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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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Postnatal cerebellar development involves extensive chromatin remodeling(a) Immunostaining of cerebellar cortex in sagittal section at postnatal day 7 (P7), 14 (P14), and 60 (P60). Ki67 marks active cell proliferation, doublecortin (Dcx) marks immature/migrating neurons, and GABAA receptor subunit α6 (GABAARα6) marks mature neurons. Nuclei stained with Hoechst (blue). White scale bar is 200 μm. This experiment was performed once. (b) Example region of mm9 genome showing reproducible DNase-seq chromatin accessibility profiles across biological triplicates of P7, P14, and P60 cerebella. Gray boxes denote DHS sites that change between developmental stages. Y-axes fixed across samples. (c) Number of total peak calls and significantly opened or closed DHS sites between P7, P14, and P60 cerebella pairwise comparisons (FDR < 0.05, n = 3 biological replicates of pooled cerebella). (d) Heatmap of DNase-seq accessibility across three timepoints for regions marked significant in DESeq analysis. Mean pairwise Pearson correlation between replicates listed above heatmap. (e) Distribution of gene features that opened, closed, and all identified DHS sites map to.
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Figure 1: Postnatal cerebellar development involves extensive chromatin remodeling(a) Immunostaining of cerebellar cortex in sagittal section at postnatal day 7 (P7), 14 (P14), and 60 (P60). Ki67 marks active cell proliferation, doublecortin (Dcx) marks immature/migrating neurons, and GABAA receptor subunit α6 (GABAARα6) marks mature neurons. Nuclei stained with Hoechst (blue). White scale bar is 200 μm. This experiment was performed once. (b) Example region of mm9 genome showing reproducible DNase-seq chromatin accessibility profiles across biological triplicates of P7, P14, and P60 cerebella. Gray boxes denote DHS sites that change between developmental stages. Y-axes fixed across samples. (c) Number of total peak calls and significantly opened or closed DHS sites between P7, P14, and P60 cerebella pairwise comparisons (FDR < 0.05, n = 3 biological replicates of pooled cerebella). (d) Heatmap of DNase-seq accessibility across three timepoints for regions marked significant in DESeq analysis. Mean pairwise Pearson correlation between replicates listed above heatmap. (e) Distribution of gene features that opened, closed, and all identified DHS sites map to.

Mentions: We used DNase-seq to globally map chromatin accessibility at three key stages in the development of the mouse cerebellum: 1) postnatal day 7 (P7), when the external granular layer (EGL) of the mouse cerebellar cortex has reached its maximal thickness12 due to the proliferation of granule neuron precursors (GNPs) (Fig. 1a); 2) P14, when newborn postmitotic CGNs begin to populate the internal granule layer (IGL)13; and 3) P60, when CGNs of the IGL express gene products that mediate mature synaptic functions8 (Fig. 1b, Supplementary Fig. 1a, Supplementary Table 1). We identified approximately 70,000 DHS sites at each of the three developmental stages, and found that these elements mapped to promoters, gene bodies, and intergenic regions (Supplementary Fig. 1b). The majority of DHS sites (~77%) are found outside of annotated gene promoter regions, highlighting the ability of DNase-seq to identify distal regulatory elements in the genome.


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)

Postnatal cerebellar development involves extensive chromatin remodeling(a) Immunostaining of cerebellar cortex in sagittal section at postnatal day 7 (P7), 14 (P14), and 60 (P60). Ki67 marks active cell proliferation, doublecortin (Dcx) marks immature/migrating neurons, and GABAA receptor subunit α6 (GABAARα6) marks mature neurons. Nuclei stained with Hoechst (blue). White scale bar is 200 μm. This experiment was performed once. (b) Example region of mm9 genome showing reproducible DNase-seq chromatin accessibility profiles across biological triplicates of P7, P14, and P60 cerebella. Gray boxes denote DHS sites that change between developmental stages. Y-axes fixed across samples. (c) Number of total peak calls and significantly opened or closed DHS sites between P7, P14, and P60 cerebella pairwise comparisons (FDR < 0.05, n = 3 biological replicates of pooled cerebella). (d) Heatmap of DNase-seq accessibility across three timepoints for regions marked significant in DESeq analysis. Mean pairwise Pearson correlation between replicates listed above heatmap. (e) Distribution of gene features that opened, closed, and all identified DHS sites map to.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414887&req=5

Figure 1: Postnatal cerebellar development involves extensive chromatin remodeling(a) Immunostaining of cerebellar cortex in sagittal section at postnatal day 7 (P7), 14 (P14), and 60 (P60). Ki67 marks active cell proliferation, doublecortin (Dcx) marks immature/migrating neurons, and GABAA receptor subunit α6 (GABAARα6) marks mature neurons. Nuclei stained with Hoechst (blue). White scale bar is 200 μm. This experiment was performed once. (b) Example region of mm9 genome showing reproducible DNase-seq chromatin accessibility profiles across biological triplicates of P7, P14, and P60 cerebella. Gray boxes denote DHS sites that change between developmental stages. Y-axes fixed across samples. (c) Number of total peak calls and significantly opened or closed DHS sites between P7, P14, and P60 cerebella pairwise comparisons (FDR < 0.05, n = 3 biological replicates of pooled cerebella). (d) Heatmap of DNase-seq accessibility across three timepoints for regions marked significant in DESeq analysis. Mean pairwise Pearson correlation between replicates listed above heatmap. (e) Distribution of gene features that opened, closed, and all identified DHS sites map to.
Mentions: We used DNase-seq to globally map chromatin accessibility at three key stages in the development of the mouse cerebellum: 1) postnatal day 7 (P7), when the external granular layer (EGL) of the mouse cerebellar cortex has reached its maximal thickness12 due to the proliferation of granule neuron precursors (GNPs) (Fig. 1a); 2) P14, when newborn postmitotic CGNs begin to populate the internal granule layer (IGL)13; and 3) P60, when CGNs of the IGL express gene products that mediate mature synaptic functions8 (Fig. 1b, Supplementary Fig. 1a, Supplementary Table 1). We identified approximately 70,000 DHS sites at each of the three developmental stages, and found that these elements mapped to promoters, gene bodies, and intergenic regions (Supplementary Fig. 1b). The majority of DHS sites (~77%) are found outside of annotated gene promoter regions, highlighting the ability of DNase-seq to identify distal regulatory elements in the genome.

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