Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Smchd1 dependent and independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome.

ABSTRACT: X chromosome inactivation involves multiple levels of chromatin modification, established progressively and in a stepwise manner during early development. The chromosomal protein Smchd1 has recently been shown to play an important role in DNA methylation of CpG islands (CGIs), a late step in the X inactivation pathway required for long-term stability of gene silencing. Here we show that inactive X chromosome (Xi) CGI methylation can occur via either Smchd1 dependent or independent pathways. Smchd1 dependent CGI methylation, the primary pathway, is acquired gradually over an extended period, whereas Smchd1 independent CGI methylation occurs rapidly after the onset of X inactivation. The de novo methyltransferase Dnmt3b is required for methylation of both classes of CGI, whereas Dnmt3a and Dnmt3L are dispensable. Xi CGIs methylated by these distinct pathways differ in respect to their sequence characteristics and immediate chromosomal environment. We discuss the implications of these results for understanding CGI methylation during development. Examination of CpG methylated DNA from wild type and smchd1 null male and female somatic cells and female ES cells differentiated for either 7 or 10 days. Duplicate samples of each type were generated.

ORGANISM(S): Mus musculus

SUBMITTER: Stephen Taylor

PROVIDER: E-GEOD-37333 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Smchd1-dependent and -independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome.

Gendrel Anne-Valerie AV Apedaile Anwyn A Coker Heather H Termanis Ausma A Zvetkova Ilona I Godwin Jonathan J Tang Y Amy YA Huntley Derek D Montana Giovanni G Taylor Steven S Giannoulatou Eleni E Heard Edith E Stancheva Irina I Brockdorff Neil N

Developmental cell 20120726 2

X chromosome inactivation involves multiple levels of chromatin modification, established progressively and in a stepwise manner during early development. The chromosomal protein Smchd1 was recently shown to play an important role in DNA methylation of CpG islands (CGIs), a late step in the X inactivation pathway that is required for long-term maintenance of gene silencing. Here we show that inactive X chromosome (Xi) CGI methylation can occur via either Smchd1-dependent or -independent pathways ...[more]

PMID: 22841499

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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.

Dataset Information

Smchd1 dependent and independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome.

Publications

Smchd1-dependent and -independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome.

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