Histone Modifications Associated with DNA Methylation and Transcriptional Repression of p15INK4b in Acute Myeloid Leukemia.

Abstract

p15INK4b is a cyclin-dependent kinase inhibitor known to regulate the G1-to-S transition of the cell cycle and to be involved in negatively regulating myeloid progenitor cell production. DNA hypermethylation leading to transcriptional silencing of p15INK4b has been reported in up to 70% of acute myeloid leukemia (AML) patient samples. In our study we sought to determine if p15INK4b DNA methylation in AML is accompanied by repressive histone modifications that contribute to the transcriptional repression of the gene at the chromatin level. Chromatin immunoprecipitation and DNA tiling microarrays (ChIP-on-chip) with 20bp resolution were utilized to assess the distribution of histone modifications over a 1.2 megabase region of human chromosome 9 including p15INK4b and adjacent tumor suppressor genes p14ARF and p16INK4a. We found that AML cell lines with p15INK4b hypermethylation (Kasumi-1, KG-1, and KG-1a) had high levels of the repressive histone modification trimethylation of lysine 27 of histone H3 (H3K27me3). Remarkably, this modification spanned the entire INK4b-ARF-INK4a region while little binding was observed in adjacent regions of chromosome 9. Binding of EZH2, the polycomb associated H3K27 histone methyltransferase, co-localized with H3K27me3 distribution over the INK locus. H3K27me3 was not identified at this region in AML cell lines without p15INK4b DNA methylation (U937 and HL-60). In contrast, histone modifications associated gene activation, trimethylation of lysine 4 of H3 (H3K4me3) and acetylation of lysine 9 (H3K9Ac), were found at the p15INK4b promoter in these cells. Enrichment of another repressive histone modification, trimethylation of histone H3 on lysine 9 (H3K9me3), did not correlate with the DNA methylation status of p15INK4b and appeared highest in exons 2 and 3 of p16INK4a in most cell lines. Since p15INK4b reactivation has been described as a component of a patient's response to epigenetic therapies in AML treatment, we sought to determine the dynamics of histone modifications following treatment with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine and histone deacetylase (HDAC) inhibitor tricostatin A. In KG-1 cells, a reduction in p15INK4b DNA methylation was observed following treatment with DNMT inhibitors. Unexpectedly, treatment with HDAC inhibitors alone was also capable of reducing p15INK4b DNA methylation suggesting that a repressive chromatin structure contributes to the DNA methylation in this cell line. Loss of DNA methylation was not sufficient for reactivation of p15INK4b expression as detectible expression was only observed following the combined treatment of DNMT and HDAC inhibitors. Reactivation was associated with an increase in the activation-associated histone modifications H3K4me3 and H3K9Ac at the promoter region and, unexpectedly, maintenance of the repressive modification H3K27me3. This “bivalent” histone modification pattern is characteristic of many developmentally poised genes in embryonic stem cells and correlates with the histone methylation status of p15INK4b we observed in CD34+ bone marrow progenitor/ stem cells. This data indicates that optimal epigenetic therapies targeted to reactivate p15INK4b expression should be designed to induce activating histone modifications in addition to reducing DNA methylation.