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 291

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 1G 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 an antibody against H3K4me3 (histone-H3-lysine 4-trimethylation). H3K4me3 is a chromatin mark of gene activation that localizes to active gene promoter regions. CHIP-Seq was performed in CD34+, CD34 neg cells and whole bone marrow (WBM) from 6 patients with MDS and 4 normal controls. In total 30 samples were sequenced. Patients samples were obtained at the time of initial referral at MDACC and were sorted immediately using standard separation procedures. When compared to normal controls for each cellular compartment, we identified 36, 156 and 32 potential active gene promoters associated with H3K4me3 in CD34+, CD34 neg cells and WBM respectively. Of importance, gene promoter regions identified did not overlap among the different cellular compartments analyzed (differences were observed comparing normal vs MDS but also among different MDS compartments), indicating that chromatin structure and gene expression profiles are aberrant and distinct in non-CD34+ cells that may also contribute to the pathobiology of MDS. Here we focus on H3K4me3-associated gene promoters in CD34+ cells. To confirm the results obtained with the CHIP-seq approach, we studied the expression levels of the top 9 CHIP-Seq identified genes in an independent cohort of in CD34+ cells obtained from 54 MDS at the time of initial diagnosis. Patient characteristics have been previously reported (Leukemia, in press): 11 (20%) low risk, 20 (37%) int-1, 15 (27%) int-2 and 8 (14%) high risk by IPSS. We confirmed gene expression up-regulation of 7 (C5AR1, FPR1, FPR2, AQ9, FYB, FCAR, IL8RA) of 9 genes detected by CHIP-Seq. Using Ingenuity Pathway Analysis of the 36 genes identified in CD34+ cells revealed NF-κB as central activated knot in CD34+ cells. This was confirmed by phospho-p65 immuno-staining in primary cells. Furthermore up-regulation of all 10 NF-κB activation associated genes was confirmed in MDS CD34+ cells by Q-RT-PCR. Transfection of OCI-AML3 cells with a siRNAs cocktail targeting 4 of the CD34+ NF-κB activation genes dramatically repressed NF-κB activation as well as expression and promoter NF-κB association of JMJD3 gene, a known NF-κB transcriptional target. JMJD3 encodes a Jmjc-domain K27me3 demethylase, which positively regulates H3K4me3. We further characterized expression levels of 17 known histone demethylases known in human in 35 patients with MDS and identified JMJD3 as the only histone demethylase overexpressed in MDS CD34+ cells. siRNA targeting JMJD3 reduced expression and promoter H3K4me3 levels of several CHIP-Seq detected MDS- CD34+-NF-κB activation genes. Finally expression profile of JMJD3 and the panel CD34+-NF-κB activation genes in the 54 patients with MDS indicated that expression levels were consistently overexpressed in patients with higher-risk (high and int-2) disease compared to patients with lower (low and int-1) risk disease. In view of the known antiapoptotic and proliferative role of the NF-κB pathway, this data indicates that expression of upstream and downstream modulators of NF-κB signaling, regulated at the chromatin level by JMJD3, have a role in MDS progression and could serve as therapeutic targets. Through this novel in vivo CHIP-Seq analysis, we demonstrated that a positive regulatory loop exists in MDS CD34+ cells. This loop contains JMJD3 promoted gene activation through positive regulation of H3K4me3, which leads to NF-κB signaling activation, and then further promotion of JMJD3 expression and activation of the whole signaling cascade. Our study also demonstrates that in vivo CHIP-Seq can be used to discover disease specific targets.