-expressing cells, demonstrating that signaling regulated by EN1 occurs across the D/V border. Finally, fate mapping of AER domains in these mutants showed that En1 plays a part in positioning and maintaining the two lineage borders.
Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint
Authors: Liu Qinghua , Guntuku Saritha , Cui Xian-Shu , Matsuoka Shuhei , Cortez David , Tamai Katsuyuki , Luo Guangbin , Carattini-Rivera Sandra , DeMayo Francisco , Bradley Allan , Donehower Larry A. , Elledge Stephen J.
Published: November 2021

Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G2/M DNA damage checkpoint in response to γ-irradiation (IR). CHK1heterozygosity modestly enhances the tumorigenesis phenotype ofWNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.
Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion
Authors: Bour Barbara A. , Chakravarti Malabika , West Joshua M. , Abmayr Susan M.
Published: November 2021

The Drosophila sticks-and-stones (sns) locus was identified on the basis of its mutant phenotype, the complete absence of body wall muscles and corresponding presence of unfused myoblasts. The genetic location of the mutation responsible for this apparent defect in myoblast fusion was determined by recombination and deficiency mapping, and the corresponding wild-type gene was isolated in a molecular walk. Identification of the SNS coding sequence revealed a putative member of the immunoglobulin superfamily (IgSF) of cell adhesion molecules. As anticipated from this homology, SNS is enriched at the membrane and clusters at discrete sites, coincident with the occurrence of myoblast fusion. Both the sns transcript and the encoded protein are expressed in precursors of the somatic and visceral musculature of the embryo. Within the presumptive somatic musculature, SNS expression is restricted to the putative fusion-competent cells and is not detected in unfused founder cells. Thus, SNS represents the first known marker for this subgroup of myoblasts, and provides an opportunity to identify pathways specifying this cell type as well as transcriptional regulators of fusion-specific genes. To these ends, we demonstrate that the presence of SNS-expressing cells is absolutely dependent on Notch, and that expression of SNS does not require the myogenic regulatory protein MEF2.
Rearrangement of chromatin domains during development inXenopus
Authors: Vassetzky Yegor , Hair Alan , Méchali Marcel
Published: November 2021

A dynamic change in the organization of different gene domains transcribed by RNA polymerase I, II, or III occurs during the progression from quiescent [pre-midblastula transition (pre-MBT)] to active (post-MBT) embryos duringXenopusdevelopment. In the rDNA, c-myc, and somatic 5S gene domains, a transition from random to specific anchorage to the nuclear matrix occurs when chromatin domains become active. The keratin gene domain was also randomly associated to the nuclear matrix before MBT, whereas a defined attachment site was found in keratinocytes. In agreement with this specification, ligation-mediated (LM)-PCR genomic footprinting carried out on the subpopulation of 5S domains specifically attached to the matrix reveals the hallmarks of determined chromatin after the midblastula transition. In contrast, the same analysis performed on the total 5S gene population does not reveal specific chromatin organization, validating the use of nuclear matrix fractionation to unveil active chromatin domains. These data provide a means for the determination of active chromosomal territories in the embryo and emphasize the role of nuclear architecture in regulated gene expression during development.
A conserved family of calcineurin regulators
Authors: Kingsbury Tami J. , Cunningham Kyle W.
Published: November 2021

The protein phosphatase calcineurin mediates many cellular responses to calcium signals. Using a genetic screen in yeast, we identified a new family of proteins conserved in fungi and animals that inhibit calcineurin function when overexpressed. Overexpression of the yeast protein Rcn1p or the human homologs DSCR1 or ZAKI-4 inhibited two independent functions of calcineurin in yeast: The activation of the transcription factor Tcn1p and the inhibition of the H+/Ca2+ exchanger Vcx1p. Purified recombinant Rcn1p and DSCR1 bound calcineurin in vitro and inhibited its protein phosphatase activity. Signaling via calmodulin, calcineurin, and Tcn1p induced Rcn1p expression, suggesting that Rcn1p operates as an endogenous feedback inhibitor of calcineurin. Surprisingly, rcn1 null mutants exhibited phenotypes similar to those of Rcn1p-overexpressing cells. This effect may be due to lower expression of calcineurin in rcn1 mutants during signaling conditions. Thus, Rcn1p levels may fine-tune calcineurin signaling in yeast. The structural and functional conservation between Rcn1p and DSCR1 suggests that the mammalian Rcn1p-related proteins, termed calcipressins, will modulate calcineurin signaling in humans and potentially contribute to disorders such as Down Syndrome.
Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers
Authors: Zhu Lingyang , Wilken Jill , Phillips Nelson B. , Narendra Umadevi , Chan Ging , Stratton Stephen M. , Kent Stephen B. , Weiss Michael A.
Published: November 2021

Sex determination is regulated by diverse pathways. Although upstream signals vary, a cysteine-rich DNA-binding domain (the DM motif) is conserved within downstream transcription factors ofDrosophila melanogaster (Doublesex) and Caenorhabditis elegans (MAB-3). Vertebrate DM genes have likewise been identified and, remarkably, are associated with human sex reversal (46, XY gonadal dysgenesis). Here we demonstrate that the structure of the Doublesex domain contains a novel zinc module and disordered tail. The module consists of intertwined CCHC and HCCC Zn2+-binding sites; the tail functions as a nascent recognition α-helix. Mutations in either Zn2+-binding site or tail can lead to an intersex phenotype. The motif binds in the DNA minor groove without sharp DNA bending. These molecular features, unusual among zinc fingers and zinc modules, underlie the organization of a Drosophila enhancer that integrates sex- and tissue-specific signals. The structure provides a foundation for analysis of DM mutations affecting sexual dimorphism and courtship behavior.
Drosophila Double parked: a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts
Authors: Whittaker Allyson J. , Royzman Irena , Orr-Weaver Terry L.
Published: November 2021

We identified a Drosophila gene, double parked(dup), that is essential for DNA replication and belongs to a new family of replication proteins conserved fromSchizosaccharomyces pombe to humans. Strong mutations indup cause embryonic lethality, preceded by a failure to undergo S phase during the postblastoderm divisions. dup is required also for DNA replication in the adult ovary, establishing thatdup is needed for DNA replication at multiple stages of development. Strikingly, DUP protein colocalizes with the origin recognition complex to specific sites in the ovarian follicle cells. This suggests that DUP plays a direct role in DNA replication. Thedup transcript is cell cycle regulated and is under the control of E2F and Cyclin E. Interestingly, dup mutant embryos fail both to downregulate S phase genes and to engage a checkpoint preventing mitosis until completion of S phase. This could be either because these events depend on progression of S phase beyond the point blocked in the dup mutants or because DUP is needed directly for these feedback mechanisms.
Disruption of the ARF transcriptional activator DMP1 facilitates cell immortalization, Ras transformation, and tumorigenesis
Authors: Inoue Kazushi , Wen Renren , Rehg Jerold E. , Adachi Masashi , Cleveland John L. , Roussel Martine F. , Sherr Charles J.
Published: November 2021

The DMP1 transcription factor induces the ARF tumor suppressor gene in mouse fibroblasts, leading to cell cycle arrest in a p53-dependent manner. We disrupted sequences encoding the DNA-binding domain of DMP1 in mouse embryonic stem cells and derived animals lacking the functional protein. DMP1-null animals are small at birth, and males develop more slowly than their wild-type littermates. Some adult animals exhibit seizures and/or obstuctive uropathy, each of unknown cause. The growth of explantedDMP1-null mouse embryo fibroblasts (MEFs) is progressively retarded as cells are passaged in culture on defined transfer protocols; but, unlike the behavior of normal cells, p19ARF, Mdm2, and p53 levels remain relatively low and DMP1-null MEFs do not senesce. Whereas the establishment of cell lines from MEFs is usually always accompanied by either p53 or ARF loss of function, continuously passaged DMP1-null cells readily give rise to established 3T3 and 3T9 cell lines that retain wild-type ARFand functional p53 genes. Early-passage DMP1-null cells, like MEFs from either ARF-null or p53-null mice, can be morphologically transformed by oncogenic Ha-Ras (Val-12) alone. Splenic lymphocytes harvested from both DMP1-null andARF-null mice exhibit enhanced proliferative responses in long-term cultures when stimulated to divide with antibody to CD3 and interleukin-2. Although only 1 of 40 DMP1-null animals spontaneously developed a tumor in the first year of life, neonatal treatment with dimethylbenzanthracene or ionizing radiation induced tumors of various histologic types that were not observed in similarly treated DMP1+/+ animals. Karyotypic analyses of MEFs and lymphomas from DMP1-null animals revealed pseudodiploid chromosome numbers, consistent with the retention of wild-type p53. Together, these data suggest that ARF function is compromised, but not eliminated, in animals lacking functional DMP1.
Translational control of C/EBPα and C/EBPβ isoform expression
Authors: Calkhoven Cor F. , Müller Christine , Leutz Achim
Published: November 2021

Transcription factors derived from CCAAT/enhancer binding protein (C/EBP)α and C/EBPβ genes control differentiation and proliferation in a number of cell types. Various C/EBP isoforms arise from unique C/EBPβ and C/EBPα mRNAs by differential initiation of translation. These isoforms retain different parts of the amino terminus and therefore display different functions in gene regulation and proliferation control. We show that PKR and mTOR signaling pathways control the ratio of C/EBP isoform expression through the eukaryotic translation initiation factors eIF-2α and eIF-4E, respectively. An evolutionary conserved upstream open reading frame in C/EBPα and C/EBPβ mRNAs is a prerequisite for regulated initiation from the different translation initiation sites and integrates translation factor activity. Deregulated translational control leading to aberrant C/EBPα and C/EBPβ isoform expression or ectopic expression of truncated isoforms disrupts terminal differentiation and induces a transformed phenotype in 3T3-L1 cells. Our results demonstrate that the translational controlled ratio of C/EBPα and C/EBPβ isoform expression determines cell fate.
FIN219, an auxin-regulated gene, defines a link between phytochrome A and the downstream regulator COP1 in light control of Arabidopsis development
Authors: Hsieh Hsu-Liang , Okamoto Haruko , Wang Mingli , Ang Lay-Hong , Matsui Minami , Goodman Horward , Deng Xing Wang
Published: November 2021

Light signals perceived by photoreceptors are transduced to negatively regulate COP1, a key repressor of photomorphogenic development. To identify genes involved in light inactivation of COP1, a genetic screen was employed to identify extragenic modifier mutations of a temperature-sensitive cop1 allele. One suppressor mutation isolated also exhibited a far-red-specific long hypocotyl phenotype in a wild-type background. Further phenotypic analyses of this new mutation, named fin219, suggested that it defines a novel phytochrome A signaling component. Genetic analysis indicated that FIN219 interacts closely with another phytochrome A signaling component, FHY1. Molecular characterization of FIN219 indicated that it encodes a cytoplasmic localized protein highly similar to the GH3 family of proteins and its expression is rapidly induced by auxin. In contrast to its loss-of-function mutant phenotype, overexpression of FIN219 results in a far-red-specific hyperphotomorphogenic response. Our data suggest that FIN219 may define a critical link for phytochrome A-mediated far-red inactivation of COP1 and a possible cross-talk juncture between auxin regulation and phytochrome signaling.
Defective processing of methylated single-stranded DNA by E. coli alkB mutants
Authors: Dinglay Suneet , Trewick Sarah C. , Lindahl Tomas , Sedgwick Barbara
Published: November 2021

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agentN-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. ArecA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells.
An essential role in liver development for transcription factor XBP-1
Authors: Reimold Andreas M. , Etkin Amit , Clauss Isabelle , Perkins Andrew , Friend Daniel S. , Zhang John , Horton Heidi F. , Scott Andrew , Orkin Stuart H. , Byrne Michael C. , Grusby Michael J. , Glimcher Laurie H.
Published: November 2021

XBP-1 is a CREB/ATF family transcription factor highly expressed in hepatocellular carcinomas. Here we report that XBP-1 is essential for liver growth. Mice lacking XBP-1 displayed hypoplastic fetal livers, whose reduced hematopoiesis resulted in death from anemia. Nevertheless, XBP-1-deficient hematopoietic progenitors had no cell-autonomous defect in differentiation. Rather, hepatocyte development itself was severely impaired by two measures: diminished growth rate and prominent apoptosis. Specific target genes of XBP-1 in the liver were identified as αFP, which may be a regulator of hepatocyte growth, and three acute phase protein family members. Therefore, XBP-1 is a transcription factor essential for hepatocyte growth.
Rhomboid and Star facilitate presentation and processing of the Drosophila TGF-α homolog Spitz
Authors: Bang Anne G. , Kintner Chris
Published: November 2021

Activation of the Drosophila epidermal growth factor receptor (DER) by the transmembrane ligand, Spitz (Spi), requires two additional transmembrane proteins, Rhomboid and Star. Genetic evidence suggests that Rhomboid and Star facilitate DER signaling by processing membrane-bound Spi (mSpi) to an active, soluble form. To test this model, we use an assay based on Xenopus animal cap explants in which Spi activation of DER is Rhomboid and Star dependent. We show that Spi is on the cell surface but is kept in an inactive state by its cytoplasmic and transmembrane domains; Rhomboid and Star relieve this inhibition, allowing Spi to signal. We show further that Spi is likely to be cleaved within its transmembrane domain. However, a mutant form of mSpi that is not cleaved still signals to DER in a Rhomboid and Star-dependent manner. These results suggest strongly that Rhomboid and Star act primarily to present an active form of Spi to DER, leading secondarily to the processing of Spi into a secreted form.
c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi
Authors: Wu Min , Hemesath Timothy J. , Takemoto Clifford M. , Horstmann Martin A. , Wells Audrey G. , Price E. Roydon , Fisher Daniel Z. , Fisher David E.
Published: November 2021

Microphthalmia (Mi) is a bHLHZip transcription factor that is essential for melanocyte development and postnatal function. It is thought to regulate both differentiated features of melanocytes such as pigmentation as well as proliferation/survival, based on phenotypes of mutant mouse alleles. Mi activity is controlled by at least two signaling pathways. Melanocyte-stimulating hormone (MSH) promotes transcription of the Mi gene through cAMP elevation, resulting in sustained Mi up-regulation over many hours. c-Kit signaling up-regulates Mi function through MAP kinase phosphorylation of Mi, thereby recruiting the p300 transcriptional coactivator. The current study reveals that c-Kit signaling triggers two phosphorylation events on Mi, which up-regulate transactivation potential yet simultaneously target Mi for ubiquitin-dependent proteolysis. The specific activation/degradation signals derive from MAPK/ERK targeting of serine 73, whereas serine 409 serves as a substrate for p90 Rsk-1. An unphosphorylatable double mutant at these two residues is at once profoundly stable and transcriptionally inert. These c-Kit-induced phosphorylations couple transactivation to proteasome-mediated degradation. c-Kit signaling thus triggers short-lived Mi activation and net Mi degradation, in contrast to the profoundly increased Mi expression after MSH signaling, potentially explaining the functional diversity of this transcription factor in regulating proliferation, survival, and differentiation in melanocytes.
Activation mutants in yeast RNA polymerase II subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria
Authors: Tan Qian , Linask K. Liisi , Ebright Richard H. , Woychik Nancy A.
Published: November 2021

We have identified a mutant in RPB3, the third-largest subunit of yeast RNA polymerase II, that is defective in activator-dependent transcription, but not defective in activator-independent, basal transcription. The mutant contains two amino-acid substitutions, C92R and A159G, that are both required for pronounced defects in activator-dependent transcription. Synthetic enhancement of phenotypes of C92R and A159G, and of several other pairs of substitutions, is consistent with a functional relationship between residues 92–95 and 159–161. Homology modeling of RPB3 on the basis of the crystallographic structure of αNTD indicates that residues 92–95 and 159–162 are likely to be adjacent within the structure of RPB3. In addition, homology modeling indicates that the location of residues 159–162 within RPB3 corresponds to the location of an activation target within αNTD (the target of activating region 2 of catabolite activator protein, an activation target involved in a protein–protein interaction that facilitates isomerization of the RNA polymerase promoter closed complex to the RNA polymerase promoter open complex). The apparent finding of a conserved surface required for activation in eukaryotes and bacteria raises the possibility of conserved mechanisms of activation in eukaryotes and bacteria.
Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition
Authors: Cowley Dale O. , Graves Barbara J.
Published: November 2021

Phosphorylation of transcription factors is a key link between cell signaling and the control of gene expression. Here we report that phosphorylation regulates DNA binding of the Ets-1 transcription factor by reinforcing an autoinhibitory mechanism. Quantitative DNA-binding assays show that calcium-dependent phosphorylation inhibits Ets-1 DNA binding 50-fold. The four serines that mediate this inhibitory effect are distant from the DNA-binding domain but near structural elements required for autoinhibition. Mutational analyses demonstrate that an intact inhibitory module is required for phosphorylation-dependent regulation. Partial proteolysis studies indicate that phosphorylation stabilizes an inhibitory conformation. These findings provide a structural mechanism for phosphorylation-dependent inhibition of Ets-1 DNA binding and demonstrate a new function for inhibitory modules as structural mediators of negative signaling events.
Repression by Suppressor of Hairless and activation by Notch are required to define a single row of single-minded expressing cells in the Drosophila embryo
Authors: Morel Véronique , Schweisguth François
Published: November 2021

Notch signal transduction appears to involve the ligand-induced intracellular processing of Notch, and the formation of a processed Notch-Suppressor of Hairless complex that binds DNA and activates the transcription of Notch target genes. This suggests that loss of eitherNotch or Su(H) activities should lead to similar cell fate changes. However, previous data indicate that, in theDrosophila blastoderm embryo, mesectoderm specification requires Notch but not Su(H) activity. The determination of the mesectodermal fate is specified by Single-minded (Sim), a transcription factor expressed in a single row of cells abutting the mesoderm. The molecular mechanisms by which the dorsoventral gradient of nuclear Dorsal establishes the single-cell wide territory of sim expression are not fully understood. We have found that Notch activity is required for simexpression in cellularizing embryos. In contrast, at this stage,Su(H) has a dual function. Su(H) activity was required to up-regulate sim expression in the mesectoderm, and to prevent the ectopic expression of sim dorsally in the neuroectoderm. We have shown that repression of simtranscription by Su(H) is direct and independent of Notchactivity. Conversely, activation of sim transcription by Notch requires the Su(H)-binding sites. Thus, Notch signalling appears to relieve the repression exerted by Su(H) and to up-regulate simtranscription in the mesectoderm. We propose a model in which repression by Su(H) and derepression by Notch are essential to allow for the definition of a single row of mesectodermal cells in the blastoderm embryo.
Induction of terminal differentiation by constitutive activation of p38 MAP kinase in human rhabdomyosarcoma cells
Authors: Puri Pier Lorenzo , Wu Zhenguo , Zhang Peilin , Wood Lauren D. , Bhakta Kunjan S. , Han Jiahuai , Feramisco James R. , Karin Michael , Wang Jean Y.J.
Published: November 2021

MyoD inhibits cell proliferation and promotes muscle differentiation. A paradoxical feature of rhabdomyosarcoma (RMS), a tumor arising from muscle precursors, is the block of the differentiation program and the deregulated proliferation despite MyoD expression. A deficiency in RMS of a factor required for MyoD activity has been implicated by previous studies. We report here that p38 MAP kinase (MAPK) activation, which is essential for muscle differentiation, is deficient in RMS cells. Enforced induction of p38 MAPK by an activated MAPK kinase 6 (MKK6EE) restored MyoD function and enhanced MEF2 activity in RMS deficient for p38 MAPK activation, leading to growth arrest and terminal differentiation. Stress and cytokines could activate the p38 MAPK in RMS cells, however, these stimuli did not promote differentiation, possibly because they activated p38 MAPK only transiently and they also activated JNK, which could antagonize differentiation. Thus, the selective and sustained p38 MAPK activation, which is distinct from the stress-activated response, is required for differentiation and can be disrupted in human tumors.
The Drosophila Shark tyrosine kinase is required for embryonic dorsal closure
Authors: Fernandez Rafael , Takahashi Fumitaka , Liu Zhao , Steward Ruth , Stein David , Stanley E. Richard
Published: November 2021

Dorsal closure (DC) in the Drosophila embryo requires the coordinated interaction of two different functional domains of the epidermal cell layer—the leading edge (LE) and the lateral epidermis. In response to activation of a conserved c-Jun amino-terminal kinase (JNK) signaling module, the dorsal-most layer of cells, which constitute the LE of the stretching epithelial sheet, secrete Dpp, a member of the TGFβ superfamily. Dpp and other LE cell-derived signaling molecules stimulate the bilateral dorsal elongation of cells of the dorsolateral epidermis over the underlaying amnioserosa and the eventual fusion of their LEs along the dorsal midline. We have found that flies bearing a Shark tyrosine kinase gene mutation,shark1, exhibit a DC-defective phenotype. Dpp fails to be expressed in shark1 mutant LE cells. Consistent with these observations, epidermal-specific reconstitution ofshark function or overexpression of an activated form of c-Jun in the shark1 mutant background, rescues the DC defect. Thus, Shark regulates the JNK signaling pathway leading to Dpp expression in LE cells. Furthermore, constitutive activation of the Dpp pathway throughout the epidermis fails to rescue theshark1 DC defect, suggesting that Shark may function in additional pathways in the LE and/or lateral epithelium.
Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock
Authors: Akashi Makoto , Nishida Eisuke
Published: November 2021

Although the suprachiasmatic nucleus (SCN) is the major pacemaker in mammals, the peripheral cells or immortalized cells also contain a circadian clock. The SCN and the periphery may use different entraining signals—light and some humoral factors, respectively. We show that induction of the circadian oscillation of gene expression is triggered by TPA treatment of NIH-3T3 fibroblasts, which is inhibited by a MEK inhibitor, and that prolonged activation of the MAPK cascade is sufficient to trigger circadian gene expression. Therefore, such prolonged activation of MAPK by entraining cues may be involved in the resetting of the circadian clock.
The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1
Authors: Pfleger Cathie M. , Kirschner Marc W.
Published: November 2021

The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G1, by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20–APC requires a well-defined destruction box (D box), whereas Cdh1–APC confers a different and as yet unidentified specificity. We have determined the sequence specificity for Cdh1–APC using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1–APC in Xenopus extracts. Cdc20 is itself a Cdh1–APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1–APC through a different sequence. By analysis of Cdc20 as a substrate, we have identified a new recognition signal. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1–APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, we have identified cell cycle genes Nek2 and B99 as additional Cdh1–APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation.
E2f3 is critical for normal cellular proliferation
Authors: Humbert Patrick O. , Verona Raluca , Trimarchi Jeffrey M. , Rogers Catherine , Dandapani Savita , Lees Jacqueline A.
Published: November 2021

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, we generated an E2f3 mutant mouse strain. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, we analyzed the properties of embryonic fibroblasts derived from E2f3 mutant mice. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. We have been able to identify a number of genes, including B-myb,cyclin A, cdc2, cdc6, and DHFR, whose expression is dependent on the presence of E2F3 but not E2F1. We further show that a critical threshold level of one or more of the E2F3-regulated genes determines the timing of the G1/S transition, the rate of DNA synthesis, and thereby the rate of cellular proliferation. Finally, we show that E2F3 is not required for cellular immortalization but is rate limiting for the proliferation of the resulting tumor cell lines. We conclude that E2F3 is critical for the transcriptional activation of genes that control the rate of proliferation of both primary and tumor cells.
PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family
Authors: Attardi Laura D. , Reczek Elizabeth E. , Cosmas Corinna , Demicco Elizabeth G. , McCurrach Mila E. , Lowe Scott W. , Jacks Tyler
Published: November 2021

The p53 tumor suppressor activates either cell cycle arrest or apoptosis in response to cellular stress. Mouse embryo fibroblasts (MEFs) provide a powerful primary cell system to study both p53-dependent pathways. Specifically, in response to DNA damage, MEFs undergo p53-dependent G1 arrest, whereas MEFs expressing the adenovirus E1A oncoprotein undergo p53-dependent apoptosis. As the p53-dependent apoptosis pathway is not well understood, we sought to identify apoptosis-specific p53 target genes using a subtractive cloning strategy. Here, we describe the characterization of a gene identified in this screen, PERP, which is expressed in a p53-dependent manner and at high levels in apoptotic cells compared with G1-arrested cells. PERP induction is linked to p53-dependent apoptosis, including in response to E2F-1-driven hyperproliferation. Furthermore, analysis of the PERP promoter suggests that PERP is directly activated by p53. PERP shows sequence similarity to the PMP-22/gas3 tetraspan membrane protein implicated in hereditary human neuropathies such as Charcot–Marie–Tooth. Like PMP-22/gas3, PERP is a plasma membrane protein, and importantly, its expression causes cell death in fibroblasts. Taken together, these data suggest that PERP is a novel effector of p53-dependent apoptosis.
P-TEFb kinase recruitment and function at heat shock loci
Authors: Lis John T. , Mason Paul , Peng J. , Price David H. , Werner Janis
Published: November 2021

P-TEFb, a heterodimer of the kinase Cdk9 and cyclin T, was isolated as a factor that stimulates formation of productive transcription elongation complexes in vitro. Here, we show that P-TEFb is located at >200 distinct sites on Drosophila polytene chromosomes. Upon heat shock, P-TEFb, like the regulatory factor HSF, is rapidly recruited to heat shock loci, and this recruitment is blocked in an HSF mutant. Yet, HSF binding to DNA is not sufficient to recruit P-TEFb in vivo, and HSF and P-TEFb immunostainings within a heat shock locus are not coincident. Insight to the function of P-TEFb is offered by experiments showing that the direct recruitment of a Gal4-binding domain P-TEFb hybrid to an hsp70 promoter in Drosophilacells is sufficient to activate transcription in the absence of heat shock. Analyses of point mutants show this P-TEFb stimulation is dependent on Cdk9 kinase activity and on Cdk9's interaction with cyclin T. These results, coupled with the frequent colocalization of P-TEFb and the hypophosphorylated form of RNA polymerase II (Pol II) found at promoter-pause sites, support a model in which P-TEFb acts to stimulate promoter-paused Pol II to enter into productive elongation.
The Caenorhabditis elegans APC-related gene apr-1 is required for epithelial cell migration and Hox gene expression
Authors: Hoier Erika Fröhli , Mohler William A. , Kim Stuart K. , Hajnal Alex
Published: November 2021

Inactivation of the Caenorhabditis elegans APC-related gene (apr-1) has pointed at two separate functions of apr-1. First, apr-1 is required for the migration of epithelial cells during morphogenesis of the embryo. In this process, APR-1 may act in a Cadherin/α-Catenin/β-Catenin complex as a component of adherens junctions. Second, apr-1 is required for Hox gene expression, most likely by positively regulating the activity of the Wingless signaling pathway. During embryogenesis, apr-1 is required for the expression ofceh-13 labial in anterior seam and muscle cells and during larval development, apr-1 is necessary for the expression of lin-39 deformed in the vulval precursor cells. Thus, APR-1 may positively regulate the activity of the β-Catenin/Armadillo-related proteins HMP-2 in migrating epithelial cells and BAR-1 in the vulval precursor cells.
Inhibitor of striateconditionally suppresses cell proliferation in variegated maize
Authors: Park Sung Han , Park Su Hyun , Chin Hang Gyeong , Cho Moo Je , Martienssen Robert A. , Han Chang-deok
Published: November 2021

Since the work done by R.A. Emerson in the 1930s,Inhibitor of striate(Isr) has been recognized as a dose-dependent genetic modifier of variegation in chlorotic leaf striping mutants of maize such asstriate2(sr2). We have shown thatIsrspecifically inhibits proliferation and differentiation of plastid defective cells insr2mutants. Leaf narrowing is due to loss of intermediate veins and ground tissue located at leaf margins, and the few remaining plastid defective cells are of irregular size and aberrant organization. TheIsrgene has been cloned by targeted transposon tagging.IsrmRNA is expressed throughout young leaves, butIsrchimeras indicate that the expression ofIsrat leaf margins is sufficient to suppress both the lateral expansion ofsr2leaves and the extent of striping. Isr protein appears to encode a chloroplast protein with sequence similarity to a family of bacterial phosphatases involved in carbon catabolite repression or in carbon metabolism. We propose that the action ofIsrin nuclear and plastid communication could be triggered by carbon stress.
BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures
Authors: Wang Yi , Cortez David , Yazdi Parvin , Neff Norma , Elledge Stephen J. , Qin Jun
Published: November 2021

We report the identities of the members of a group of proteins that associate with BRCA1 to form a large complex that we have named BASC (BRCA1-associated genomesurveillance complex). This complex includes tumor suppressors and DNA damage repair proteins MSH2, MSH6, MLH1, ATM, BLM, and the RAD50–MRE11–NBS1 protein complex. In addition, DNA replication factor C (RFC), a protein complex that facilitates the loading of PCNA onto DNA, is also part of BASC. We find that BRCA1, the BLM helicase, and the RAD50–MRE11–NBS1 complex colocalize to large nuclear foci that contain PCNA when cells are treated with agents that interfere with DNA synthesis. The association of BRCA1 with MSH2 and MSH6, which are required for transcription-coupled repair, provides a possible explanation for the role of BRCA1 in this pathway. Strikingly, all members of this complex have roles in recognition of abnormal DNA structures or damaged DNA, suggesting that BASC may serve as a sensor for DNA damage. Several of these proteins also have roles in DNA replication-associated repair. Collectively, these results suggest that BRCA1 may function as a coordinator of multiple activities required for maintenance of genomic integrity during the process of DNA replication and point to a central role for BRCA1 in DNA repair.
Analysis of p53-regulated gene expression patterns using oligonucleotide arrays
Authors: Zhao Renbin , Gish Kurt , Murphy Maureen , Yin Yuxin , Notterman Daniel , Hoffman William H. , Tom Edward , Mack David H. , Levine Arnold J.
Published: November 2021

Oligonucleotide microarrays were employed to quantitate mRNA levels from a large number of genes regulated by the p53 transcription factor. Responses to DNA damage and to zinc-inducible p53 were compared for their transcription patterns in cell culture. A cluster analysis of these data demonstrates that genes induced by γ radiation, UV radiation, and the zinc-induced p53 form distinct sets and subsets with a few genes in common to all these treatments. Cell type- or cell line-specific p53 responses were detected. When p53 proteins were induced with zinc, the kinetics of induction or repression of mRNAs from p53-responsive genes fell into eight distinct classes, five different kinetics of induction, and three different kinetics of repression. In addition, low levels of p53 in a cell induced or repressed only a subset of genes observed at higher p53 levels. The results of this study demonstrate that the nature of the p53 response in diverse mRNA species depends on the levels of p53 protein in a cell, the type of inducing agent or event, and the cell type employed. Of 6000 genes examined for p53 regulatory responses, 107 induced and 54 repressed genes fell into categories of apoptosis and growth arrest, cytoskeletal functions, growth factors and their inhibitors, extracellular matrix, and adhesion genes.
MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis
Authors: Srayko Martin , Buster Dan W. , Bazirgan Omar A. , McNally Francis J. , Mains Paul E.
Published: November 2021

The Caenorhabditis elegans meiotic spindle is morphologically distinct from the first mitotic spindle, yet both structures form in the same cytoplasm ∼20 minutes apart. Themei-1 and mei-2 genes of C. elegans are required for the establishment of the oocyte meiotic spindle but are not required for mitotic spindle function. mei-1 encodes an AAA ATPase family member with similarity to the p60 catalytic subunit of the heterodimeric sea urchin microtubule-severing protein, katanin. We report that mei-2 encodes a 280-amino acid protein containing a region similar to the p80-targeting subunit of katanin. MEI-1 and MEI-2 antibodies decorate the polar ends of meiotic spindle microtubules and meiotic chromatin. We find that the subcellular location of MEI-2 depends on wild-type mei-1 activity and vice versa. These experiments, combined with MEI-1 and MEI-2's similarity to p60 and p80 katanin, suggest that the C. elegans proteins function as a complex. In support of this idea, MEI-1 and MEI-2 physically associate in HeLa cells. Furthermore, co-expression of MEI-1 and MEI-2 in HeLa cells results in the disassembly of microtubules. These data lead us to conclude that MEI-1/MEI-2 microtubule-severing activity is required for meiotic spindle organization in C. elegans.