Analysis of histone modification around the CpG island region of the p15 gene in acute myeloblastic leukemia
Introduction
DNA methylation and histone modifications are the major epigenetic mechanisms for gene expression. Hypermethylation of DNA in the promoter CpG islands of tumor suppressor genes that causes transcriptional silencing is thought to play an important role in carcinogenesis [1], [2], [3]. Histone proteins assemble into nucleosomes, which function as both DNA packaging units and transcriptional regulators. The amino-terminal tails of histones protrude from the nucleosome and are subject to chemical modifications including acetylation, methylation and phosphorylation [4]. Acetylated histones are associated with transcriptionally active regions of euchromatin, whereas hypoacetylated histones are associated with transcriptionally inert regions of heterochromatin [5], [6], [7], [8]. Histone H3 methylated at lysine 9 is enriched in deacetylated, transcriptionally silent heterochromatic regions, whereas histone H3 acetylated at lysine 9 is enriched in euchromatic domains and correlates with active gene expressions [9], [10], [11]. Recent reports have identified a mechanistic pathway of epigenetic silencing by cytosine methylation, histone hypoacetylation and chromatin condensation suggesting that these mechanisms act together to inactivate gene transcription [5], [12].
The p15 (also known as INK4b or MTS2) gene is a candidate tumor suppressor gene with structural and functional similarity to the p16 gene. Both p15 and p16 specifically inhibit cyclin-dependent kinase (CDK)4 and CDK6 [13], [14], which are key regulators of the progression of eukaryotic cells through the G1 phase of the cell cycle. Cameron et al. [15] reported that the degree of methylation varies in primary acute leukemia, whereas the entire CpG island region of p15 in normal lymphocytes is largely devoid of methylation. We have shown a dynamic change in methylation at the p15 CpG island and the inverse expression of p15 during normal myeloid development under stimulation with granulocyte-macrophage colony-stimulating factor alone (GM-CSF) or in combination with stem cell factor (SCF) [16]. More recently, we reported that GM-CSF can induce de novo methylation of the p15 gene, using histone deacetylase as well as DNA methyltransferase [17].
However, histone modifications regulating expression of the genes which encode CDK inhibitors in normal peripheral blood (PB)/bone marrow (BM) 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.
Section snippets
Cell preparation
BM cells were aspirated in heparinized plastic syringes from 11 children with AML after obtaining the informed consent of each patient and/or the parents. Mononuclear cells (MNCs) were separated by density centrifugation over Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden), and were frozen with liquid nitrogen until the experiment. Fifty-two to 97% of the isolated cells were leukemic blasts. The patients were classified into FAB subtypes on the basis of morphologic and cytochemical
Histone modifications around the CpG island of the p15 gene in leukemic cell lines
To elucidate DNA methylation and histone modifications around the CpG island of the p15 gene in leukemic cells, we performed bisulfite genomic sequencing and ChIP assays on leukemic cell lines (HL60 cells and Molt4 cells). The results are presented in Fig. 1. The CpG island region of p15 is completely unmethylated in HL60 cells, whereas it is almost fully methylated in Molt4 cells, being consistent with the previous results [16], [20]. Nucleoprotein complexes were sonicated to reduce the size
Discussion
Frequent p15 hypermethylation was demonstrated in children and adults with AML, whereas p16 methylation has rarely been observed [21]. When at least half of 27 CpG sites in the p15 gene were methylated in an individual allele, the allele was defined as methylated. Given this criterion, 7 (63.6%) of 11 patients with AML had allele(s) with methylated p15 CpG islands. These results are consistent with those reported previously [21], [22], [23]. There have been controversies regarding a
Acknowledgments
This work was supported by Grants-in-Aid Nos. 15790521, 11670753, and 09041178 from the Ministry of Education of Japan.
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