Unknown,Transcriptomics,Genomics,Proteomics

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The role of SMCHD1 in the formation of constitutive heterochromatin

ABSTRACT: A large portion of the mammalian genome is assembled into constitutive heterochromatin which is highly compact and transcriptionally silent throughout the cell cycle. On the molecular level it is characterized by DNA methylation, “repressive” histone marks (hypoacethylation, H3K9 trimethylation, H4K20 trimethylation) and the presence of heterochromatin protein 1 (HP1). Regions of constitutive heterochromatin such as telomeres, pericentromeres and centromeres play a critical role in the maintenance of the genome integrity. Using a technique called Proteomics of Isolated Chromatin Segments (PICh) we have identified SMCHD1 as a novel component of telomeres and pericentomeres. SMCHD1 was previously shown to be involved in the inactivaction of the X chromosome and imprinting, but the its exact role in these processes is not understood. Our study aims to unravel the role of SMCHD1 in the formation and/or maintenance of constitutive chromatin. Using human cancer cells as research model we are trying to discover its mechanism of action by identifying its interacting partners, characterizing its genome wide binding sites and characterizing the effect of SMCHD1 knockout on the heterochromatin function. Characterization of SMCHD1 binding sites in HCT-116 cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Jérôme Déjardin

PROVIDER: E-GEOD-46462 | 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.

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The role of SMCHD1 in the formation of constitutive heterochromatin

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