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Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis

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ABSTRACT

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

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Genome-wide Mapping of Ascl1 Binding Sites in Differentiating NS Cells(A) Location of Ascl1 BEs respective to various genomic features.(B) Location of Ascl1 BEs in relation to the closest annotated TSS.(C) Chromatin state in differentiating NS cells, at Ascl1 bound regions in the vicinity of Fbxw7, Olig2, and Dll3 genes.(D) Heat maps of chromatin states for H3K27ac and H3K4me1 within ±2 kb of Ascl1-ERT2 peak summits in proliferating or differentiating NS cells (before and 24 hr after the addition of tamoxifen, respectively).(E) DNA motif enrichment for Ascl1 E-box within a 20-bp region centered at Ascl1 peak summits (R, enrichment over local genomic background; S, motif score).See also Figure S2.
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fig2: Genome-wide Mapping of Ascl1 Binding Sites in Differentiating NS Cells(A) Location of Ascl1 BEs respective to various genomic features.(B) Location of Ascl1 BEs in relation to the closest annotated TSS.(C) Chromatin state in differentiating NS cells, at Ascl1 bound regions in the vicinity of Fbxw7, Olig2, and Dll3 genes.(D) Heat maps of chromatin states for H3K27ac and H3K4me1 within ±2 kb of Ascl1-ERT2 peak summits in proliferating or differentiating NS cells (before and 24 hr after the addition of tamoxifen, respectively).(E) DNA motif enrichment for Ascl1 E-box within a 20-bp region centered at Ascl1 peak summits (R, enrichment over local genomic background; S, motif score).See also Figure S2.

Mentions: In order to validate binding events (BEs) mediated by the Ascl1-ERT2 protein, we used ChIP-PCR against WT Ascl1, with chromatin extracted from embryonic E14.5 ventral telencephalic progenitors, or WT NS5 cells (Figure S2A). We reasoned that most BEs in differentiating NS cells should be identified in at least one of the chromatin samples. Indeed, this is the case for all ChIP-seq peaks associated with p < 10−18 (with the majority of BEs validated in both chromatins), indicating a good match between Ascl1-ERT2 and WT Ascl1. Most of the 11,782 Ascl1 BEs defined by this high-confidence list (p < 10−18) are located within intergenic regions (63%) and at long distances from the nearest identified transcription start site (TSS). Less than one third of Ascl1 binding occurs inside genes (30%) or their promoter regions (7%) (Figures 2A and 2B), suggesting that Ascl1 binds predominantly to distal enhancer regions. We next used a Hidden-Markov model to characterize the chromatin states at Ascl1-bound regions, using genome-wide profiles of histone modifications generated from proliferating and differentiating NS cells (before or 24 hr after addition of tamoxifen, respectively) (Ernst and Kellis, 2012). Ascl1 BEs fall mostly within regions of chromatin highly co-enriched for H3K4me1 and H3K27ac, characteristic of active enhancers (same result for p < 10−10; data not shown). Moreover, the same association with this chromatin state is found in proliferating NS cells, indicating that during differentiation Ascl1 binds predominantly to regions that are already marked as active enhancers in proliferating NS cells (Figures 2C, 2D, and S2B).


Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis
Genome-wide Mapping of Ascl1 Binding Sites in Differentiating NS Cells(A) Location of Ascl1 BEs respective to various genomic features.(B) Location of Ascl1 BEs in relation to the closest annotated TSS.(C) Chromatin state in differentiating NS cells, at Ascl1 bound regions in the vicinity of Fbxw7, Olig2, and Dll3 genes.(D) Heat maps of chromatin states for H3K27ac and H3K4me1 within ±2 kb of Ascl1-ERT2 peak summits in proliferating or differentiating NS cells (before and 24 hr after the addition of tamoxifen, respectively).(E) DNA motif enrichment for Ascl1 E-box within a 20-bp region centered at Ascl1 peak summits (R, enrichment over local genomic background; S, motif score).See also Figure S2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5383937&req=5

fig2: Genome-wide Mapping of Ascl1 Binding Sites in Differentiating NS Cells(A) Location of Ascl1 BEs respective to various genomic features.(B) Location of Ascl1 BEs in relation to the closest annotated TSS.(C) Chromatin state in differentiating NS cells, at Ascl1 bound regions in the vicinity of Fbxw7, Olig2, and Dll3 genes.(D) Heat maps of chromatin states for H3K27ac and H3K4me1 within ±2 kb of Ascl1-ERT2 peak summits in proliferating or differentiating NS cells (before and 24 hr after the addition of tamoxifen, respectively).(E) DNA motif enrichment for Ascl1 E-box within a 20-bp region centered at Ascl1 peak summits (R, enrichment over local genomic background; S, motif score).See also Figure S2.
Mentions: In order to validate binding events (BEs) mediated by the Ascl1-ERT2 protein, we used ChIP-PCR against WT Ascl1, with chromatin extracted from embryonic E14.5 ventral telencephalic progenitors, or WT NS5 cells (Figure S2A). We reasoned that most BEs in differentiating NS cells should be identified in at least one of the chromatin samples. Indeed, this is the case for all ChIP-seq peaks associated with p < 10−18 (with the majority of BEs validated in both chromatins), indicating a good match between Ascl1-ERT2 and WT Ascl1. Most of the 11,782 Ascl1 BEs defined by this high-confidence list (p < 10−18) are located within intergenic regions (63%) and at long distances from the nearest identified transcription start site (TSS). Less than one third of Ascl1 binding occurs inside genes (30%) or their promoter regions (7%) (Figures 2A and 2B), suggesting that Ascl1 binds predominantly to distal enhancer regions. We next used a Hidden-Markov model to characterize the chromatin states at Ascl1-bound regions, using genome-wide profiles of histone modifications generated from proliferating and differentiating NS cells (before or 24 hr after addition of tamoxifen, respectively) (Ernst and Kellis, 2012). Ascl1 BEs fall mostly within regions of chromatin highly co-enriched for H3K4me1 and H3K27ac, characteristic of active enhancers (same result for p < 10−10; data not shown). Moreover, the same association with this chromatin state is found in proliferating NS cells, indicating that during differentiation Ascl1 binds predominantly to regions that are already marked as active enhancers in proliferating NS cells (Figures 2C, 2D, and S2B).

View Article: PubMed Central - PubMed

ABSTRACT

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

No MeSH data available.


Related in: MedlinePlus