Setdb1-mediated H3K9 methylation is enriched on the inactive X and plays a role in its epigenetic silencing
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE66526 | GEO | 2016/05/18
SECONDARY ACCESSION(S): PRJNA277193
REPOSITORIES: GEO
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Project description:BackgroundThe presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored.ResultsHere we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements.ConclusionsHere we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome.
| S-EPMC4870784 | biostudies-literature
Project description:Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Setdb1 in mouse embryonic fibroblasts (MEF); (2) to profile key epigenetic marks H3K9me2, H3K9me3 and H3K27me3; utilizing wildtype cells with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Chromatin immunoprecipitation for Setdb1, H3K9me2, H3K9me3 and H3K27me3 was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked MEFs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Setdb1, H3K9me2, H3K9me3 and H3K27me3. DNA was extracted from the immunoprecipitated fraction following reverse-crosslinking. Isolated DNA was used to generate sequencing libraries with Illumina's TruSeq DNA Sample Preparation Kit according to manufacturer's instruction. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. Chromatin occupancy of the epigenetic regulator Setdb1, H3K9me2, H3K9me3 and H3K27me3 in mouse embryonic fibroblasts (MEFs) with wildtype MEFs and nonsilencing shRNA mediated knockdown or Setdb1 geneTrap heterozygous MEFs with Setdb1 shRNA mediated knockdown was determined by Setdb1, H3K9me2, H3K9me3 and H3K27me3 ChIP-seq, respectively.
2016-05-18 | E-GEOD-66522 | biostudies-arrayexpress
Project description:Purpose: The aim of this study was to profile the transcriptome of differentiating embryonic stem cells (ES) with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Female Setdb1+/+ XistâA/+ ES cells were induced to differentiate and RNA sampled on day 0, day 3 and day 5 of differentiation. RNAseq libraries were prepared with TruSeq RNA sample preparation v2 kit. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. Total RNA was extracted and purified from each cell type and their transcriptomes analysed by RNA-Seq.
2016-05-18 | E-GEOD-66523 | biostudies-arrayexpress
Project description:Purpose: The aim of this study was to determine the DNA methylation state of wildtype female mouse embryonic fibroblasts with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Enhanced Reduced Representation Bisulfite Libraries (eRRBS) were produced as previously descirbed (Akalin et al. 2012). Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads with dark cycle parameters (Boyle et al. 2012). Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. Determine the DNA methylation state of mouse embryonic fibroblasts (MEFs) with wildtype MEFs and nonsilencing shRNA mediated knockdown or Setdb1 geneTrap heterozygous MEFs with Setdb1 shRNA mediated knockdown. Single-end with dark cycle protocol (Boyle et al. 2012)
2016-05-18 | E-GEOD-66524 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2016-05-18 | E-GEOD-66526 | biostudies-arrayexpress
Project description:Purpose: The aim of this study was to determine the DNA methylation state of wildtype female mouse embryonic fibroblasts with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Enhanced Reduced Representation Bisulfite Libraries (eRRBS) were produced as previously descirbed (Akalin et al. 2012). Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads with dark cycle parameters (Boyle et al. 2012). Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data.
2016-05-18 | GSE66524 | GEO
Project description:Purpose: The aim of this study was to profile the transcriptome of differentiating embryonic stem cells (ES) with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Female Setdb1+/+ Xist∆A/+ ES cells were induced to differentiate and RNA sampled on day 0, day 3 and day 5 of differentiation. RNAseq libraries were prepared with TruSeq RNA sample preparation v2 kit. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data.
2016-05-18 | GSE66523 | GEO
Project description:Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Setdb1 in mouse embryonic fibroblasts (MEF); (2) to profile key epigenetic marks H3K9me2, H3K9me3 and H3K27me3; utilizing wildtype cells with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Chromatin immunoprecipitation for Setdb1, H3K9me2, H3K9me3 and H3K27me3 was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked MEFs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Setdb1, H3K9me2, H3K9me3 and H3K27me3. DNA was extracted from the immunoprecipitated fraction following reverse-crosslinking. Isolated DNA was used to generate sequencing libraries with Illumina's TruSeq DNA Sample Preparation Kit according to manufacturer's instruction. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data.
2016-05-18 | GSE66522 | GEO
Project description:Setdb1-mediated H3K9 methylation is enriched on the inactive X and plays a role in its epigenetic silencing
| PRJNA277193 | ENA
Project description:Epigenetic inheritance mechanisms play fundamental roles in maintaining cellular memory of gene expression states. In fission yeast, histone H3 lysine 9 (H3K9) is methylated (H3K9me) at heterochromatic domains. These domains can be epigenetically inherited when epe1+ , encoding an enzyme that promotes H3K9 demethylation, is deleted. How native epigenetic states are stably maintained in epe1+ cells remains unknown. Here, we developed a system to examine the role of DNA sequence and genomic context in propagation of a cis-heritable H3K9me-dependent silenced state. We show that in epe1+ cells, in addition to sequence-independent mechanisms that propagate H3K9me, epigenetic inheritance of silencing requires binding sites for sequence-dependent activating transcription factor (ATF)-adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) family transcription factors within their native chromosomal context. Thus, specific DNA sequences contribute to cis inheritance of H3K9me and silent epigenetic states.
| S-EPMC5718040 | biostudies-literature