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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.

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Ascl1 Binds Closed Chromatin and Promotes Chromatin Accessibility(A) Visual representation of ChIP-seq and DNase-seq enrichment profiles in the vicinity of various Ascl1 targets. Examples are shown of differentiation-induced and constant DHSs localized at Ascl1 binding sites (green and red arrows, respectively) and DHSs with no apparent association with Ascl1 binding (blue arrows).(B) Induction of expression of genes upon Ascl1-induced differentiation as quantified by real-time PCR. Data are represented as mean ± SD.(C) FAIRE-PCR validation of differentiation-induced DHSs at Ascl1 binding sites (green arrows in A). Bars show quantification of genomic DNA obtained from proliferating and differentiating NS cells (before and 24 hr after induction, respectively). Data are represented as mean ± SD.(D) DNase-seq signal profile at genome-wide Ascl1 BEs at t = 30 min (top left) or restricted to BEs falling within differentiation-induced DHSs (top right). Profile determined as the median read count of DNase-seq reads mapped to the 4-kb regions centered at the peak summits in proliferating (blue) and differentiating cells (red). (Bottom) Corresponding fold change for the median profiles.(E) Ascl1 BEs located at 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 Ascl1 BEs annotated to each set of genes; boxplots, distribution of Ascl1 BEs associations with 1,000 random sets of genes. Test data represented as box with median of test and first and third quartiles; whiskers, ±1.5 × IQR. See also Figure S6.(F) In vivo binding of Ascl1 to the NeuroD4 regulatory region assessed by ChIP-PCR using chromatin extracted from mouse ventral telencephalon. Data are represented as mean ± SD.(G) Chromatin structure analysis by FAIRE-PCR of NeuroD4 and Dll1 regulatory regions bound by Ascl1, in mouse ventral telencephalon. Data are represented as mean ± SD.
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fig6: Ascl1 Binds Closed Chromatin and Promotes Chromatin Accessibility(A) Visual representation of ChIP-seq and DNase-seq enrichment profiles in the vicinity of various Ascl1 targets. Examples are shown of differentiation-induced and constant DHSs localized at Ascl1 binding sites (green and red arrows, respectively) and DHSs with no apparent association with Ascl1 binding (blue arrows).(B) Induction of expression of genes upon Ascl1-induced differentiation as quantified by real-time PCR. Data are represented as mean ± SD.(C) FAIRE-PCR validation of differentiation-induced DHSs at Ascl1 binding sites (green arrows in A). Bars show quantification of genomic DNA obtained from proliferating and differentiating NS cells (before and 24 hr after induction, respectively). Data are represented as mean ± SD.(D) DNase-seq signal profile at genome-wide Ascl1 BEs at t = 30 min (top left) or restricted to BEs falling within differentiation-induced DHSs (top right). Profile determined as the median read count of DNase-seq reads mapped to the 4-kb regions centered at the peak summits in proliferating (blue) and differentiating cells (red). (Bottom) Corresponding fold change for the median profiles.(E) Ascl1 BEs located at 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 Ascl1 BEs annotated to each set of genes; boxplots, distribution of Ascl1 BEs associations with 1,000 random sets of genes. Test data represented as box with median of test and first and third quartiles; whiskers, ±1.5 × IQR. See also Figure S6.(F) In vivo binding of Ascl1 to the NeuroD4 regulatory region assessed by ChIP-PCR using chromatin extracted from mouse ventral telencephalon. Data are represented as mean ± SD.(G) Chromatin structure analysis by FAIRE-PCR of NeuroD4 and Dll1 regulatory regions bound by Ascl1, in mouse ventral telencephalon. Data are represented as mean ± SD.

Mentions: Next, we investigated how regions of chromatin newly opened during differentiation may be associated with the observed changes in gene expression. We find a statistically significant enrichment of differentiation-induced DHSs in the vicinity of upregulated genes (the sum of clusters 1–4; p < 1.3 × 10−29), in sharp contrast with downregulated genes (clusters 5–7) (Figure 5C). Moreover, a large fraction of all upregulated genes (413 of 760) is associated with at least one differentiation-induced DHS, suggesting the importance of these putative regulatory regions in activating gene expression during neurogenesis. Notably, upregulated genes associated with differentiation-induced DHSs are either not expressed in proliferating NS cells or expressed at a low level when compared with upregulated genes near constant DHSs (Figure 5D). This is well exemplified by the induction of NeuroD4, a pro-differentiation TF only expressed in post-mitotic precursors (Ohsawa et al., 2005), and which is associated with newly open DHSs (Figure 6B). Overall, our results indicate that activation of differentiation genes is strongly associated with the appearance of new DHSs.


Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis
Ascl1 Binds Closed Chromatin and Promotes Chromatin Accessibility(A) Visual representation of ChIP-seq and DNase-seq enrichment profiles in the vicinity of various Ascl1 targets. Examples are shown of differentiation-induced and constant DHSs localized at Ascl1 binding sites (green and red arrows, respectively) and DHSs with no apparent association with Ascl1 binding (blue arrows).(B) Induction of expression of genes upon Ascl1-induced differentiation as quantified by real-time PCR. Data are represented as mean ± SD.(C) FAIRE-PCR validation of differentiation-induced DHSs at Ascl1 binding sites (green arrows in A). Bars show quantification of genomic DNA obtained from proliferating and differentiating NS cells (before and 24 hr after induction, respectively). Data are represented as mean ± SD.(D) DNase-seq signal profile at genome-wide Ascl1 BEs at t = 30 min (top left) or restricted to BEs falling within differentiation-induced DHSs (top right). Profile determined as the median read count of DNase-seq reads mapped to the 4-kb regions centered at the peak summits in proliferating (blue) and differentiating cells (red). (Bottom) Corresponding fold change for the median profiles.(E) Ascl1 BEs located at 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 Ascl1 BEs annotated to each set of genes; boxplots, distribution of Ascl1 BEs associations with 1,000 random sets of genes. Test data represented as box with median of test and first and third quartiles; whiskers, ±1.5 × IQR. See also Figure S6.(F) In vivo binding of Ascl1 to the NeuroD4 regulatory region assessed by ChIP-PCR using chromatin extracted from mouse ventral telencephalon. Data are represented as mean ± SD.(G) Chromatin structure analysis by FAIRE-PCR of NeuroD4 and Dll1 regulatory regions bound by Ascl1, in mouse ventral telencephalon. Data are represented as mean ± SD.
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fig6: Ascl1 Binds Closed Chromatin and Promotes Chromatin Accessibility(A) Visual representation of ChIP-seq and DNase-seq enrichment profiles in the vicinity of various Ascl1 targets. Examples are shown of differentiation-induced and constant DHSs localized at Ascl1 binding sites (green and red arrows, respectively) and DHSs with no apparent association with Ascl1 binding (blue arrows).(B) Induction of expression of genes upon Ascl1-induced differentiation as quantified by real-time PCR. Data are represented as mean ± SD.(C) FAIRE-PCR validation of differentiation-induced DHSs at Ascl1 binding sites (green arrows in A). Bars show quantification of genomic DNA obtained from proliferating and differentiating NS cells (before and 24 hr after induction, respectively). Data are represented as mean ± SD.(D) DNase-seq signal profile at genome-wide Ascl1 BEs at t = 30 min (top left) or restricted to BEs falling within differentiation-induced DHSs (top right). Profile determined as the median read count of DNase-seq reads mapped to the 4-kb regions centered at the peak summits in proliferating (blue) and differentiating cells (red). (Bottom) Corresponding fold change for the median profiles.(E) Ascl1 BEs located at 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 Ascl1 BEs annotated to each set of genes; boxplots, distribution of Ascl1 BEs associations with 1,000 random sets of genes. Test data represented as box with median of test and first and third quartiles; whiskers, ±1.5 × IQR. See also Figure S6.(F) In vivo binding of Ascl1 to the NeuroD4 regulatory region assessed by ChIP-PCR using chromatin extracted from mouse ventral telencephalon. Data are represented as mean ± SD.(G) Chromatin structure analysis by FAIRE-PCR of NeuroD4 and Dll1 regulatory regions bound by Ascl1, in mouse ventral telencephalon. Data are represented as mean ± SD.
Mentions: Next, we investigated how regions of chromatin newly opened during differentiation may be associated with the observed changes in gene expression. We find a statistically significant enrichment of differentiation-induced DHSs in the vicinity of upregulated genes (the sum of clusters 1–4; p < 1.3 × 10−29), in sharp contrast with downregulated genes (clusters 5–7) (Figure 5C). Moreover, a large fraction of all upregulated genes (413 of 760) is associated with at least one differentiation-induced DHS, suggesting the importance of these putative regulatory regions in activating gene expression during neurogenesis. Notably, upregulated genes associated with differentiation-induced DHSs are either not expressed in proliferating NS cells or expressed at a low level when compared with upregulated genes near constant DHSs (Figure 5D). This is well exemplified by the induction of NeuroD4, a pro-differentiation TF only expressed in post-mitotic precursors (Ohsawa et al., 2005), and which is associated with newly open DHSs (Figure 6B). Overall, our results indicate that activation of differentiation genes is strongly associated with the appearance of new DHSs.

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