Author response: The REST remodeling complex protects genomic integrity during embryonic neurogenesis (2015)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation. https://doi.org/10.7554/eLife.09584.001 eLife digest In the brain, cells called neurons connect to each other to form complex networks through which information is rapidly processed. These cells start to form in the developing brains of animal embryos when “neural” stem cells divide in a process called neurogenesis. For this process to proceed normally, particular genes in the stem cells have to be switched on or off at different times. This ensures that the protein products of the genes are only made when they are needed. Proteins called transcription factors can bind to DNA to activate or inactivate particular genes; for example, a transcription factor called REST inactivates thousands of genes that are needed by neurons. During neurogenesis, the production of REST normally declines, and some studies have shown that if the production of this protein is artificially increased, the formation of neurons is delayed. However, other studies suggest that REST may not play a major role in neurogenesis. Here, Nechiporuk et al. re-examine the role of REST in mice. The experiments used genetically modified mice in which the gene that encodes REST was prematurely switched off in neural stem cells. Compared with normal mice, these mutant mice had much smaller brains that contained fewer neurons because the stem cells stopped dividing earlier than normal. Unexpectedly, many genes that are normally switched off by REST, were not significantly changed, while genes that are not normally regulated by REST – such as the gene that encodes a protein called p53 – were active. It is known from previous work that p53 is expressed when cells are exposed to harmful conditions that can damage DNA. This helps to prevent cells from becoming cancerous. Nechiporuk et al. found that cells that lacked REST had higher levels of DNA damage than normal cells due to errors during the process of copying DNA before a cell divides. Furthermore, when both REST and p53 were absent, the neural stem cells became cancerous and formed tumors in the mice. Nechiporuk et al.’s findings suggest that REST protects the DNA of genes that are needed for neurons to form and work properly. The new challenge is to understand where in the genome the damage is occurring. https://doi.org/10.7554/eLife.09584.002 Introduction The transcriptional repressor REST (also called NRSF; Schoenherr and Anderson, 1995) binds to thousands of coding and non-coding genes that, as an ensemble, are required for the terminally differentiated neuronal phenotype (Bruce et al., 2004; Conaco et al., 2006; Otto et al., 2007; Mortazavi et al., 2006). In situ hybridization analysis shows a striking reciprocal pattern of REST expression to genes expressed within the developing nervous system, consistent with its role as a repressor (Schoenherr and Anderson, 1995; Chong et al., 1995). In differentiating cells in culture, REST is down-regulated during the transition to a mature neuron (Ballas et al., 2005) and overexpression of REST by in utero electroporation leads to delay in neuronal maturation (Mandel et al., 2011). These results suggest that REST serves as a timer of terminal neuronal differentiation. Counter to this model, in vivo loss-of-function analyses in mice have not shown evidence for precocious differentiation or de-repression of genes responsible for the mature neuronal phenotype in the embryonic nervous system. A global germline knockout (KO) of REST is early embryonic lethal, but no obvious morphological defects in neural tube formation were noted in that study (Chen et al., 1998). Later, in embryogenesis, brain-specific loss of REST, by conventional Cre lox technology, also lacks an obvious nervous system phenotype (Aoki et al., 2012), while conditional loss of REST from adult neural progenitors shows only transient and subtle precocious neuronal differentiation (Gao et al., 2011). Despite these results, a recent study points to a role for REST in human neurogenesis and microcephaly through regulation of REST by a factor, ZNF335, mutated in patients with a severe form of microcephaly (Yang et al., 2012). Additionally, microcephaly results from dysregulation of a REST/BAF170/Pax6 repressor complex during neurogenesis (Tuoc et al., 2013). Thus, the role of REST in embryonic neurogenesis remains an open question. To re-examine the role of REST during embryonic neurogenesis, we use mice containing a conditional gene trap (GT) cassette in an intron of the endogenous Rest gene that terminates transcription upstream from the initiator codon. Using this line, we generate mice with a REST deficiency in nestin-positive neural progenitors, prior to the time when REST is dismissed normally from chromatin during neurogenesis. We examined these mice for embryonic and adult brain phenotypes and found DNA damage, apoptosis, and smaller brain size as prominent defects. The DNA damage persisted and caused glioblastoma (GBM) in mice also lacking the tumor suppressor, p53. We also characterized REST binding properties and embryonic phenotypes in a conventional brain-specific Rest KO line (Gao et al., 2011), targeting Rest exon 2, which we show still expresses a C-terminal REST peptide, for comparison with our Rest GT mice. Our results indicate that REST is required to protect genomic integrity, supervised by S phase surveillance, and that this function is key for regulating proper timing of terminal neuronal differentiation. Results Global Rest loss using a GT approach We exploited a mouse line carrying a GT in the Rest intron (RestGT) between non-coding exon 1a–c and the first coding exon, exon 2 (Figure 1A). The GT cassette contains a splice acceptor site upstream of a promoter-less β-galactosidase and neomycin gene fusion (β-geo) and a polyadenylation sequence (Schnutgen et al., 2005). Thus, β-geo expression in this line is under the control of endogenous Rest regulatory elements. The GT was confirmed in the Rest locus by Southern blot analysis (Figure 1B) and we verified the insertion of a single GT in the genome by additional Southern blot and DNA sequence analysis (data not shown). RestGT/GT mice, like Rest KO mice generated by germ-line deletions of Rest exons 2 and 4 (Chen et al., 1998; Aoki et al., 2012), are growth-arrested (Figure 1C) and die between E9.5 and 11.5, validating the RestGTallele as a model for loss-of-function. Figure 1 Download asset Open asset A GT in the Rest gene (RestGT) causes REST deficiency and embryonic lethality. (A) Schematic showing the GT in the first Rest intron. Red and green boxes indicate alternative 5’ untranslated exons (1a–c) and first coding exon (2), respectively. The GT cassette contains an SA site, a reporter gene encoding a β-galactosidase neomycin fusion gene (β-geo), and a pA sequence. Arrowheads depict target sites for Flpe and Cre recombinases. Dashed line indicates probe location for Southern blot in B. (B) Southern blot of genomic DNA from indicated genotypes. (C) E10.5 wild type (Rest+/+) and mutant (RestGT/GT) embryos. (D) Western blot analysis of REST protein in E10.5 embryos. Ecad (E-cadherin), loading control. (E) qRT-PCR analyses of Rest mRNA levels, normalized to 18S RNA, in E9.5 embryos, n=6 mice/genotype. Means and SD are shown. (F) qRT-PCR analysis, normalized to 18S RNA, for Rest targets, n=3 mice/genotype. Means and SD are shown. Syp and Snap25 values in Rest+/+ are 4.3×10-4 ± 2.0×10−4 and 4.1×10−4 ± 2.0×10−4, correspondingly. Statistical significance was determined by ANOVA with Tukey posthoc (E) and by unpaired t-test with Welch correction. (F) (G) Whole mount X-gal staining of E11.5 embryo. (H) Left, in situ hybridization analysis for Rest transcripts in E12.5 embryo. Arrowhead indicates region magnified in adjacent image. Counterstain (pink) is nuclear fast red. Right panel, Immuno-labeling showing location of TuJ1+ neurons used to determine PP and VZ boundaries. (I) Immuno-labeling of cortical section from E13.5 embryo using indicated antibodies and DAPI stain for nuclei. *, p<0.05, **, p<0.01, ***, p<0.001. ANOVA, analysis of variance; GT, gene trap; mRNA, messenger RNA; PP, preplate; qRT-PCR, quantitative real-time polymerase chain reaction; SA splice acceptor; SD, standard deviations; VZ, ventricular zone. https://doi.org/10.7554/eLife.09584.003 In RestGT/GT mice, we also observed loss of both REST protein (Figure 1D) and messenger RNA (mRNA; Figure 1E) (Rest+/+, 0.95, standard deviation [SD], 0.27; RestGT/+, 0.47, SD 0.18; RestGT/GT, 0.7×10−2, SD 0.003). This was accompanied by the predicted reciprocal up-regulation of select REST target genes (Figure 1F). The β-gal activity programmed from the GT correlated tightly with the pattern of endogenous Rest mRNA. For example, β-gal activity (Figure 1G) and endogenous Rest expression (Figure 1H, left panel) were both detected in non-neural tissues outside the developing nervous system. In the embryonic brain, endogenous Rest mRNA was confined largely to neural progenitors in the ventricular zone (VZ) and mostly absent from preplate cells that were populated with TuJ1+ neurons (Figure 1H). Correspondingly, REST protein expression was confined predominantly to SOX2+ neural progenitors (Figure 1I). We were not able to detect REST protein in the subventricular zone (SVZ) occupied by the more committed TBR2+ basal progenitors (not shown), indicating that the down-regulation of REST occurred most robustly prior to the generation of mature neurons and the transition to basal progenitors. Conditional REST gene deficiency in neural progenitors Early embryonic lethality, coincident with the onset of neurogenesis, precluded analysis of REST function in RestGT/GT neural progenitors. Therefore, we used a two-step breeding scheme to remove REST specifically from neural progenitors. In the first step, RestGT mice (Figure 1A) were crossed to mice expressing the Flpe recombinase transgene (Dymecki et al., 2000). This resulted in inversion of the GT cassette (GTinv) to restore normal splicing of Rest exons 1a–c to exon 2 (Figure 2A, top). In the second step, RestGTi/+ mice, heterozygous for the inverted allele, were bred to mice expressing a Nestin Cre recombinase transgene. This resulted in re-inversion of the GTinv cassette to create a mutant Rest allele in which exons 1a–c were spliced to the β-geo gene instead of exon 2 (Figure 2A, bottom), terminating transcription upstream of remaining Rest sequences. All genotypes were confirmed by DNA sequence analysis. Figure 2 Download asset Open asset A conditional RestGT allele results in REST-deficiency in Nestin+ progenitors. (A) KO strategy. Top, mice bearing an inverted GT cassette (RestGTinv), resulting in normal splicing, were generated by mating RestGT mice (Figure 1A) to mice containing the Flpe transgene. Bottom, conditional mutants, resulting in splicing of exon 1 to the GT cassette (Cre+, RestGTi), were generated by mating RestGTi mice (top) to mice bearing the Nestin Cre transgene. It is noteworthy that β-geo expression was still under the control of Rest regulatory elements. (B) Whole-mount X-gal staining of E11.5 RestGTi/+ (left) and Cre+, RestGTi/+ (right) embryos. (C) X-gal staining of a coronal section of E13.5 cortex from Cre+, RestGTi/+ mice. (D) Western blot of nuclear extracts from neuroepithelia of E13.5 mice. HDAC, histone deacetylase 2, loading control. (E) qRT-PCR analysis of Rest transcripts, normalized to 18S RNA, in E13.5 brain (n=6 mice/genotype) and NPCs grown as neurospheres for 5 days (n=3 mice/genotype). (F) Quantitative chromatin immunoprecipitation analysis of REST enrichment at RE1 sites in the glycine receptor (Glra; +275 bp from TSS) and Snap25 genes (+867 bp from TSS) (n=4 mice/genotype). Amplicons were designed within 100 bp of RE1 binding sites. Snap25 CDS and Myf5 transcriptional start site lack RE1 sites. Means and SD are shown in (E) and (F). Statistical significance was determined by Mann–Whitney test in E and by unpaired t-test with Welch correction in F. (G) Immuno-labeling of E13.5 telencephalon using indicated antibodies. Boxes indicate regions of higher magnification in images at right. White arrowheads indicate DAPI+ cells that express both proteins. *, p<0.05, **, p<0.01, ***, p<0.001. CDS, coding sequence; CP, cortical plate; GT, gene trap; KO, knockout; MZ, marginal zone; NPC, neural progenitor cells; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation; VZ, Ventricular zone. https://doi.org/10.7554/eLife.09584.004 We examined β-gal activity in the developing brains of Cre+, RestGTi /GTi and RestGTi/GTi (controls hereafter) mice. Consistent with the global GT, β-gal activity matched expression of the endogenous Rest gene. Specifically, β-gal+ cells in the Cre+, RestGTi/GTi mice were confined to neurogenic areas of E11.5 embryos (Figure 2B). Within the cortical VZ at E13.5, β-gal activity (Figure 2C) and endogenous Rest mRNA (data not shown) were detected primarily in the apical region, which is occupied by progenitors (Götz and Huttner, 2005). Interestingly, we also detected β-gal+ cells in some presumably mature cells in the marginal zone (MZ) of the cortical plate (CP) indicating Rest promoter activity in a subset of neurons (Figure 2C). Ninety-five percent of the Cre+, RestGTi /GTi mice survived into adulthood, but had significantly reduced REST protein and mRNA levels in neural progenitors and E13.5 brains compared with controls (Figure 2D and E). Chromatin immunoprecipitation (ChIP) analysis of E13.5 brains from Cre+, RestGTi /GTi mice indicated ~four-fold reduction in REST occupancy at consensus RE1 sites within 1kb of the Glycine receptor and Snap25 transcriptional start sites, in the first exon and intron, respectively (Glycine receptor: RestGTi /GTi, 0.64, SD 0.13; Cre+, RestGTi /GTi, 0.16, SD 0.07), Snap 25: RestGTi /GTi, 0.32, SD 0.04; Cre+, RestGTi /GT, 0.07, SD 0.04) (Figure 2F). There was no significant change in REST occupancy in the Snap25 coding sequence or the myf5 promoter region that lacked RE1 binding sites (Figure 2F). The very low levels of glycine receptor mRNA in control mice was not within the linear range of detection. Levels of this mRNA in the mutant, normalized to 18S RNA, were consistently within the linear range, but still very low (0.13, SD 0.08). For SNAP25, we observed an ~2-fold increase in mutant mRNA compared with control levels (RestGTi/GTi: 0.09, SD 0.06; Cre+, RestGTi/GTi: 0.2, SD 0.05; p <0.05, unpaired t-test, n=7–8, E13.5 brain). It is possible that the low mRNA levels for both genes reflect the absence of transcriptional activators at this time. In control mice, SOX2 progenitors of the VZ stained positively for REST, but the majority of TuJ1+ neurons were not immuno-positive (Figure 1I and 2G), consistent with the known down-regulation of REST during neurogenesis (Ballas et al., 2005). Interestingly, however, we could detect REST protein in a small number of TuJ1+ neurons in the outmost MZ of the CP (Figure 2G, right images), corroborating β-gal staining (Figure 2C), suggesting either that REST is re-expressed at later stages of differentiation or that REST expression has not yet been extinguished completely in these neurons. Whether REST is bound to the RE1 sequence in the TuJ1 gene at this stage cannot be determined given the small number of labeled neurons. Cre+, RestGTi /GTi mutants have smaller brains, thinner cerebral cortices, and reduced numbers of upper layer neurons Because a previous study indicated that loss of REST was associated with microcephaly (Yang et al., 2012), we asked whether this was also true for our RestGT mice. Indeed, significantly smaller brains were evident at birth (data not shown) and in postnatal (P45) Cre+, RestGTi /GTi mice when compared with control mice of two different control genotypes, RestGTi/GTi and Nestin Cre (Figure 3A and B left panel). The brain size in the mutant was reduced to 71% of the brain size of RestGTi/GTi mice (RestGTi/GTi, 0.46, 95% confidence interval [CI] 0.45–0.46; Cre+, RestGTi/GTi 0.33, CI 0.32–0.34), similar to the difference from Nestin Cre mice (Nestin Cre+, Rest+/+: 0.43, CI 0.42 to 0.44). A third potential control line is Cre+, RestGTi/+ mice. However, we measured a significant brain size reduction Cre+, RestGTi/+ mice (0.36, CI 0.35–0.37, p value <0.001) compared to RestGTi/GTi (reduction to 78%) or Cre+, Rest+/+(reduction to 83%) mice, likely due to the combination of the slightly reduced brain size of the Cre recombinase background and reduced REST levels (Figure 3B and 1E). The hypomorphic effect is consistent with previous studies, indicating that gene expression levels are sensitive to small changes in REST levels (Ballas et al., 2005; Ballas and Mandel, 2005), so the RestGTi/GTi mice were used as controls in the remaining experiments. Figure 3 Download asset Open asset Reduced brain size, thinner cortex, and reduced numbers of upper layer neurons in Nestin Cre+, RestGTi/GTi mice. (A) Representative P45 brains from control and Cre+, RestGTi/GTi littermates. (B) Comparison of brain mass in P45 mice deleted for REST using Nestin (left panel) and hGFAP (right panel) promoter driven Cre recombinases. The numbers of mice analyzed for each genotype are shown. Statistical significance determined by ANOVA with Tukey posthoc (left) and Kruskal–Wallis ANOVA test with Dunn posthoc (right). (C) Nissl-stained P45 coronal brain sections. Boxes denote higher magnification views in right hand panels. *, corpus collosum. Scale bar for right panel, 0.5mm. (D) Measurements of cortical thickness in P45 brain (n=8–11 mice/genotype). Statistical significance determined by ANOVA with Tukey posthoc. (E) and (F) Representative immuno-labeling of P1 cortical sections with indicated antibodies revealing cortical layering in control and Cre+, RestGTi/GTi mice. The presence of both low and high expressing CTIP2 cells in layers 6 and 5, respectively, is noteworthy. (G) Quantification of (E) and (F) numbers over brackets denote cortical layers where the cells were counted. Cells were counted in 400 μm of cortical thickness (n=8–10 mice/genotype). Note that only 200 μm images are shown in E, F. Low (layer 6) and high (layer cells were used to between cells of layer 5 and cells in layer cells were counted layer of cells. (H) cell from areas in each cortical mice/genotype. Statistical significance was determined by Mann–Whitney t-test for (G) and Means and 95% CI are shown in and *, **, ***, p<0.001. ANOVA, analysis of variance; We also inversion of the RestGTi allele using mice expressing an hGFAP Cre recombinase transgene at slightly later than Nestin Cre et al., In these mice, was a smaller reduction in brain mass to only of control levels (Figure 3B right suggesting a for REST function at neurogenic This in utero electroporation experiments revealing an phenotype of REST at E13.5 that is not observed at (Yang et al., et al., 2012). The reduction in brain mass in REST mutant mice correlated with reduced cortical both and and reduced corpus thickness (Figure and To determine whether these phenotypes were associated with an of progenitor we counted neurons in the cortical layers that are at different during with layer neurons the birth of upper layer neurons. To this we for transcription factor for adjacent layers et al., et al., et al., 2005; et al., There was a reduction to of control numbers of upper layer neurons (RestGTi/GTi, CI – Cre+, RestGTi/GTi: CI but no significant between mutant and control in layer 5 and 6 neurons that were earlier in neurogenesis (Figure and However, CTIP2 which layer 5, into layer 6 and in mutant brain (Figure and to some due to the loss of REST. Despite this the of loss of REST during neurogenesis was significantly fewer postnatal neurons in the upper cortical layers during neurogenesis. loss of REST from neural progenitors leads to cell The small brain size and numbers of neurons at birth could reflect of progenitor cells cell To the we first apical and basal progenitors by immuno-labeling with antibodies to and respectively (Figure of apical progenitors in RestGTi/GTi mice were reduced to of control values (RestGTi/GTi, CI Cre+, RestGTi/GTi: CI Figure There were also reduced numbers of TBR2+ basal progenitors, which are of the apical progenitors, at E13.5 /GTi, CI Cre+, RestGTi/GTi: CI (Figure Figure 4 Download asset Open asset of apical progenitors and cell in Cre+, RestGTi/GTi mice. (A) Representative immuno-labeling apical and basal progenitors in E13.5 by is shown in images at right. (B) Quantification of Measurements from 100 μm cortical from the VZ to mice Statistical significance was determined by Mann–Whitney Red show of the images shown in (C) Representative immuno-labeling for and in the cortical section of mouse brain with for The by is magnified in the adjacent (D) Top, showing percent stained cells to the number of DAPI+ nuclei. Bottom, of cells the cell as of cells in cell Measurements were made in 200 μm cortical areas from ventricular to mice/genotype. Statistical significance was determined by Mann–Whitney Means and 95% CI are shown in B and *, **, zone; subventricular zone. To determine whether the of apical progenitors correlated with cell we E13.5 embryos with to cells that were DNA we for a of and and the results to the number of DAPI+ nuclei. The staining in Cre+, RestGTi/GTi cells was reduced to of the control values (RestGTi/GTi, CI Cre+, CI with no change in the of cells (RestGTi/GTi, CI Cre+, CI (Figure and panel). To determine the of cells that the cell in a we the of cells in the cell Our results indicated that between E13.5 and the progenitor were as more progenitors the cell in Cre+, RestGTi/GTi compared with controls (RestGTi/GTi, CI Cre+, CI (Figure panel). This indicates a progenitor to generate the upper layer neurons (Figure and neural progenitors cell to neuronal differentiation analysis of E12.5 brain from Cre+, RestGTi/GTi and control mice not show significant up-regulation in REST neuronal target genes However, our analysis significant up-regulation of genes et al., and genes and and in brain and progenitors 1 and Figure To test for cell we stained E13.5 brain sections from the cortex and with the form of a of the which is a of and required for chromatin and DNA 1998). in control cortices, was prominent in cells in Cre+, RestGTi/GTi mice, at the between the and CP (Figure Interestingly, cells in the are populated with TBR2+ basal progenitors, the cells were not for TBR2+ (Figure 1A). Figure 5 with 3 all Download asset Open asset and neurons in cortex of Cre+, RestGTi/GTi mice that is by of p53. (A) qRT-PCR analyses normalized to 18S RNA, of p53 target and progenitor and neuronal in progenitors from E13.5 mice, Cre+, n=6 mice. (B) Representative immuno-labeling for cells with and neuronal TuJ1 in coronal sections from E13.5 mice. (C) of in cortex and of Cre+, RestGTi/GTi mice and cortex of RestGTi/GTi mice, in areas of 100 μm ventricular from VZ to the Means and are shown. (D) of and SOX2+ cells in all cells in E13.5 of Cre+, RestGTi/GTi mice in ventricular mice. (E) Quantification of in E13.5 cortex and The data for Cre+, RestGTi/GTi cortex and from is for (F) qRT-PCR analysis of mRNA levels, to 18S RNA, of p53 and p53 in E12.5 brain mice/genotype). (G) Measurements of brain mass in P45 mice. of mice are indicated in the the data from Cre+, Figure 3B is for Means and 95% CI are shown in E, Statistical significance was determined by Mann–Whitney t-test ANOVA test with Tukey posthoc and and Kruskal–Wallis ANOVA test with Dunn posthoc (F). *, p<0.05, **, p<0.01, ***, ANOVA, analysis of variance; confidence mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation at in the cortex, the time of for this brain region and the of upper layer neuronal et al., and to control levels by (Figure The of was earlier in at E13.5 (Figure The number of cells in RestGTi/GTi was similar to the low numbers in the cortex (not shown). The of for the cortex and coincident with time of differentiation and et al., and indicate that was to the timing of cell of progenitor in these brain and not to a effect on all progenitors or effects