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MEK and GSK3β inhibition drives ES cells into a naive pluripotent state irrespective of their global methylation status

ABSTRACT: We carry out methyl DNA immunoprecipitation (meDIP), hydroxymethyl DNA immunoprecipitaion (hmeDIP) and H3K27me3 chromatin imminoprecipitation (ChIP) prior to sequencing on the Ion Proton semiconductor sequencing platform to report on the genome-wide epigenetic patterns in J1 mESC and mutant mESCs (hyper active DNMT3b/3l or DNMT1/3a/3b triple knock out) grown under serum conditions or in 2i meadia for 14 days

ORGANISM(S): Mus musculus

PROVIDER: GSE72555 | GEO | 2019/09/24

REPOSITORIES: GEO

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Genome wide 5hmC patterns in the mouse liver, embryonic fibroblasts and induced pluripontent stem cells.

Project description:We carry out hydroxymethyl DNA immunoprecipitation (hmeDIP) prior to sequencing on the Ion Proton semiconductor sequencing platform to demonstate the strategy as an alternative method to Illumina sequencing.

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Genome-wide epigenetic maps in the liver from male and female Wistar and Sprague-Daley rats

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MEK and GSK3β inhibition drives ES cells into a naive pluripotent state irrespective of their global methylation status

Project description:Mouse embryonic stem cells (mESCs) can convert to a ground state by dual inhibition of MEK and GSK3β signalling in defined media in the presence of LIF (2i). The cellular transition in 2i leads to uniform expression of pluripotency markers and global DNA hypomethylation. Whether hypomethylation is required for achieving ground state or is an outcome of the conversion process is still not clear. Here we show that J1 wild type and hypomethylated mESCs (dnmt3a-/-, dnmt3b-/-, dnmt1-/- [TKO]) lacking DNA methyltransferases undergo similar morphological and expression changes upon culturing in 2i that are consistent with conversion of both to a more naive state. Maintenance of global DNA methylation levels in 2i media by constitutive expression of de novo methyltransferases is also not a barrier to conversion. Hence, signalling pathways in mES cells regulate pluripotency networks and cell state independently of their global DNA methylation status. Indicated mESCs were cultured in 2i/LIF for 2 weeks and then harvested along with serum/LIF counterparts for microarray analysis. 1ug of total RNA was used to prepare Cy3 labelled aRNA using an Amino Allyl MessageAmp II aRNA kit (Ambion) following manufacturers protocol. The samples were hybridised to SurePrint G3 mouse GE 8x60k microarrays (Agilent) and were scanned using NimbleGen MS 200 (Roche). Results were analysed with custom-written scripts implemented in the statistical programming language R (http://www.R-project.org).

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3D genome organisation in Yeast with or without induced DNA methylation

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2020-09-30 | E-MTAB-9257 | biostudies-arrayexpress

Comparative high density microarray analysis of global 5hmC patterns in mouse brain and liver following independent affinity based enrichment protocols.

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RNA-Seq of WT and constitutively methylated mESCs

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Identify genomic binding sites of Tfcp2l1 in mouse embryonic stem cells grown in 2i medium

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2021-03-01 | GSE159877 | GEO

DNA IP for 5hmC modified CpGs in the liver of control mice and a Tet1 -/- mouse liver [hmeDIP-seq]

Project description:Through the analysis of mouse liver tumours promoted by distinct routes (DEN exposure alone, DEN exposure plus non-genotoxic insult with phenobarbital and non-alcoholic fatty liver disease); we report that the cancer associated hyper-methylated CGI events in mice are also predicated by silent promoters that are enriched for both the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone modification H3K27me3 in normal liver. During cancer progression these CGIs undergo hypo-hydroxymethylation, prior to subsequent hyper-methylation; whilst retaining H3K27me3. A similar loss of promoter-core 5hmC is observed in Tet1 deficient mouse livers indicating that reduced Tet1 binding at CGIs may be responsible for the epigenetic dysregulation observed during hepatocarcinogenesis. Consistent with this reduced Tet1 protein levels are observed in mouse liver tumour lesions. As in human, DNA methylation changes at CGIs do not appear to be direct drivers of hepatocellular carcinoma progression in mice. Instead dynamic changes in H3K27me3 promoter deposition are strongly associated with tumour-specific activation and repression of transcription. Our data suggests that loss of promoter associated 5hmC in diverse liver tumours licences DNA methylation reprogramming at silent CGIs during cancer progression. We carry out 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) prior to sequencing Ion Proton P1 to report on the genome-wide 5hmC patterns. Heterozygote pairs of Tet1 B6;129S4-Tet1tm1.1Jae/J mice were bought from The Jackson Laboratory (Maine USA). Heterozygotes were interbred to produce homozygous knock out males with colony mate wild type controls. Genome-wide 5hmC patterns were generated by hydroxymethyl-DNA immuoprecipitation (hmeDIP) followed by genome wide sequencing on the Ion Proton P1 sequencer.

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