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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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CRISPR-VP64 based activation confirms enhancer activity of late-opening DHS sites nearby Grin2c(a) DNase-seq and H3K27ac ChIP-seq signal in vicinity of Grin2c with two DHS sites that open across development and increase in H3K27ac signal marked in gray. (b) RNA-seq expression of Grin2c across developmental time in vivo and in culture. BDNF = Brain-derived neurotrophic factor. Error bars = 95% CI. (c) Cultured CGNs were either left uninfected (none), or were infected with dCas9-VP64 activator, three gRNAs targeting site #1 from (a), three gRNAs targeting site #2, or a combination of dCas9-VP64 and one of the two sets of gRNAs on +1DIV, and harvested for qPCR on +7DIV. All data normalized to expression of Gapdh and scaled to average expression in control conditions (dCas9-VP64 or gRNAs only). P = 0.0028 for site 1 and P = 0.00022 for site 2 by two-sided Student’s t-test for dCas9-VP64 plus gRNAs vs. dCas9-VP64 or gRNAs alone (n = 5 for dCas9-VP64 with gRNAs, n = 8 for site 1 controls, n = 10 for site 2 controls; t = 5.5 for site 1, t = 12.3 for site 2). Error bars = s.e.m.
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Figure 3: CRISPR-VP64 based activation confirms enhancer activity of late-opening DHS sites nearby Grin2c(a) DNase-seq and H3K27ac ChIP-seq signal in vicinity of Grin2c with two DHS sites that open across development and increase in H3K27ac signal marked in gray. (b) RNA-seq expression of Grin2c across developmental time in vivo and in culture. BDNF = Brain-derived neurotrophic factor. Error bars = 95% CI. (c) Cultured CGNs were either left uninfected (none), or were infected with dCas9-VP64 activator, three gRNAs targeting site #1 from (a), three gRNAs targeting site #2, or a combination of dCas9-VP64 and one of the two sets of gRNAs on +1DIV, and harvested for qPCR on +7DIV. All data normalized to expression of Gapdh and scaled to average expression in control conditions (dCas9-VP64 or gRNAs only). P = 0.0028 for site 1 and P = 0.00022 for site 2 by two-sided Student’s t-test for dCas9-VP64 plus gRNAs vs. dCas9-VP64 or gRNAs alone (n = 5 for dCas9-VP64 with gRNAs, n = 8 for site 1 controls, n = 10 for site 2 controls; t = 5.5 for site 1, t = 12.3 for site 2). Error bars = s.e.m.

Mentions: Next, to directly test the hypothesis that opening DHS sites represent enhancer elements in their native genomic context, we examined the locus surrounding the Grin2c gene on chromosome 11 in detail (Fig. 3a). Within this interval we identified two DHS sites that both open and gain H3K27ac signal across postnatal development – one just upstream of the Grin2c promoter and the second in an intron of the nearby Tmem104 gene. Of the four annotated genes in this region (Tmem104, Grin2c, Fdxr, Fads6), all are expressed in cerebellum, but only Grin2c expression is highly upregulated in differentiating CGNs, where it is a NMDA-type glutamate receptor subunit that mediates mature synaptic functions19 (Fig. 3b, Supplementary Table 6). Grin2c expression can also be robustly induced by culturing CGNs for seven days (+7DIV) in the presence of Brain-Derived Neurotrophic Factor (BDNF), offering an opportunity to experimentally test transcriptional mechanisms of Grin2c regulation28. Notably, BDNF-induced gene expression changes occur largely independent of changes in chromatin accessibility as we observed only 33 opened and 6 closed DHS sites in response to BDNF exposure (Supplementary Table 7).


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)

CRISPR-VP64 based activation confirms enhancer activity of late-opening DHS sites nearby Grin2c(a) DNase-seq and H3K27ac ChIP-seq signal in vicinity of Grin2c with two DHS sites that open across development and increase in H3K27ac signal marked in gray. (b) RNA-seq expression of Grin2c across developmental time in vivo and in culture. BDNF = Brain-derived neurotrophic factor. Error bars = 95% CI. (c) Cultured CGNs were either left uninfected (none), or were infected with dCas9-VP64 activator, three gRNAs targeting site #1 from (a), three gRNAs targeting site #2, or a combination of dCas9-VP64 and one of the two sets of gRNAs on +1DIV, and harvested for qPCR on +7DIV. All data normalized to expression of Gapdh and scaled to average expression in control conditions (dCas9-VP64 or gRNAs only). P = 0.0028 for site 1 and P = 0.00022 for site 2 by two-sided Student’s t-test for dCas9-VP64 plus gRNAs vs. dCas9-VP64 or gRNAs alone (n = 5 for dCas9-VP64 with gRNAs, n = 8 for site 1 controls, n = 10 for site 2 controls; t = 5.5 for site 1, t = 12.3 for site 2). Error bars = s.e.m.
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Figure 3: CRISPR-VP64 based activation confirms enhancer activity of late-opening DHS sites nearby Grin2c(a) DNase-seq and H3K27ac ChIP-seq signal in vicinity of Grin2c with two DHS sites that open across development and increase in H3K27ac signal marked in gray. (b) RNA-seq expression of Grin2c across developmental time in vivo and in culture. BDNF = Brain-derived neurotrophic factor. Error bars = 95% CI. (c) Cultured CGNs were either left uninfected (none), or were infected with dCas9-VP64 activator, three gRNAs targeting site #1 from (a), three gRNAs targeting site #2, or a combination of dCas9-VP64 and one of the two sets of gRNAs on +1DIV, and harvested for qPCR on +7DIV. All data normalized to expression of Gapdh and scaled to average expression in control conditions (dCas9-VP64 or gRNAs only). P = 0.0028 for site 1 and P = 0.00022 for site 2 by two-sided Student’s t-test for dCas9-VP64 plus gRNAs vs. dCas9-VP64 or gRNAs alone (n = 5 for dCas9-VP64 with gRNAs, n = 8 for site 1 controls, n = 10 for site 2 controls; t = 5.5 for site 1, t = 12.3 for site 2). Error bars = s.e.m.
Mentions: Next, to directly test the hypothesis that opening DHS sites represent enhancer elements in their native genomic context, we examined the locus surrounding the Grin2c gene on chromosome 11 in detail (Fig. 3a). Within this interval we identified two DHS sites that both open and gain H3K27ac signal across postnatal development – one just upstream of the Grin2c promoter and the second in an intron of the nearby Tmem104 gene. Of the four annotated genes in this region (Tmem104, Grin2c, Fdxr, Fads6), all are expressed in cerebellum, but only Grin2c expression is highly upregulated in differentiating CGNs, where it is a NMDA-type glutamate receptor subunit that mediates mature synaptic functions19 (Fig. 3b, Supplementary Table 6). Grin2c expression can also be robustly induced by culturing CGNs for seven days (+7DIV) in the presence of Brain-Derived Neurotrophic Factor (BDNF), offering an opportunity to experimentally test transcriptional mechanisms of Grin2c regulation28. Notably, BDNF-induced gene expression changes occur largely independent of changes in chromatin accessibility as we observed only 33 opened and 6 closed DHS sites in response to BDNF exposure (Supplementary Table 7).

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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