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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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Zic1/2 binding is highly dynamic across postnatal cerebellar development(a) Zic1 and Zic2 RNA-seq expression across in vivo and cultured CGN development (n = 3). (b) Zic binding changes between P7 and P60 cerebellum. Red points represent ChIP-seq peaks with significantly increased Zic binding from P7 to P60, blue points are peaks with decreased binding (FDR < 0.05, n = 2 biological replicates of pooled cerebella). UCSC genome browser images to the right show examples of increased and decreased Zic binding. Note the peak located in the 3′UTR of Cbln3 coincides with opened enhancer element tested in Fig. 2g. (c) Overlap between differential Zic binding sites and DHS sites that open or close from P7 to P60. (d) Overlap between differential Zic binding sites and H3K27ac ChIP-seq peaks identified in P7 or P60 cerebellum.
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Figure 6: Zic1/2 binding is highly dynamic across postnatal cerebellar development(a) Zic1 and Zic2 RNA-seq expression across in vivo and cultured CGN development (n = 3). (b) Zic binding changes between P7 and P60 cerebellum. Red points represent ChIP-seq peaks with significantly increased Zic binding from P7 to P60, blue points are peaks with decreased binding (FDR < 0.05, n = 2 biological replicates of pooled cerebella). UCSC genome browser images to the right show examples of increased and decreased Zic binding. Note the peak located in the 3′UTR of Cbln3 coincides with opened enhancer element tested in Fig. 2g. (c) Overlap between differential Zic binding sites and DHS sites that open or close from P7 to P60. (d) Overlap between differential Zic binding sites and H3K27ac ChIP-seq peaks identified in P7 or P60 cerebellum.

Mentions: As expected, motifs for the MEF2 and NF1 families, which have been shown to play important roles in CGN differentiation9,36, were enriched in both opening and closing DHS sites (Supplementary Fig. 8). However we were surprised to find the zinc-finger in cerebellum (Zic) transcription factor family motifs enriched in the set of opening DHS sites. Mutations in the human ZIC genes have been associated with cerebellar development disorders, suggesting their importance in CGN differentiation, and mouse knockout studies have indicated that the Zics function in GNPs to prevent their premature exit from the cell cycle37,38. However the Zic transcription factors remain highly expressed in differentiated CGNs (Fig. 6a), raising the possibility that these factors also contribute to later stages in CGN maturation. Given our observation that the Zic motif was enriched in opening DHS sites, we hypothesized that the Zic transcription factors might change their gene targets over time by binding to developmentally regulated DHS sites.


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)

Zic1/2 binding is highly dynamic across postnatal cerebellar development(a) Zic1 and Zic2 RNA-seq expression across in vivo and cultured CGN development (n = 3). (b) Zic binding changes between P7 and P60 cerebellum. Red points represent ChIP-seq peaks with significantly increased Zic binding from P7 to P60, blue points are peaks with decreased binding (FDR < 0.05, n = 2 biological replicates of pooled cerebella). UCSC genome browser images to the right show examples of increased and decreased Zic binding. Note the peak located in the 3′UTR of Cbln3 coincides with opened enhancer element tested in Fig. 2g. (c) Overlap between differential Zic binding sites and DHS sites that open or close from P7 to P60. (d) Overlap between differential Zic binding sites and H3K27ac ChIP-seq peaks identified in P7 or P60 cerebellum.
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Figure 6: Zic1/2 binding is highly dynamic across postnatal cerebellar development(a) Zic1 and Zic2 RNA-seq expression across in vivo and cultured CGN development (n = 3). (b) Zic binding changes between P7 and P60 cerebellum. Red points represent ChIP-seq peaks with significantly increased Zic binding from P7 to P60, blue points are peaks with decreased binding (FDR < 0.05, n = 2 biological replicates of pooled cerebella). UCSC genome browser images to the right show examples of increased and decreased Zic binding. Note the peak located in the 3′UTR of Cbln3 coincides with opened enhancer element tested in Fig. 2g. (c) Overlap between differential Zic binding sites and DHS sites that open or close from P7 to P60. (d) Overlap between differential Zic binding sites and H3K27ac ChIP-seq peaks identified in P7 or P60 cerebellum.
Mentions: As expected, motifs for the MEF2 and NF1 families, which have been shown to play important roles in CGN differentiation9,36, were enriched in both opening and closing DHS sites (Supplementary Fig. 8). However we were surprised to find the zinc-finger in cerebellum (Zic) transcription factor family motifs enriched in the set of opening DHS sites. Mutations in the human ZIC genes have been associated with cerebellar development disorders, suggesting their importance in CGN differentiation, and mouse knockout studies have indicated that the Zics function in GNPs to prevent their premature exit from the cell cycle37,38. However the Zic transcription factors remain highly expressed in differentiated CGNs (Fig. 6a), raising the possibility that these factors also contribute to later stages in CGN maturation. Given our observation that the Zic motif was enriched in opening DHS sites, we hypothesized that the Zic transcription factors might change their gene targets over time by binding to developmentally regulated DHS sites.

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