Epigenetic regulation of p15 mRNA expression in juvenile myelomonocytic

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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