Abstract 4800: Genome-wide CHIP-Seq analysis of histone methylation reveals modulators of NF-κB signaling and the histone demethylase JMJD3 as implicated in disease progression in myelodysplastic syndrome (MDS)

Abstract

Abstract Although cytogenetic abnormalities are common in MDS, search for genetic alterations has been less informative with few prevalent abnormalities thus far known. To identify genes aberrantly activated in MDS, we developed a novel approach based on chromatin immuno-precipitation combined with massive parallel sequencing (CHIP-Seq) using the Solexa sequencing technology. To our knowledge this is the first example of the use of this technology in primary human samples. For CHIP analysis we used H3K4me3 (histone-H3-lysine 4-trimethylation), which is a chromatin mark of gene activation that localizes to gene promoter regions. CHIP-Seq was performed in CD34pos and CD34neg from 5 patients with MDS and 4 normal controls. When compared to normal controls for each cellular compartment, we identified 36 and 156 active gene promoters associated with high levels of H3K4me3 in MDS CD34pos and CD34neg cells respectively. Here we focus on H3K4me3-associated gene promoters in CD34pos cells. To confirm the results obtained with the CHIP-seq approach, we first performed Q-PCR in a cohort of CD34pos cells obtained from 57 MDS. We confirmed the positive association between upregulation of H3K4me3 and activation of expression for the top 9 CHIP-Seq identified genes. Then, using Ingenuity Pathway Analysis (IPA) tool, we associated the 11 of the 36 genes identified in CD34pos cells with activation of NF-κB. This was confirmed by immuno-staining of phosphor-p65, a marker of NF-κB activation, and by the Q-PCR revealed upregulation of all 11 CHIP-Seq identified NF-κB activation related genes. Further, transfection of OCI-AML3 cells with a siRNAs cocktail targeting 4 of the genes identified in CD34pos that encode NF-κB activators dramatically repressed NF-κB signal. Next, we characterized 17 histone demethylases in MDS CD34pos cells. JMJD3, a Jmjc-domain K27me3 demethylase and a known positive regulator of H3K4me3, was found to be the only one significantly upregulated. SiRNA targeting JMJD3 repressed NF-κB activation. For the 9 CHIP-Seq identified genes encoding NF-κB activators, targeting JMJD3 repressed the expression of 7 of them and reduced promoter H3K4me3 was detected for 2 of them. Of importance, JMJD3 is a known direct transcriptional target of NF-κB. When we transfected cells with the siRNA pool targeting CHIP-Seq identified genes encoding NF-κB activators, JMJD3 expression was reduced, accompanied with displacement of NF-κB p65 from JMJD3 gene promoter. In summary, through this novel in vivo CHIP-Seq analysis, we demonstrated that a positive regulatory circuitry exists in MDS CD34pos cells between JMJD3 and NF-κB signaling activation. Our study also demonstrates that in vivo CHIP-Seq can be used to discover disease specific and/or therapy related histone codes in MDS and leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4800.