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The chromatin Remodeler CHD8 is required for activation of progesterone receptor-dependent enhancers.

Ceballos-Chávez M, Subtil-Rodríguez A, Giannopoulou EG, Soronellas D, Vázquez-Chávez E, Vicent GP, Elemento O, Beato M, Reyes JC - PLoS Genet. (2015)

Bottom Line: We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI.Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs).Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Department, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain.

ABSTRACT
While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

No MeSH data available.


Related in: MedlinePlus

Genome-wide analysis of CHD8 binding sites under normal proliferation conditions.(A) Distribution of CHD8 peaks at low (P < 10–10), middle (P < 10–15), and high (P < 10–20) confidence thresholds, in proliferating T47D-MTVL cells relative to known RefSeq genes. Promoters: ± 2 kb around transcription start site (TSS); Downstream extremities: ± 2 kb around transcription end site; Exons: exonic regions; Introns: intronic regions; Intergenic > 2 kb away from RefSeq TSS. (B) Meta-gene representation of CHD8 ChIP-seq signal at the low confidence threshold. Log2 normalized ratios versus IgG signal are represented. (C) Genome Browser view of IgG, CHD8, H3K4me3 and RNAPII occupancy in a region of chromosome 8. Numbers in the y-axis are reads per million mapped reads. (D) CHD8 occupancy around the centre of the CHD8 binding sites at TSS (red), introns (black) or intergenic regions (blue). (E) Overlapping between CHD8 identified peaks at the low confidence threshold (12655), RNAPII peaks (46586) and H3K4me3 peaks (25210).
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pgen.1005174.g001: Genome-wide analysis of CHD8 binding sites under normal proliferation conditions.(A) Distribution of CHD8 peaks at low (P < 10–10), middle (P < 10–15), and high (P < 10–20) confidence thresholds, in proliferating T47D-MTVL cells relative to known RefSeq genes. Promoters: ± 2 kb around transcription start site (TSS); Downstream extremities: ± 2 kb around transcription end site; Exons: exonic regions; Introns: intronic regions; Intergenic > 2 kb away from RefSeq TSS. (B) Meta-gene representation of CHD8 ChIP-seq signal at the low confidence threshold. Log2 normalized ratios versus IgG signal are represented. (C) Genome Browser view of IgG, CHD8, H3K4me3 and RNAPII occupancy in a region of chromosome 8. Numbers in the y-axis are reads per million mapped reads. (D) CHD8 occupancy around the centre of the CHD8 binding sites at TSS (red), introns (black) or intergenic regions (blue). (E) Overlapping between CHD8 identified peaks at the low confidence threshold (12655), RNAPII peaks (46586) and H3K4me3 peaks (25210).

Mentions: To identify the genome-wide distribution of CHD8 in proliferating human breast cancer cells T47D-MTVL [28], we performed chromatin immunoprecipitation of CHD8 followed by deep sequencing (ChIP-seq). We found 12655, 4900 and 2500 peaks of CHD8 by using three different confidence threshold values, respectively (ChIPseeqer threshold level 10–10, 10–15 and 10–20). In all cases, false discovery rates (FDR) were lower than 0.03 (see Materials and Methods). At the lowest threshold about 48% of the peaks (6257) were located within promoters, with a strong enrichment around TSS (Fig 1A and 1B) and about 50% of the peaks were distributed between introns (2187) and intergenic regions (3748). Interestingly, if confidence threshold for peaks identification is increased, the percentage of peaks associated with promoters raise to about 78% (Fig 1A). This is due to differences in CHD8 signal intensity. In fact, CHD8 signals at TSSs are higher than at intergenic or intronic regions (Fig 1C and 1D). Therefore, when only highly significant peaks are analyzed most of them map at promoters. Consistently, a strong overlap between CHD8 and RNA polymerase II (RNAPII) or H3K4me3 ChIP-seq signals was observed (Fig 1C and 1E). CHD8 occupancy was confirmed by ChIP-qPCR in three selected target promoters (CCND1, HDS11B2 and CCNE2) using a different anti-CHD8 antibody (S1A Fig). Furthermore, as control, we also verified that knockdown of CHD8 decreased ChIP-qPCR signal (S1B Fig). All these experiments validated our ChIP-seq results. The rest of the analysis was performed using the 12655 CHD8 sites identified at the lowest, but still very significant, threshold. We have previously reported that CHD8 binds 1965 promoters in a ChIP-on-chip analysis of proliferating cervical carcinoma C33 cells [17]. Approximately 60% of the promoters identified as CHD8 targets by ChIP-on-chip (1175) were also identified by ChIP-seq, despite the different cell lines used. CHD8 bound genes were enriched in Gene Ontology categories related to macromolecular biosynthetic processes (transcription, mRNA processing) and cell cycle (S2A Fig). Consistently with our previous data from C33 cells [17] CHD8 target promoters were strongly enriched in E2F (p-value = 3.6 × 10–135), ELK-1 (p-value = 2.2 × 10–122), AP-2 (p-value = 4.7 × 10–100), and SP1 (p-value = 4.2 × 10–80) transcription factors binding sites (S2B Fig). Taken together these results indicate that CHD8 is mostly bound to promoters in proliferating T47D-MTVL cells.


The chromatin Remodeler CHD8 is required for activation of progesterone receptor-dependent enhancers.

Ceballos-Chávez M, Subtil-Rodríguez A, Giannopoulou EG, Soronellas D, Vázquez-Chávez E, Vicent GP, Elemento O, Beato M, Reyes JC - PLoS Genet. (2015)

Genome-wide analysis of CHD8 binding sites under normal proliferation conditions.(A) Distribution of CHD8 peaks at low (P < 10–10), middle (P < 10–15), and high (P < 10–20) confidence thresholds, in proliferating T47D-MTVL cells relative to known RefSeq genes. Promoters: ± 2 kb around transcription start site (TSS); Downstream extremities: ± 2 kb around transcription end site; Exons: exonic regions; Introns: intronic regions; Intergenic > 2 kb away from RefSeq TSS. (B) Meta-gene representation of CHD8 ChIP-seq signal at the low confidence threshold. Log2 normalized ratios versus IgG signal are represented. (C) Genome Browser view of IgG, CHD8, H3K4me3 and RNAPII occupancy in a region of chromosome 8. Numbers in the y-axis are reads per million mapped reads. (D) CHD8 occupancy around the centre of the CHD8 binding sites at TSS (red), introns (black) or intergenic regions (blue). (E) Overlapping between CHD8 identified peaks at the low confidence threshold (12655), RNAPII peaks (46586) and H3K4me3 peaks (25210).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403880&req=5

pgen.1005174.g001: Genome-wide analysis of CHD8 binding sites under normal proliferation conditions.(A) Distribution of CHD8 peaks at low (P < 10–10), middle (P < 10–15), and high (P < 10–20) confidence thresholds, in proliferating T47D-MTVL cells relative to known RefSeq genes. Promoters: ± 2 kb around transcription start site (TSS); Downstream extremities: ± 2 kb around transcription end site; Exons: exonic regions; Introns: intronic regions; Intergenic > 2 kb away from RefSeq TSS. (B) Meta-gene representation of CHD8 ChIP-seq signal at the low confidence threshold. Log2 normalized ratios versus IgG signal are represented. (C) Genome Browser view of IgG, CHD8, H3K4me3 and RNAPII occupancy in a region of chromosome 8. Numbers in the y-axis are reads per million mapped reads. (D) CHD8 occupancy around the centre of the CHD8 binding sites at TSS (red), introns (black) or intergenic regions (blue). (E) Overlapping between CHD8 identified peaks at the low confidence threshold (12655), RNAPII peaks (46586) and H3K4me3 peaks (25210).
Mentions: To identify the genome-wide distribution of CHD8 in proliferating human breast cancer cells T47D-MTVL [28], we performed chromatin immunoprecipitation of CHD8 followed by deep sequencing (ChIP-seq). We found 12655, 4900 and 2500 peaks of CHD8 by using three different confidence threshold values, respectively (ChIPseeqer threshold level 10–10, 10–15 and 10–20). In all cases, false discovery rates (FDR) were lower than 0.03 (see Materials and Methods). At the lowest threshold about 48% of the peaks (6257) were located within promoters, with a strong enrichment around TSS (Fig 1A and 1B) and about 50% of the peaks were distributed between introns (2187) and intergenic regions (3748). Interestingly, if confidence threshold for peaks identification is increased, the percentage of peaks associated with promoters raise to about 78% (Fig 1A). This is due to differences in CHD8 signal intensity. In fact, CHD8 signals at TSSs are higher than at intergenic or intronic regions (Fig 1C and 1D). Therefore, when only highly significant peaks are analyzed most of them map at promoters. Consistently, a strong overlap between CHD8 and RNA polymerase II (RNAPII) or H3K4me3 ChIP-seq signals was observed (Fig 1C and 1E). CHD8 occupancy was confirmed by ChIP-qPCR in three selected target promoters (CCND1, HDS11B2 and CCNE2) using a different anti-CHD8 antibody (S1A Fig). Furthermore, as control, we also verified that knockdown of CHD8 decreased ChIP-qPCR signal (S1B Fig). All these experiments validated our ChIP-seq results. The rest of the analysis was performed using the 12655 CHD8 sites identified at the lowest, but still very significant, threshold. We have previously reported that CHD8 binds 1965 promoters in a ChIP-on-chip analysis of proliferating cervical carcinoma C33 cells [17]. Approximately 60% of the promoters identified as CHD8 targets by ChIP-on-chip (1175) were also identified by ChIP-seq, despite the different cell lines used. CHD8 bound genes were enriched in Gene Ontology categories related to macromolecular biosynthetic processes (transcription, mRNA processing) and cell cycle (S2A Fig). Consistently with our previous data from C33 cells [17] CHD8 target promoters were strongly enriched in E2F (p-value = 3.6 × 10–135), ELK-1 (p-value = 2.2 × 10–122), AP-2 (p-value = 4.7 × 10–100), and SP1 (p-value = 4.2 × 10–80) transcription factors binding sites (S2B Fig). Taken together these results indicate that CHD8 is mostly bound to promoters in proliferating T47D-MTVL cells.

Bottom Line: We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI.Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs).Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Department, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain.

ABSTRACT
While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

No MeSH data available.


Related in: MedlinePlus