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Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis

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.


Characterization of Changes in Chromatin Accessibility during Differentiation of NS Cells(A) Number of DHSs in proliferating NS cells and 24 hr upon Ascl1-mediated differentiation.(B) FAIRE-PCR validation of differentiation-induced DHSs. Bars show fold enrichment of genomic DNA obtained from NS cells 24 hr after induction over cells prior to addition of tamoxifen. Data are represented as mean ± SD.(C) Differentiation-induced DHSs are significantly associated with clusters of activated genes (left) and not with clusters of repressed genes (right). Red bars, total number of DHSs annotated to each set of genes; boxplots, distribution of DHSs associations with 1,000 random sets of genes. Test data are represented as box with median of test and first and third quartiles; whiskers, ±1.5 × IQR.(D) Activated genes associated with differentiation-induced DHSs have low or no expression in proliferating cells. Gene expression levels in proliferating cells are significantly lower (p < 10−11, Wilcoxon test) for activated genes associated with induced DHSs (right) than for activated genes with no such association (left). Red bar, level of expression of NeuroD4 gene. Data distribution represented as box with median and first and third quartiles; whiskers, ±1.5 × IQR; notches, ±1.58 × IQR/n1/2).(E) Frequency of motif occurrence at high occupancy sites found within differentiation-induced DHSs by Digital Genomic Footprinting (overrepresentation ratio indicated between brackets).(F) Comparison of Ascl1 BEs at t = 30 min (left) or t = 18 hr (right) with the DNase-seq profile. Color shows the proportion of Ascl1 BEs by cumulative bins of increasing p value (bin = 1,842), which fall within regions of open chromatin (DHSs) in proliferating (top) or differentiating (bottom) NS cells.
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fig5: Characterization of Changes in Chromatin Accessibility during Differentiation of NS Cells(A) Number of DHSs in proliferating NS cells and 24 hr upon Ascl1-mediated differentiation.(B) FAIRE-PCR validation of differentiation-induced DHSs. Bars show fold enrichment of genomic DNA obtained from NS cells 24 hr after induction over cells prior to addition of tamoxifen. Data are represented as mean ± SD.(C) Differentiation-induced DHSs are significantly associated with clusters of activated genes (left) and not with clusters of repressed genes (right). Red bars, total number of DHSs annotated to each set of genes; boxplots, distribution of DHSs associations with 1,000 random sets of genes. Test data are represented as box with median of test and first and third quartiles; whiskers, ±1.5 × IQR.(D) Activated genes associated with differentiation-induced DHSs have low or no expression in proliferating cells. Gene expression levels in proliferating cells are significantly lower (p < 10−11, Wilcoxon test) for activated genes associated with induced DHSs (right) than for activated genes with no such association (left). Red bar, level of expression of NeuroD4 gene. Data distribution represented as box with median and first and third quartiles; whiskers, ±1.5 × IQR; notches, ±1.58 × IQR/n1/2).(E) Frequency of motif occurrence at high occupancy sites found within differentiation-induced DHSs by Digital Genomic Footprinting (overrepresentation ratio indicated between brackets).(F) Comparison of Ascl1 BEs at t = 30 min (left) or t = 18 hr (right) with the DNase-seq profile. Color shows the proportion of Ascl1 BEs by cumulative bins of increasing p value (bin = 1,842), which fall within regions of open chromatin (DHSs) in proliferating (top) or differentiating (bottom) NS cells.

Mentions: We next asked what impact Ascl1 may have on the chromatin landscape when it promotes neuronal differentiation of NS cells. We started by characterizing the chromatin landscape of proliferating NS cells and of NS cells undergoing differentiation by Ascl1, using a DNase I hypersensitivity assay coupled to massive parallel sequencing (DNase-seq). This technique identifies regions of decreased nucleosome occupancy (herein referred as “open chromatin”) on a genome-wide scale, which correspond mostly to active regulatory elements such as promoters, enhancers, insulators, and silencers (Boyle et al., 2008, Natarajan et al., 2012, Thurman et al., 2012). The density of mapped reads for each genome position was computed to generate a comprehensive list of DNase I hypersensitivity sites (DHSs). Using a constant threshold of p < 10−5, we identified ∼87,000 and ∼94,000 DHSs in proliferating and differentiating NS cells, respectively (Tables S5 and S6), numbers with a magnitude consistent with those obtained in other cell types (Song et al., 2011). Although the majority of these sites is shared by both experimental conditions, each cell state exhibits a specific set of ∼20,000 DHSs (Figure 5A).


Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis
Characterization of Changes in Chromatin Accessibility during Differentiation of NS Cells(A) Number of DHSs in proliferating NS cells and 24 hr upon Ascl1-mediated differentiation.(B) FAIRE-PCR validation of differentiation-induced DHSs. Bars show fold enrichment of genomic DNA obtained from NS cells 24 hr after induction over cells prior to addition of tamoxifen. Data are represented as mean ± SD.(C) Differentiation-induced DHSs are significantly associated with clusters of activated genes (left) and not with clusters of repressed genes (right). Red bars, total number of DHSs annotated to each set of genes; boxplots, distribution of DHSs associations with 1,000 random sets of genes. Test data are represented as box with median of test and first and third quartiles; whiskers, ±1.5 × IQR.(D) Activated genes associated with differentiation-induced DHSs have low or no expression in proliferating cells. Gene expression levels in proliferating cells are significantly lower (p < 10−11, Wilcoxon test) for activated genes associated with induced DHSs (right) than for activated genes with no such association (left). Red bar, level of expression of NeuroD4 gene. Data distribution represented as box with median and first and third quartiles; whiskers, ±1.5 × IQR; notches, ±1.58 × IQR/n1/2).(E) Frequency of motif occurrence at high occupancy sites found within differentiation-induced DHSs by Digital Genomic Footprinting (overrepresentation ratio indicated between brackets).(F) Comparison of Ascl1 BEs at t = 30 min (left) or t = 18 hr (right) with the DNase-seq profile. Color shows the proportion of Ascl1 BEs by cumulative bins of increasing p value (bin = 1,842), which fall within regions of open chromatin (DHSs) in proliferating (top) or differentiating (bottom) NS cells.
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fig5: Characterization of Changes in Chromatin Accessibility during Differentiation of NS Cells(A) Number of DHSs in proliferating NS cells and 24 hr upon Ascl1-mediated differentiation.(B) FAIRE-PCR validation of differentiation-induced DHSs. Bars show fold enrichment of genomic DNA obtained from NS cells 24 hr after induction over cells prior to addition of tamoxifen. Data are represented as mean ± SD.(C) Differentiation-induced DHSs are significantly associated with clusters of activated genes (left) and not with clusters of repressed genes (right). Red bars, total number of DHSs annotated to each set of genes; boxplots, distribution of DHSs associations with 1,000 random sets of genes. Test data are represented as box with median of test and first and third quartiles; whiskers, ±1.5 × IQR.(D) Activated genes associated with differentiation-induced DHSs have low or no expression in proliferating cells. Gene expression levels in proliferating cells are significantly lower (p < 10−11, Wilcoxon test) for activated genes associated with induced DHSs (right) than for activated genes with no such association (left). Red bar, level of expression of NeuroD4 gene. Data distribution represented as box with median and first and third quartiles; whiskers, ±1.5 × IQR; notches, ±1.58 × IQR/n1/2).(E) Frequency of motif occurrence at high occupancy sites found within differentiation-induced DHSs by Digital Genomic Footprinting (overrepresentation ratio indicated between brackets).(F) Comparison of Ascl1 BEs at t = 30 min (left) or t = 18 hr (right) with the DNase-seq profile. Color shows the proportion of Ascl1 BEs by cumulative bins of increasing p value (bin = 1,842), which fall within regions of open chromatin (DHSs) in proliferating (top) or differentiating (bottom) NS cells.
Mentions: We next asked what impact Ascl1 may have on the chromatin landscape when it promotes neuronal differentiation of NS cells. We started by characterizing the chromatin landscape of proliferating NS cells and of NS cells undergoing differentiation by Ascl1, using a DNase I hypersensitivity assay coupled to massive parallel sequencing (DNase-seq). This technique identifies regions of decreased nucleosome occupancy (herein referred as “open chromatin”) on a genome-wide scale, which correspond mostly to active regulatory elements such as promoters, enhancers, insulators, and silencers (Boyle et al., 2008, Natarajan et al., 2012, Thurman et al., 2012). The density of mapped reads for each genome position was computed to generate a comprehensive list of DNase I hypersensitivity sites (DHSs). Using a constant threshold of p < 10−5, we identified ∼87,000 and ∼94,000 DHSs in proliferating and differentiating NS cells, respectively (Tables S5 and S6), numbers with a magnitude consistent with those obtained in other cell types (Song et al., 2011). Although the majority of these sites is shared by both experimental conditions, each cell state exhibits a specific set of ∼20,000 DHSs (Figure 5A).

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.