Epigenetic regulation of p15 mRNA expression in juvenile myelomonocytic

青少年粒单核细胞白血病（JMML）是一种罕见的骨髓增生性疾病，发生在婴儿和儿童早期。Emanuel等提出，JMML骨髓增殖的主要机制是GM祖细胞对粒细胞-巨噬细胞集落刺激因子（GM-CSF）的超敏反应。在我们以前的研究中，JMML GM祖细胞对GM-CSF+干细胞因子显示出良好的反应。虽然1/3的JMML患者存在染色体数目畸变，但结构异常如缺失和易位是罕见的。在本研究中，我们检测了GM-CSF依赖型JMML髓系发育过程中p15甲基化和表达的动力学，我们的甲基化特异性聚合酶链反应和测序显示，正常的CD 34 ^+骨髓细胞在p15 CpG岛完全未甲基化，而6例JMML患者中有2例的部分CD 34 ^+骨髓细胞有甲基化的CpG位点。GM-CSF刺激7天诱导甲基化 ...更多信息 在正常骨髓细胞中的频率为40%到70%。相比之下，在所有6名JMML患者中，第7天髓样细胞几乎没有甲基化CpG位点。实时荧光定量PCR分析显示，JMML患者p15 mRNA水平显著高于正常对照组。在流式细胞术分析中，观察到更高水平的细胞内p15蛋白含量在第7天与GM-CSF生长的骨髓细胞从一个病人与JMML患者，当从正常对照组中获得的值相比，在第7天与GM-CSF产生的骨髓细胞表达的DNMT 1，DNMT 3a的mRNA在JMML患者的显着高于正常对照组的程度，根据实时PCR分析。DNMT 3b是两种细胞中含量最低的DNMT。核蛋白复合物经超声处理后，DNA片段大小降至约1kb。然后，洗脱基因组DNA的乙酰基-组蛋白H3和H4富集级分，并用p15启动子区的引物（相对于转录起始点为-49至+270）通过PCR进行分析。300bp的PCR产物从富含乙酰组蛋白H3和乙酰组蛋白H4的基因组DNA中扩增出来，这些基因组DNA来自与来自正常CD 34 ^+骨髓细胞的GM-CSF一起生长的第7天培养的髓样细胞。同时，从抗甲基-H3-K9 Ab富集级分扩增p15启动子。相反，在JMML中，从抗甲基组蛋白H3-K9 Ab富集的基因组DNA级分中未检测到p15 PCR产物。与p15 CpG岛相关的组蛋白H3和H4均被乙酰化，提示在该疾病中存在p15 mRNA表达的表观遗传失调。少

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disorder that occurs in infancy and early childhood. Emanuel et al. proposed that the primary mechanism for the myeloproliferation in JMML is hypersensitivity of the GM progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF). In our previous study, JMML GM progenitors showed the favorable response to GM-CSF plus stem cell factor. While one-third of JMML patients have numerical aberration of karyotype, but structural abnormalities such as deletion and translocation are rare. In the present study, we examined the kinetics of p15 methylation and expression during GM-CSF dependent-myeloid development in JMML.Our methylation-specific polymerase chain reaction and sequencing showed that normal CD34^+ bone marrow cells were completely unmethylated at the p15 CpG island, whereas part of CD34^+ bone marrow cells had methylated CpG sites in 2 of 6 JMML patients. GM-CSF stimulation for 7 days induced methylation … More at a frequency of 40% to 70% on normal bone marrow cells. In contrast, Day 7-myeloid cells were nearly devoid of methylated CpG sites in all of 6 JMML patients. Real-time PCR analysis revealed that p15 mRNA levels were significantly higher in JMML patients than in normal controls. In flow cytometric analysis, a greater level of intracellular p15 protein content was observed in Day 7 myeloid cells grown with GM-CSF from a patient with JMML, when compared with the value obtained from normal control.Day 7 myeloid cells generated with GM-CSF expressed mRNA for DNMT1, DNMT3a in JMML patients to a significantly higher extent than normal controls, according to real-time PCR analyses. DNMT3b was the least abundant DNMT in the both cells.Nucleoprotein complexes were sonicated to reduce the size of DNA fragments to approximately 1 kb. Then, the acetyl-histone H3 and H4 enriched fraction of genomic DNA was eluted, and analyzed by PCR with the primers for the p15 promoter region (from -49 to +270 relative to the transcriptional starting point). The 300 by PCR product was amplified from both the acetyl-histone H3 and acetyl-histone H4 enriched genomic DNA from Day 7 cultured myeloid cells grown with GM-CSF from normal CD34^+ bone marrow cells. Simultaneously, the p15 promoter was amplified from anti-methyl-H3-K9 Ab enriched fraction. In contrast, p15 PCR product was not detected from anti-methyl-histone H3-K9 Ab enriched fraction of genomic DNA in JMML. Both histones H3 and H4 associated with the p15 CpG island were acetylated.These results suggest epigenetic dysregulation of p15 mRNA expression in this disorder. Less