Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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DNA methylation changes induced by overexpression of IDH1mut or treatment with 2HG in the sorted mouse bone marrow cells

ABSTRACT: Mutations in the enzymes IDH1 and IDH2 have been identified in a wide variety of tumors like glioma, chondrosarcoma, thyroid cancer, lymphoma, melanoma, and in acute myeloid leukemia. Mutated IDH1/2 produces the metabolite 2-hydroxyglutarate (2HG), which interferes with epigenetic regulation of gene expression, and thus may promote tumorigenesis. HoxA9 immortalised bone marrow cells from C57BJ/6 mice were tranduced with either empty vector or pSF91-IDH1wt-IRES-EGFP, or pSF91-IDH1mut-IRES-GFP and transplanted in irradiated recipient mice. Alternatively, HoxA9 immortalised cells transduced with empty vector transplanted mice were treated with R-2HG at dose of 1mg/day for four weeks. Four weeks after transplantation/treatment GFP+ cells were sorted from mouse bone marrow, from which total DNA was extracted and subjected to Reduced Representation Bisulfite Sequencing (RRBS) to determine the genome-wide methylation signature.

ORGANISM(S): Mus musculus

SUBMITTER: Anuhar Chaturvedi

PROVIDER: E-GEOD-77828 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The activation-induced cytidine deaminase enzyme (AID) is required in germinal center (GC) B cells for somatic hyper-mutation and class switch recombination at the immunoglobulin locus. In GC-B cells, AID is highly expressed, with inherent mutator activity that helps generate antibody diversity. However, AID may also regulate gene expression epigenetically, irrespective of mutator activity, by directly deaminating 5-methylcytosine (5mC) in concert with base excision repair glycosylases to exchange unmethylated cytosine. This pathway promotes gene demethylation, thereby removing epigenetic memory. For example, AID promotes active demethylation of the genome in primordial germ cells. However, the range and mechanism by which AID promotes pluripotency is not known. Different studies have suggested either a requirement or a lack of function for promoting pluripotency in somatic nuclei following fusion with embryonic stem cells (ESC). Here we tested directly whether AID regulates epigenetic memory, by comparing the relative ability of cells lacking AID to reprogram from a differentiated cell type to an induced pluripotent stem cell (iPSC). We show that loss of AID impacts two distinct steps of reprogramming: First, AID-null cells are transiently hyper-responsive to the reprogramming process. Second, although they initiate expression of pluripotency genes, they fail to stabilize the pluripotent state. The genome of AID-null cells remains hypermethylated in reprogramming cells, and hypermethylated genes associated with pluripotency fail to be stably up-regulated. MYC target genes are highly enriched in the set of genes hypermethylated and under-expressed in reprogramming cells lacking AID. Recent studies identified a distinctive late step of reprogramming associated with methylation status. AID appears to regulate this step to stabilize the pluripotent state, removing epigenetic memory to promote expression of secondary pluripotency network genes. Transcriptome sequencing of AID-null tail fibroblasts, wildtype tail fibroblasts, AID-null and wildtype tail fibroblasts reprogrammed for three weeks by ectopic expression of transcription factors Oct4, Sox2, KLf4 and cMyc. Methylation profiling by reduced representation bisulphite seuencing of AID-null tail fibroblasts, wildtype tail fibroblasts, AID-null and wildtype tail fibroblasts reprogrammed for three weeks and AID-null and wildtype clones after three weeks of reprogramming (Picked at two weeks)

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DNA methylation changes induced by overexpression of IDH1mut or treatment with 2HG in the sorted mouse bone marrow cells

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