Epigenetic regulation of proliferation and differentiation of hematopoietic stem cells

本研究旨在了解GM-CSF诱导CpG甲基化的过程。p15岛区域，我们研究了甲基化状态的基因在人GM-CSF依赖性巨核细胞白血病细胞系，MO7_c，使用亚硫酸氢盐基因组测序。本研究以MO7_c细胞为研究对象，观察了粒细胞-巨噬细胞集落刺激因子（GM-CSF）诱导p15 CpG岛甲基化的过程。在GM-CSF+胎牛血清（FBS）刺激下增殖的细胞p15 CpG岛几乎完全甲基化。将GM-CSF和IBS两者撤回48小时降低了细胞活力，并且使具有完全或部分去甲基化CpG位点的等位基因的频率增加了约50%。活细胞是造成这种表观遗传变化的原因。GM-CSF的回加恢复了甲基化。停用GM-CSF + FBS 72小时，然后暴露于DNA甲基转移酶（DNMT）和组蛋白脱乙酰酶（HDAC）抑制剂24小时， ...更多信息 在测序的每个等位基因上使用p15 CpG岛中几乎所有CpG位点的去甲基化。当在96小时处理后重新加入GM-CSF时，细胞通过甲基化表现出p15转录沉默。初始甲基化事件涵盖整个CpG岛。在DNMT和HDAC抑制剂共存的情况下，未出现新的甲基化等位基因。总之，GM-CSF可能能够使用11 DAC以及DNMT诱导p15基因的从头甲基化。在正常外周血/骨髓细胞和急性髓细胞性白血病（AML）中，调节编码CDK抑制剂的基因表达的组蛋白修饰尚不清楚。我们试图，第一次，阐明组蛋白修饰周围的CpG岛区域的p15基因在AML细胞中，使用染色质免疫沉淀（ChIP）试验和甲基化特异性PCR与亚硫酸氢盐基因组测序。11例AML患者中有7例具有等位基因，其中p15基因的27个CpG位点中超过一半被甲基化。AML患者骨髓细胞p15 CpG岛区域被乙酰化组蛋白H3（A_cH3）和二甲基化组蛋白H3-赖氨酸9（M_eH3K9）包围，而正常骨髓细胞仅被A_cH3包围。抗A_cH_3抗体和抗M_eH_3K_9抗体免疫沉淀的p15 CpG岛在患者中并不总是非甲基化和甲基化的。这些结果表明AML中p15 CpG岛区域周围组蛋白H3的扰动修饰。少

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