Genome-wide binding and mechanistic analyses of Smchd1 mediated epigenetic regulation
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Ruijie Liu
PROVIDER: E-GEOD-65749 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain; (2) to profile key epigenetic marks H3K4me3, H3K27me3 and DNA methylation in wild type and Smchd1 null NSCs; (3) to identify the chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs. Methods: Chromatin immunoprecipitation for Smchd1, H3K4me3, H3K27me3 and Ctcf was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked NSCs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Smchd1, H3K4me3 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 or Ovation Ultralow system (NuGen) 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. To examine the level of DNA methylation, genomic DNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen) and methylated DNA was isolated via binding to the methyl-CpG binding domain of human MBD2 protein coupled beads using the MethylMiner methylated DNA enrichment kit (Life Technologies) according to the manufacturer’s instructions. Isolated DNA was used to generate sequencing libraries as for the ChIP-seq experiment with Illumina’s TruSeq DNA Sample Preparation Kit according to manufacturer's instruction and sequenced on the Illumina HiSeq 2000 platform for 49 bp single-end reads. Sequencing analysis was performed as described for the ChIP-seq experiments. Chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain was determined by Smchd1 ChIP-seq. Enrichment of H3K4me3 and H3K27me3 in wild type and Smchd1 null NSCs were assessed by H3K4me3 and H3K27me3 ChIP-seq, respectively. DNA methylation in wild type and Smchd1 null NSCs was assessed by MBD-seq. Chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs was determined by by Ctcf ChIP-seq.
2015-06-11 | E-GEOD-65748 | biostudies-arrayexpress
Project description:Purpose: The aim of this study is to identify genes that are under the transcriptional control of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain Methods: Total RNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen) from cultured neural stem cells derived from male mouse E14.5 brains either wild-type or null for Smchd1. 1 µg total RNA was used to generate sequencing libraries for whole transcriptome analysis with Illumina’s TruSeq RNA Sample Preparation Kit v2 as per standard protocols. Libraries were sequenced on HiSeq 2000 with Illumina TruSeq SBS Kit v3-HS reagents as either 100 bp single-end or paired-end reads at the Australian Genome Research Facility (AGRF), Melbourne. Reads were aligned to the mouse reference genome mm10 and mapped to known genomic features at the gene level using the Rsubread package (version 1.10.5) (Liao et al. 2013). Mapped reads were then summarized into gene-level counts using FeatureCounts (Liao et al. 2014). Total RNA was extracted and purified from each cell line and their transcriptomes analyzed by RNA-Seq.
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Project description:Tissue fibrosis is a serious complication of Crohnâs disease (CD) as well as of a variety of other complex, chronic pathologies. Understanding the underlying pathophysiology of tissue fibrosis is crucial for the development of tissue-specific prevention and interventional treatment strategies. To identify molecular states specific to fibrotic disease, we employed deep sequencing to define the genome-wide DNA methylome and the whole transcriptome of purified human intestinal fibrotic fibroblasts (HIFs) isolated from the colon of patients with fibrotic CD. Integration of this information, via computational tools, identified candidate molecular interactions that could lead to fibrosis pathology. Our definition of a genome-wide fibrosis-specific DNA methylome provides a new paradigm for understanding mechanisms of pathological gene expression that lead to intestinal fibrosis and may have relevance to fibrogenesis in other organs. Human intestinal fibroblasts (HIFs) were extracted and cultured from colon specimens of two groups: Crohnâs disease with associated fibrosis (n=3) and normal fibroblasts from patients with Diverticulitis (n=3). Both RNA-seq and MBD-isolated genome sequencing (MiGS) were performed on every sample.
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Project description:HDAC inhibitors are thought to regulate gene expression by post-translational modification of histone as well as non-histone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action, however, little is known of the extent of genome-wide changes of the mammalian genome when stimulated by the hydroxamic acids, TSA and SAHA. In primary human vascular endothelial cells we map the chromatin modifications, histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation mediated gene expression is often associated with modification of other lysine residues we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). Genome-wide mRNA sequencing indicates the differential expression of about 30% of genes, with almost equal numbers being up- and down- regulated. We observe deacetylation conferred by TSA and SAHA that are associated with decreased gene expression. Histone deacetylation is associated with the loss of p300/CBP binding at gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation. HAEC MethylMiner profiles of TSA treated and control samples were generated by deep sequencing, in triplicate, using Illumina GAIIx.
2014-02-23 | E-GEOD-54911 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-11-04 | E-GEOD-38217 | biostudies-arrayexpress
Project description:DNA methylation is thought to induce a transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators that do not recognize their binding sites when methylated, and the recruitment of transcriptional repressors that specifically bind methylated DNA. Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. However, the exact contribution of each protein in the DNA methylation dependent transcriptional repression occurring during development and diseases remains elusive. Here we present MBD2 ChIPseq data generated from the endogenous protein in an isogenic cellular model of human mammary oncogenic transformation. In immortalized or transformed cells, MBD2 was found in one fourth of methylated regions and associated with transcriptional silencing. Depletion of MBD2 induces upregulations of genes bound by MBD2 and methylated in their transcriptional start site regions. MBD2 was partially redistributed on methylated DNA during oncogenic transformation, independently of DNA methylation changes. Genes downregulated during this transformation preferentially gained MBD2 binding sites on their promoter. Depletion of MBD2 in transformed cells induced the upregulation of some of these repressed genes, independently of the strategy used for the abrogation of oncosuppressive barriers. Our data confirm that MBD2 is a major interpret of DNA methylation, and show an unreported dynamic in this interpretation during oncogenic transformation. Methylated DNA precipitation (MeDP) and Endogenous MBD2 Chromatin Immunoprecipitation (MBD2 ChIP) followed by hight-througput sequencing in HMEC-hTERT and HMLER cells.
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Project description:In mammals, dosage compensation for the sex chromosomes is achieved by transcriptional silencing of one of the two X chromosomes in females. The inactive X adopts a particular epigenetic state, characterised by specific histones, histone marks, DNA methylation and 3D chromatin structure. As allelic resolution with short-read sequencing is limited, we do not yet have chromosome-wide phased methylomes of the active and inactive X. In this study, we obtained such complete X methylomes in mouse placenta and neural stem cells (NSCs) via long-read nanopore sequencing. This accession corresponds to the RNA-seq for the NSCs.
2024-10-31 | E-MTAB-14486 | biostudies-arrayexpress
Project description:DNA methylation and hydroxymethylation have been implicated in normal development and differentiation, but our knowledge about the genome-wide distribution of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) during cellular differentiation remains limited. Using in vitro model system of gradual differentiation of human embryonic stem (hES) cells into ventral midbrain-type neural precursor (NP) cells and terminally into dopamine (DA) neurons, we explored changes in 5mC or 5hmC patterns during lineage commitment. We used three techniques, 450K DNA methylation array, MBD-seq, and hMeDIP-seq, and found combination of these methods can provide comprehensive information on the genome-wide 5mC or 5hmC patterns. We observed dramatic changes of 5mC patterns during differentiation of hES cells into NP cells. Although genome-wide 5hmC distribution was more stable than 5mC, coding exons, CpG islands and shores showed dynamic 5hmC patterns during differentiation. In addition to the role of DNA methylation as a mechanism to initiating gene silencing, we also found DNA methylation as a locking system to maintain gene silencing. More than 1,000 genes including mesoderm development related genes acquired promoter methylation during neuronal differentiation even though they were already silenced in hES cells. Finally, we found that activated genes lost 5mC in transcription start site (TSS) but acquired 5hmC around TSS and gene body during differentiation. Our findings may provide clues for elucidating the molecular mechanisms underlying lineage specific differentiation of pluripotent stem cells during human embryonic development. Examination of genome-wide DNA methylation in 3 cell types (human embryonic stem, neural precursor, and dopamine neuron cells)
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