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Regulation of chromatin accessibility and Zic binding at enhancers in the developing cerebellum.

Frank CL, Liu F, Wijayatunge R, Song L, Biegler MT, Yang MG, Vockley CM, Safi A, Gersbach CA, Crawford GE, West AE - Nat. Neurosci. (2015)

Bottom Line: Motif discovery in differentially accessible chromatin regions suggested a previously unknown role for the Zic family of transcription factors in CGN maturation.We confirmed the association of Zic with these elements by ChIP-seq and found, using knockdown, that Zic1 and Zic2 are required for coordinating mature neuronal gene expression patterns.Together, our data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate the gene expression patterns necessary for neuronal differentiation and function.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA. [2] Center for Genomic and Computational Biology, Duke University Medical Center, Durham, North Carolina, USA.

ABSTRACT
To identify chromatin mechanisms of neuronal differentiation, we characterized chromatin accessibility and gene expression in cerebellar granule neurons (CGNs) of the developing mouse. We used DNase-seq to map accessibility of cis-regulatory elements and RNA-seq to profile transcript abundance across postnatal stages of neuronal differentiation in vivo and in culture. We observed thousands of chromatin accessibility changes as CGNs differentiated, and verified, using H3K27ac ChIP-seq, reporter gene assays and CRISPR-mediated activation, that many of these regions function as neuronal enhancers. Motif discovery in differentially accessible chromatin regions suggested a previously unknown role for the Zic family of transcription factors in CGN maturation. We confirmed the association of Zic with these elements by ChIP-seq and found, using knockdown, that Zic1 and Zic2 are required for coordinating mature neuronal gene expression patterns. Together, our data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate the gene expression patterns necessary for neuronal differentiation and function.

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Opening DHS sites mark late-acting neuronal enhancers(a,b) Cumulative fraction of genes nearest to opened promoter-located, opened distal, and all identified DHS sites with given fold-change in RNA-seq expression from P7 to P60 cerebellum (a) or from GNPs to +7DIV (b). Rightward shift indicates genes increased in expression across developmental time. Significance assessed by Mann-Whitney U test. (n = 3 biological replicates of pooled cerebella). (c) Mean H3K27ac ChIP-seq signal (reads per million mapped) present at center of P7 to P60 opened DHS sites (4053 sites) in either P7 cerebellum (orange line) or P60 cerebellum (green line). Gray = s.e.m.. (n = 2 biological replicates of pooled cerebella). (d) Percent of opened promoter-located, opened distal, and all DHS sites overlapping H3K27ac peaks identified in P7 or P60 cerebellum. (e) UCSC browser image highlighting a DHS site found in the 3′UTR of Cbln3 (black box) that opens during development and overlaps H3K27ac and H3K4me1 enrichment in adult cerebellum. (f) RNA-seq expression of Cbln3 across developmental time in vivo and in cultured CGNs. DIV = Days In Vitro. Error bars = 95% CI. (g) Luciferase reporter assay for enhancer activity conferred by a DHS site in Cbln3 at +3DIV and +6DIV. P = 0.0012 by two-sided Student’s t-test (n = 3 transfections, t = 8.3). Error bars = s.e.m.
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Figure 2: Opening DHS sites mark late-acting neuronal enhancers(a,b) Cumulative fraction of genes nearest to opened promoter-located, opened distal, and all identified DHS sites with given fold-change in RNA-seq expression from P7 to P60 cerebellum (a) or from GNPs to +7DIV (b). Rightward shift indicates genes increased in expression across developmental time. Significance assessed by Mann-Whitney U test. (n = 3 biological replicates of pooled cerebella). (c) Mean H3K27ac ChIP-seq signal (reads per million mapped) present at center of P7 to P60 opened DHS sites (4053 sites) in either P7 cerebellum (orange line) or P60 cerebellum (green line). Gray = s.e.m.. (n = 2 biological replicates of pooled cerebella). (d) Percent of opened promoter-located, opened distal, and all DHS sites overlapping H3K27ac peaks identified in P7 or P60 cerebellum. (e) UCSC browser image highlighting a DHS site found in the 3′UTR of Cbln3 (black box) that opens during development and overlaps H3K27ac and H3K4me1 enrichment in adult cerebellum. (f) RNA-seq expression of Cbln3 across developmental time in vivo and in cultured CGNs. DIV = Days In Vitro. Error bars = 95% CI. (g) Luciferase reporter assay for enhancer activity conferred by a DHS site in Cbln3 at +3DIV and +6DIV. P = 0.0012 by two-sided Student’s t-test (n = 3 transfections, t = 8.3). Error bars = s.e.m.

Mentions: We postulated that the developmentally regulated chromatin accessibility changes we observed might mark a combination of promoter and enhancer elements that become activated at specific developmental stages to drive target gene expression. We first mapped each DHS site to its nearest gene and analyzed the relationship between that DHS site and the change in its associated gene expression value between P7 and P60. Whereas all DHS sites together exhibit a normal distribution of nearby gene expression changes centered on a fold-change of 1, both promoter and distal DHS sites that open up between P7 and P60 were significantly associated with genes that have higher expression levels at P60 (P= 2.7×10−34 and P = 5.0×10−31, respectively, Mann-Whitney test) (Fig. 2a). Similar to our in vivo data, we found a strong association between GNP to +7DIV opening DHS sites and increased nearby gene expression in cultured CGNs (Fig. 2b).


Regulation of chromatin accessibility and Zic binding at enhancers in the developing cerebellum.

Frank CL, Liu F, Wijayatunge R, Song L, Biegler MT, Yang MG, Vockley CM, Safi A, Gersbach CA, Crawford GE, West AE - Nat. Neurosci. (2015)

Opening DHS sites mark late-acting neuronal enhancers(a,b) Cumulative fraction of genes nearest to opened promoter-located, opened distal, and all identified DHS sites with given fold-change in RNA-seq expression from P7 to P60 cerebellum (a) or from GNPs to +7DIV (b). Rightward shift indicates genes increased in expression across developmental time. Significance assessed by Mann-Whitney U test. (n = 3 biological replicates of pooled cerebella). (c) Mean H3K27ac ChIP-seq signal (reads per million mapped) present at center of P7 to P60 opened DHS sites (4053 sites) in either P7 cerebellum (orange line) or P60 cerebellum (green line). Gray = s.e.m.. (n = 2 biological replicates of pooled cerebella). (d) Percent of opened promoter-located, opened distal, and all DHS sites overlapping H3K27ac peaks identified in P7 or P60 cerebellum. (e) UCSC browser image highlighting a DHS site found in the 3′UTR of Cbln3 (black box) that opens during development and overlaps H3K27ac and H3K4me1 enrichment in adult cerebellum. (f) RNA-seq expression of Cbln3 across developmental time in vivo and in cultured CGNs. DIV = Days In Vitro. Error bars = 95% CI. (g) Luciferase reporter assay for enhancer activity conferred by a DHS site in Cbln3 at +3DIV and +6DIV. P = 0.0012 by two-sided Student’s t-test (n = 3 transfections, t = 8.3). Error bars = s.e.m.
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Related In: Results  -  Collection

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Figure 2: Opening DHS sites mark late-acting neuronal enhancers(a,b) Cumulative fraction of genes nearest to opened promoter-located, opened distal, and all identified DHS sites with given fold-change in RNA-seq expression from P7 to P60 cerebellum (a) or from GNPs to +7DIV (b). Rightward shift indicates genes increased in expression across developmental time. Significance assessed by Mann-Whitney U test. (n = 3 biological replicates of pooled cerebella). (c) Mean H3K27ac ChIP-seq signal (reads per million mapped) present at center of P7 to P60 opened DHS sites (4053 sites) in either P7 cerebellum (orange line) or P60 cerebellum (green line). Gray = s.e.m.. (n = 2 biological replicates of pooled cerebella). (d) Percent of opened promoter-located, opened distal, and all DHS sites overlapping H3K27ac peaks identified in P7 or P60 cerebellum. (e) UCSC browser image highlighting a DHS site found in the 3′UTR of Cbln3 (black box) that opens during development and overlaps H3K27ac and H3K4me1 enrichment in adult cerebellum. (f) RNA-seq expression of Cbln3 across developmental time in vivo and in cultured CGNs. DIV = Days In Vitro. Error bars = 95% CI. (g) Luciferase reporter assay for enhancer activity conferred by a DHS site in Cbln3 at +3DIV and +6DIV. P = 0.0012 by two-sided Student’s t-test (n = 3 transfections, t = 8.3). Error bars = s.e.m.
Mentions: We postulated that the developmentally regulated chromatin accessibility changes we observed might mark a combination of promoter and enhancer elements that become activated at specific developmental stages to drive target gene expression. We first mapped each DHS site to its nearest gene and analyzed the relationship between that DHS site and the change in its associated gene expression value between P7 and P60. Whereas all DHS sites together exhibit a normal distribution of nearby gene expression changes centered on a fold-change of 1, both promoter and distal DHS sites that open up between P7 and P60 were significantly associated with genes that have higher expression levels at P60 (P= 2.7×10−34 and P = 5.0×10−31, respectively, Mann-Whitney test) (Fig. 2a). Similar to our in vivo data, we found a strong association between GNP to +7DIV opening DHS sites and increased nearby gene expression in cultured CGNs (Fig. 2b).

Bottom Line: Motif discovery in differentially accessible chromatin regions suggested a previously unknown role for the Zic family of transcription factors in CGN maturation.We confirmed the association of Zic with these elements by ChIP-seq and found, using knockdown, that Zic1 and Zic2 are required for coordinating mature neuronal gene expression patterns.Together, our data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate the gene expression patterns necessary for neuronal differentiation and function.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA. [2] Center for Genomic and Computational Biology, Duke University Medical Center, Durham, North Carolina, USA.

ABSTRACT
To identify chromatin mechanisms of neuronal differentiation, we characterized chromatin accessibility and gene expression in cerebellar granule neurons (CGNs) of the developing mouse. We used DNase-seq to map accessibility of cis-regulatory elements and RNA-seq to profile transcript abundance across postnatal stages of neuronal differentiation in vivo and in culture. We observed thousands of chromatin accessibility changes as CGNs differentiated, and verified, using H3K27ac ChIP-seq, reporter gene assays and CRISPR-mediated activation, that many of these regions function as neuronal enhancers. Motif discovery in differentially accessible chromatin regions suggested a previously unknown role for the Zic family of transcription factors in CGN maturation. We confirmed the association of Zic with these elements by ChIP-seq and found, using knockdown, that Zic1 and Zic2 are required for coordinating mature neuronal gene expression patterns. Together, our data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate the gene expression patterns necessary for neuronal differentiation and function.

Show MeSH