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• W1861340857 abstract "Article29 October 2015free access NeuroD1 reprograms chromatin and transcription factor landscapes to induce the neuronal program Abhijeet Pataskar Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Johannes Jung Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Pawel Smialowski Adolf Butenandt Institute and Center for Integrated Protein Science, Ludwig Maximilian University, Munich, Germany Search for more papers by this author Florian Noack DFG-Research Center for Regenerative Therapies, Cluster of Excellence, TU-Dresden, Dresden, Germany Search for more papers by this author Federico Calegari DFG-Research Center for Regenerative Therapies, Cluster of Excellence, TU-Dresden, Dresden, Germany Search for more papers by this author Tobias Straub Adolf Butenandt Institute and Center for Integrated Protein Science, Ludwig Maximilian University, Munich, Germany Search for more papers by this author Vijay K Tiwari Corresponding Author Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Abhijeet Pataskar Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Johannes Jung Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Pawel Smialowski Adolf Butenandt Institute and Center for Integrated Protein Science, Ludwig Maximilian University, Munich, Germany Search for more papers by this author Florian Noack DFG-Research Center for Regenerative Therapies, Cluster of Excellence, TU-Dresden, Dresden, Germany Search for more papers by this author Federico Calegari DFG-Research Center for Regenerative Therapies, Cluster of Excellence, TU-Dresden, Dresden, Germany Search for more papers by this author Tobias Straub Adolf Butenandt Institute and Center for Integrated Protein Science, Ludwig Maximilian University, Munich, Germany Search for more papers by this author Vijay K Tiwari Corresponding Author Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Author Information Abhijeet Pataskar1,‡, Johannes Jung1,‡, Pawel Smialowski2, Florian Noack3, Federico Calegari3, Tobias Straub2 and Vijay K Tiwari 1 1Institute of Molecular Biology (IMB), Mainz, Germany 2Adolf Butenandt Institute and Center for Integrated Protein Science, Ludwig Maximilian University, Munich, Germany 3DFG-Research Center for Regenerative Therapies, Cluster of Excellence, TU-Dresden, Dresden, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 6131 39 21460; E-mail: [email protected] EMBO J (2016)35:24-45https://doi.org/10.15252/embj.201591206 See also: A Glahs & RP Zinzen (January 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cell fate specification relies on the action of critical transcription factors that become available at distinct stages of embryonic development. One such factor is NeuroD1, which is essential for eliciting the neuronal development program and possesses the ability to reprogram other cell types into neurons. Given this capacity, it is important to understand its targets and the mechanism underlying neuronal specification. Here, we show that NeuroD1 directly binds regulatory elements of neuronal genes that are developmentally silenced by epigenetic mechanisms. This targeting is sufficient to initiate events that confer transcriptional competence, including reprogramming of transcription factor landscape, conversion of heterochromatin to euchromatin, and increased chromatin accessibility, indicating potential pioneer factor ability of NeuroD1. The transcriptional induction of neuronal fate genes is maintained via epigenetic memory despite a transient NeuroD1 induction during neurogenesis. NeuroD1 also induces genes involved in the epithelial-to-mesenchymal transition, thereby promoting neuronal migration. Our study not only reveals the NeuroD1-dependent gene regulatory program driving neurogenesis but also increases our understanding of how cell fate specification during development involves a concerted action of transcription factors and epigenetic mechanisms. Synopsis Using a genome-wide approach this study delineates the binding requirements and target sites for NeuroD1, illustrating how this factor drives the formation of a transcriptional and epigenetic program to ensure neuronal differentiation. Genome-wide assessment of NeuroD1 binding reveals its targeting to regulatory elements of critical neuronal development genes. Following expression, NeuroD1 locates to and binds its target genomic locations in a highly sequence-specific fashion. Prior to NeuroD1 binding, its target sites are developmentally silenced by epigenetic mechanisms and regulatory factors. NeuroD1 binding initiates events that confer transcriptional competence to neuronal fate genes including conversion of heterochromatin to euchromatin and reprogramming of the transcription factor landscape. The transient action of NeuroD1 during neuronal development is sufficient to induce a neuronal gene expression program that is maintained by epigenetic mechanisms. Introduction Embryonic development in mammals involves function of a plethora of transcription factors that act at various levels to generate a spatiotemporally regulated gene expression program (Guillemot, 2007; Spitz & Furlong, 2012). Basic helix-loop-helix (bHLH) transcription factors are among such key players, and they contribute to the lineage commitment and terminal differentiation of various cell types during development (Jones, 2004). In mice, the first neurons of the central nervous system (CNS) are born at mid-gestation, between embryonic day 10 (E10) and E11, heralding an important transition in the development of neural progenitor cells in the brain. Radial glial (RG) stem cells persist as the principal progenitor type during development of the embryonic and postnatal CNS (Kriegstein & Alvarez-Buylla, 2009; Taverna et al, 2014). These neural stem cells located in the ventricular zone (VZ) undergo asymmetric division generating basal progenitors and neurons that migrate toward the subventricular zone (SVZ) and cortical plate (CP), respectively (Taverna et al, 2014). This process involves a number of key factors, including neurogenin family members (Neurog 1/2), which initiate a cascade of other critical proneural transcription factors, one of which is NeuroD1 (Ma et al, 1996; Sommer et al, 1996). NeuroD1 is a bHLH transcription factor that plays an important role during neuronal differentiation (Hevner et al, 2006; Aprea et al, 2014). Given its prominent function during embryonic neurogenesis, it has also recently been used to reprogram other somatic cell types into neurons. In one such study, a combination of Pou3f2, Ascl1, and Myt1l, together with NeuroD1, was successfully used to reprogram fetal and postnatal fibroblasts into neurons (Vierbuchen et al, 2010). Furthermore, NeuroD1 alone was able to convert reactive glial cells into functional neurons in vivo as well as it could convert human astrocytes into glutamatergic neurons (Guo et al, 2014). These findings imply that NeuroD1 is a highly potent factor that promotes neuronal fate. However, no comprehensive investigation has been performed to uncover the gene regulatory program through which NeuroD1 mediates neuronal fate specification during development and reprogramming. Furthermore, NeuroD1's direct genomewide targets during neurogenesis remain unknown. In addition, despite existing knowledge that cell fate specification involves reprogramming of the epigenome (Mohn et al, 2008; Magnusdottir et al, 2012; Wamstad et al, 2012; Xie et al, 2013), very little is known regarding whether NeuroD1's function at its target sites involves chromatin remodeling. Finally, it is unknown whether the transient action of such factors during differentiation is able to induce long-term epigenetic memory. Here, we show that ectopic expression of NeuroD1 is sufficient to induce a program that closely recapitulates neuronal development in vivo. Genomewide analysis revealed that NeuroD1 activates neuronal development genes by directly binding to their regulatory elements. We found that NeuroD1 is able to bind its target heterochromatic promoters, which is followed by the loss of the Polycomb group (PcG)-associated repressive mark H3K27me3 and replacement of repressor proteins such as TBX3. This is accompanied by the gain of the active mark H3K27ac, as well as increased chromatin accessibility, resulting in induced gene expression. On the other hand, NeuroD1 target enhancers are repressed by MBD3 occupancy and histone deacetylation in its absence. NeuroD1 binding displaces MBD3 from these sites and results in increased H3K27ac levels, leading to gene activation. These observations further suggest that NeuroD1 targeting to distal sites is both necessary and sufficient to trigger events that activate enhancers. Interestingly, in addition to key neuronal development genes, NeuroD1 also induces genes involved in the epithelial-to-mesenchymal transition. We further show that a transient action of NeuroD1 during development is sufficient to induce a neuronal gene expression program that is stably maintained by epigenetic memory. Taken together, our comprehensive findings uncovered the gene regulatory program through which NeuroD1 specifies the neuronal fate and revealed how this function involves reprogramming of the transcription factor and chromatin landscapes at its target sites. Results Ectopic expression of NeuroD1 is sufficient to induce the neuronal development program A number of previous studies have shown that NeuroD1 is induced following the onset of neurogenesis (Lee et al, 1995, 2000; Schwab et al, 1998). We first investigated whether high NeuroD1 expression is a specific feature of neurogenic progenitors and newborn neurons. Using RNA-seq datasets (Table EV1) (Fietz et al, 2012; Shen et al, 2012; Yue et al, 2014) from embryonic tissues that are representative of all three lineages, as well as from various layers of embryonic cortex, we found that NeuroD1 was highly expressed in the ventricular zone (VZ), was upregulated several fold in the subventricular zone (SVZ), and then downregulated in the cortical plate (CP; Fig 1A). Except in the pancreas, where it was transcribed at low levels, NeuroD1 was not expressed in any other investigated tissue. To determine whether similar NeuroD1 expression dynamics could be observed during the neuronal differentiation of embryonic stem cells, we adapted a highly refined system that generates over 95% pure neuronal progenitor (NP) cells (“radial glial-like” cells) from embryonic stem (ES) cells that subsequently become terminally differentiated pyramidal neurons (TN) (Bibel et al, 2004, 2007). Previous studies, including our own, have demonstrated highly synchronous and reproducible changes in the epigenome and transcriptome during neuronal differentiation in this system (Mohn et al, 2008; Lienert et al, 2011; Stadler et al, 2011; Tiwari et al, 2012a,b; Thakurela et al, 2013). Moreover, in terms of the epigenome and the transcriptome, both the behavior of individual genes and genomewide analyses in mouse primary cortical neurons were in good agreement with this in vitro system, making it a highly appropriate model (Mohn et al, 2008; Tiwari et al, 2012a; Thakurela et al, 2013). We performed expression analysis of NeuroD1 in this system using qPCR which revealed that its expression was peaking immediately following the onset of neurogenesis (Fig 1B). Figure 1. Ectopic expression of NeuroD1 is sufficient to initiate a neurogenic program that mimics neuronal development in vivo A. RNA-seq datasets from in vivo embryonic samples were analyzed for NeuroD1 expression. RNA represents the normalized tag counts from biological replicates. B. RT–qPCR results for NeuroD1 expression in an in vitro neuronal differentiation time course using biological replicates. RNA reflects the relative gene expression normalized to a housekeeping gene (Rpl19). C. Immunostaining of ES cells for TUJ1 (neuronal marker) and Hoechst (nucleus) after 48 h of NeuroD1 induction (+Dox) as well as for non-induced control cells (−Dox). Scale bar, 10 μm. D, E. RT–qPCR results for the expression of neuronal markers (D) and hallmark pluripotency genes (E) after 48 h of NeuroD1 induction in ES cells. RNA fold change reflects the relative gene expression normalized to a housekeeping gene (Rpl19) plotted as the fold change of induced (+Dox) versus non-induced (−Dox) condition. The y-axis in (D) is plotted in a log10 scale. F. Volcano plot [x-axis: log2 (fold change +Dox versus −Dox), y-axis: −log10 (P-value)] depicting differential gene expression after 48 h of NeuroD1 induction in ES cells. Data points marked in green represent genes crossing our significance cutoff for differential expression P-value < 0.05 (blue dotted line) as well as abs (log2 (fold change)) ≥ 0.58 (green dotted line). G. Top GO terms that are enriched in the high-confidence upregulated genes (URG) in (F). The bar length is determined by the enrichment score calculated by GSEA. H, I. Line plot showing NeuroD1 expression as fold change over ES during in vitro neurogenesis (H, upper panel). Heat map depicting the expression of URG from (F) in an in vitro neuronal RNA-seq time course using biological triplicates (H, lower panel). Each row represents one promoter where gene expression is scaled from red (high expression) to blue (low expression). The same information is presented in (I) as a boxplot. J, K. Line plot showing NeuroD1 expression in embryonic tissues from (A) (J, upper panel). Heat map depicting the expression of URG in embryonic tissues from (A) (J, lower panel). The same information is presented in (K) as a boxplot. Data information: Error bars reflect standard error of the mean from three biological replicates if not stated otherwise. Significance was determined by t-test with *P < 0.05, **P < 0.01, ***P < 0.001. Boxplots depicting RNA-seq data contain expression values scaled between 0 and 1 on y-axis. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate; Ecto, ectoderm; Endo, endoderm; Meso, mesoderm; ES, embryonic stem cells; CA, cellular aggregates; TN, terminally differentiated neurons; DRG, downregulated genes; and URG, upregulated genes. GEO IDs for all sequencing data used are provided in Table EV1. Download figure Download PowerPoint To uncover the gene regulatory circuitry through which NeuroD1 functions to promote the neuronal fate, we ectopically induced expression of NeuroD1 in pluripotent mouse ES cells using a previously described system (Appendix Fig S1A and B) (Iacovino et al, 2011). Strikingly, within 48 h of NeuroD1 induction, many cells exhibited a neuron-like morphology and expressed the neuron-specific protein TUJ1 as revealed by immunofluorescence analysis (Fig 1C). These observations were further supported by quantitative expression analysis of several neuronal markers, all of which significantly increased in expression compared with non-induced cells (Fig 1D). This induction of neuronal markers was accompanied by a downregulation of the pluripotency genes Oct4, Nanog, and Klf4, but not Sox2 (Fig 1E). Based on these observations, we next attempted to characterize the global gene expression changes induced by NeuroD1 in ES cells after 48 h by performing RNA-sequencing on NeuroD1-induced (+Dox) and non-induced (−Dox) cells. After applying stringent criteria for the significance and fold-change (FC) cutoff values (FDR ≤ 0.05, FC ≥ 1.5), a total of 2,209 upregulated genes (referred to as “URG” from here onwards) and 1,699 downregulated genes (referred to as “DRG” from here onwards) were identified (Fig 1F, Table EV2). Interestingly, GO term analysis of the URG showed enrichment exclusively for neurogenesis-related ontologies (Fig 1G). KEGG pathway analysis of these genes revealed enrichment for neurogenic pathways (Appendix Fig S1C). Moreover, the highest upregulated genes were the most significant contributors to the neurogenesis-related GO terms (Appendix Fig S1D). Interestingly, DRG were also downregulated during in vitro neurogenesis (Appendix Fig S1E and F) and were almost exclusively expressed in non-neuronal lineages (Appendix Fig S1G and H). Furthermore, these genes were most enriched for non-neuronal GO terms such as metabolic processes and cell adhesion (Appendix Fig S1I). Importantly, NeuroD1 induced the neuronal program despite the presence of pluripotency signals (LIF) and the absence of neuron-promoting culture media, suggesting that NeuroD1 function is sufficient to override the pluripotent state and promote neuronal commitment. Given the prominent enrichment of neuronal genes among the NeuroD1-induced transcripts, we questioned whether these genes are also normally induced during neuronal differentiation. Transcriptomic analysis showed that the majority of these genes were upregulated during the transition from neuronal progenitors to neurons both in vitro and in vivo (Fig 1H–K). Furthermore, most URG were considerably more expressed in cortical layers than in tissues of other lineages (Fig 1J and K). Interestingly, the expression of a large number of these genes remained high in terminally differentiated neurons, suggesting that NeuroD1-induced transcriptional state persists following the brief period of NeuroD1 peaked expression and action during neuronal development (Fig 1H–K). We conclude that the ectopic expression of NeuroD1 is sufficient to induce a neuronal differentiation program that closely recapitulates neuronal development in vivo. NeuroD1 directly targets regulatory elements of critical neuronal genes to induce their expression Prompted by our findings, we next investigated whether this function could be directly linked to NeuroD1's DNA binding ability. To identify its genomewide targets, we ectopically expressed NeuroD1 in ES cells for 24 h and then performed a chromatin immunoprecipitation (ChIP) assay for NeuroD1 in combination with next-generation sequencing (ChIP-seq). Visual inspection of the genomic regions in these data suggested that NeuroD1 targeted distinct genomic sites including promoters and intergenic regions (Fig 2A and B). A comprehensive analysis revealed that NeuroD1 binding occurred at both promoter and non-promoter regions (Fig 2C). However, when normalized for the small size promoters constitute in the entire genome, NeuroD1 peaks showed a preferential occurrence at promoters (around 39%, n = 341) (Fig 2C). Given the regulatory roles of promoters and enhancers, we next focused on thoroughly analyzing NeuroD1 occupancy at these target sites. Acetylation of lysine 27 at histone H3 (H3K27ac) is a hallmark of active enhancers (Creyghton et al, 2010; Rada-Iglesias et al, 2011; Bonn et al, 2012; Zhu et al, 2013; Shlyueva et al, 2014). Therefore, to determine enhancers that are bound by NeuroD1, we obtained H3K27ac ChIP-seq data from an early stage of neurogenesis in vitro (TN d1), a time point immediate to the highest NeuroD1 expression, and identified NeuroD1-bound non-promoter sites enriched for H3K27ac modification. A comparison of genes associated with these non-promoter sites as well as NeuroD1-bound promoters with URG revealed that a significant number of genes induced upon NeuroD1 expression were directly bound by NeuroD1 at their regulatory elements (~25%) (Appendix Fig S2A). We further classified the non-promoter NeuroD1 targets into exonic, intronic, and intergenic enhancers (Appendix Fig S2B). We found that NeuroD1-bound intronic and exonic enhancers were associated with URG involved in neurogenesis and showed induced expression upon neuronal differentiation in vitro and in vivo (Appendix Fig S2C–L). To avoid influence of genic chromatin landscape and other transcriptional regulatory events such as elongation that occur in gene bodies, we decided to focus on intergenic enhancers for further functional analysis (referred to as “enhancers” from here onwards). Figure 2. NeuroD1 directly binds the regulatory elements of critical neuronal developmental genes A, B. UCSC genome browser screenshots showing enrichment of NeuroD1 at representative target promoters (A) or target enhancers (B) in ES cells after 24 h of induction. The merged wiggle files were generated from two biological replicates. The genes are displayed as arrows representing the direction of transcription. The baseline on y-axis represents “0” values. C. Pie chart depicting the distribution of NeuroD1 peaks (n = 2,409) reproducibly called in two biological replicates for particular genomic classes (promoters, intergenic regions, exons, and introns), normalized by genome size. The absolute number of peaks is shown in the inset. After genome size normalization, ˜39% (absolute number = 341) of total peaks are called at promoters, ˜18% (absolute number = 1,107) of peaks at intergenic, ˜28% (absolute number = 930) at intronic, and ˜16% at exonic (absolute number = 31) regions. D. Venn diagram showing the overlap of the URG (n = 2,209) and NeuroD1-bound promoters (E > 0.75, n = 478) and the genes (n = 330) associated with NeuroD1-bound enhancers (Top 500 enriched sites). Altogether, 195 URG are bound by NeuroD1 at their regulatory elements (upregulated targets, URT). E, F. Heat map (E) showing the RNA-seq expression of URT in an in vitro neuronal differentiation time course. The same information is presented in (F) as a boxplot. G, H. Heat map (G) showing the RNA-seq expression of URT in various embryonic tissues. The same information is presented in (H) as a boxplot. I, J. Bar plots showing top GO terms for URT regulated at promoters (I) and enhancers (J). The bar length is based on the enrichment score determined by GSEA. K. ChIP-qPCR results for NeuroD1 binding to selected target (black) and control (gray) promoters in ES cells after 24 h of induction. L. ChIP-qPCR results for NeuroD1 enrichment at target enhancers (black) and additional intergenic control (gray) in ES cells after 24 h of induction. M. ChIP-qPCR results for NeuroD1 enrichment at target promoters and enhancers (black) as well as control regions (gray) in early neurons derived in vitro (TN d1). N, O. Tables depicting the top three enriched motifs at NeuroD1-bound promoters (N) and enhancers (O). Data information: Error bars reflect standard error of the mean from three biological replicates if not stated otherwise. Significance shown in (D) was determined by Fischer's test with *P < 0.05, **P < 0.01, ***P < 0.001. Boxplots depicting RNA-seq data contain expression values scaled between 0 and 1 on y-axis. The y-axis of ChIP-qPCR results shows the relative ChIP enrichment plotted as the ratio of precipitated DNA (bound) to input DNA and further normalized to an intergenic control region (fold enrichment above background) from two biological replicates. URT, upregulated targets. GEO IDs for all sequencing data used are given in Table EV1. Download figure Download PowerPoint Comparing the overlap of NeuroD1-bound targets with URG, we found that a significant fraction of genes upregulated upon NeuroD1 induction were directly bound by NeuroD1 at their promoters (n = 83), enhancers (n = 107), or both (n = 5) (referred to as “URT” (upregulated targets) from here onwards) (Fig 2D). These URT (n = 195) were significantly higher upregulated as compared to the overall transcriptional induction of URG following NeuroD1 expression (Appendix Fig S3A). Additionally, a significant fraction of our NeuroD1 target enhancers overlapped with previously described E14.5 brain- or cortex-specific enhancers (Appendix Fig S3B) (Shen et al, 2012). Importantly, the majority of URT were upregulated during neurogenesis in vitro (Fig 2E and F) and in vivo (Fig 2G and H) and they were almost exclusively expressed in cortical layers but not in tissues of other lineages (Fig 2G and H). These observations were further supported by the enrichment of neurogenesis- and development-related GO terms among URT (Fig 2I and J, and Appendix Fig S3C and D). We validated a number of these promoter and enhancer target sites using ChIP-qPCRs (Fig 2K and L, data not shown). Importantly, although these targets were discovered after ectopic expression of NeuroD1 in ES cells, they were also bound by NeuroD1 in early neurons (Fig 2M). To investigate any sequence specificity in NeuroD1 targeting, we performed a motif enrichment analysis of genomic sequences underlying NeuroD1 peaks. Interestingly, the NeuroD1 motif was among the top three enriched motifs at the target promoters (Fig 2N and Appendix Fig S3E) and enhancer elements (Fig 2O and Appendix Fig S3F), suggesting a sequence-dependent targeting of NeuroD1. A de novo motif prediction further revealed that most NeuroD1 peaks (approximately 95%) exhibit an E-box motif directly at the peak summit, which is known to be associated with classical bHLH protein family members (Jones, 2004) (Appendix Fig S3G). To extend our findings to a differentiated cell type, we ectopically expressed NeuroD1 in murine fibroblasts and analyzed its binding at identified target sites by ChIP assay and influence on the expression of their associated genes. Interestingly, NeuroD1 was able to locate and bind its target sequences and induce expression of associated genes (Appendix Fig S4A–G). This was accompanied by an upregulation of the neuronal marker Tubb3 (Appendix Fig S4H), but not of the housekeeper gene Tbp (Appendix Fig S4I). These observations indicate that, irrespective of the cell type, NeuroD1 is able to trigger activation of neuronal development genes by directly binding to their regulatory elements. NeuroD1 induces the expression of transcription factors involved in neuronal development and migration Because NeuroD1 expression induces a large number of neuronal genes, many of which are not direct NeuroD1 targets (Fig 2D), we speculated that NeuroD1 activates the expression of additional transcriptional regulators that could then mediate the observed secondary gene expression responses. Indeed, a deeper analysis of URT revealed a number of transcription factors and epigenetic regulators (promoter URT: n = 27/88; enhancer URT: n = 30/112). Interestingly, these factors were induced during neurogenesis in vitro as well as in vivo and were largely repressed in non-neuronal lineages (Fig 3A–D). Importantly, although this list contained established regulators of neurogenesis (e.g. Hes6, Pou3f2, Sox11), it also harbored a number of factors not previously implicated in neurogenesis (e.g. Zfand5, Rnf182, or Aff3). Analysis of in situ hybridization (ISH) images as well as RNA-seq datasets from the developing murine cortex validated the expression pattern of many identified NeuroD1 target genes (Fig 3E–J). We also observed, using example of one such target gene Lzts1, that the expression pattern of NeuroD1 target genes during cortical development may closely mimic NeuroD1 (Fig 3K). Figure 3. NeuroD1 induces expression of transcription factors that are involved in neurogenesis and the epithelial-to-mesenchymal transition A–D. Heat map depicting the expression (RNA-seq) of promoter URT transcription factors and epigenetic regulators (A, B) or URT regulated at enhancers (C, D) during an in vitro neuronal differentiation time course (A, C) and in various embryonic tissues (B, D). E–J. In situ hybridization images of an antisense probe from the Allen Brain Atlas showing the expression of NeuroD1 (E) and representative targets [Nhlh1 (F), Nhlh2 (G), Lzts1 (H), Apc2 (I), and Pcsk2 (J)] in E15.5 cortex (upper panel). Bar plots depicting normalized tag counts from RNA-seq expression analysis for the corresponding genes in E14.5 cortical layers (lower panel). K. In situ hybridization of an antisense probe from the Allen Brain Atlas showing the expression of NeuroD1 and Lzts1 during several stages of cortical development. L. Venn diagram showing the overlap between genes that are induced during murine EMT (FC > twofold at any stage, n = 7,779) and URG (n = 2,209, overlap with EMT induced genes: ˜40%). Examples of hallmark EMT regulators are shown as inset. M. Venn diagram showing the significant overlap (˜10% of URG induced in EMT) between URG that are induced during murine EMT (n = 898) and promoter (n = 88) and enhancer URT (n = 112). Example genes are shown as inset. Data information: Error bars reflect standard error of the mean from two biological replicates. Significance was determined by Fischer's test with *P < 0.05, **P < 0.01, ***P < 0.001. GEO IDs for all sequencing data used are provided in Table EV1. Download figure Download PowerPoint NeuroD1 has also been implicated in neuronal migration (Kim, 2013), but the underlying molecular mechanism is largely unknown. Therefore, we next questioned whether classical migration genes involved in epithelial–mesenchymal transition (EMT) are induced following NeuroD1 expression. To address this question, we compared the NeuroD1-induced genes with genes that we recently identified as upregulated during EMT (Sahu et al, 2015). Interestingly, this analysis showed that URG encompass a large number of genes that are upregulated during EMT (n = 878, ~40%) and include a number of hallmark genes that are known to promote EMT (Fig 3L). Furthermore, a number of these genes exhibit direct binding of NeuroD1 at their regula" @default.
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• W1861340857 title "NeuroD1 reprograms chromatin and transcription factor landscapes to induce the neuronal program" @default.
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