Epigenetic regulation of proliferation and differentiation of hematopoietic stem cells

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In the present study, to understand the process of GM-CSF-induced methylation in the CpG. island region of p15, we examined the methylation status of the gene in a human GM-CSF-dependent megakaryoblastic leukemic cell line, MO7_c, using bisulfite genomic sequencing. The process of p15 CpG island methylation induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated, using MO7_c cells. The cells proliferating in response to GM-CSF plus fetal bovine serum (FBS) were almost fully methylated in the p15 CpG island. The withdrawal of both GM-CSF and IBS for 48 hours reduced the cell viability, and increased the frequency of alleles with completely or partially demethylated CpG sites by approximately 50%. Viable cells were responsible for this epigenetic change. The add-back of GM-CSF restored the methylation. Seventy-two hours withdrawal of GM-CSF plus FBS followed by 24 hours exposure to inhibitors for DNA methyltransferase (DNMT) and histone deacctylase (HDAC) ca … More used the demethylation of nearly all CpG sites in the p15 CpG island on every allele sequenced. When GM-CSF was re-added after the 96-hour treatment, the cells exhibited p15 transcriptional silencing via the methylation. The initial methylation event encompassed the entire CpG island. No new methylated alleles appeared in the coexistence of the DNMT and HDAC inhibitors. Taken together, GM-CSF may be able to induce de novo methylation of the p15 gene, using 11DAC(s) as well as DNMT(s).Histone modifications regulating expression of the genes which encode CDK inhibitors in normal peripheral blood/bone marrow cells and acute myeloblastic leukemia (AML) remain unclear. We attempted, for the first time, to elucidate histone modifications around the CpG island region of the p15 gene in AML cells, using the chromatin immunoprecipitation (ChIP) assay and methylation-specific PCR with bisulfite genomic sequencing. Seven of 11 patients with AML had allele(s) in which more than half of 27 CpG sites in the p15 gene were methylated. The p15 CpG island region was surrounded with both the acetylated histone H3 (A_cH3) and dimethylated histone H3-lysine 9 (M_eH3K9) in bone marrow cells of AML patients, whereas with A_cH3 alone in normal marrow cells. The p 15 CpG islands of DNA immunoprecipitated with anti-A_cH3 antibody and anti-M_eH3K9 antibody were not always unmethylated and methylated, respectively, in the patients. These results suggest perturbed modifications of histone H3 around the p15 CpG island region in AML. Less