Genome-wide profiling of histone 3 lysine 36 trimethylation in clear cell renal cell carcinoma.

Abstract

464 Background: Although the von Hippel-Lindau (VHL) tumor suppressor gene is mutated in 60% of ccRCC, deletion of VHL in mice is insufficient for tumorigenesis. Sequencing of ccRCC tumors identified mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. We hypothesize that loss of SETD2 methyltransferase activity alters the genome wide pattern of H3K36 trimethylation (H3K36me3) in ccRCC, and contributes to the cancer phenotype. Methods: To generate a genome-wide profile of H3K36me3 in frozen nephrectomy samples and RCC cell lines, we optimized a chromatin immunoprecipitation (ChIP) protocol for the isolation of DNA associated with H3K36me3. H3K36me3 is associated with open chromatin and an H3K36me3-specific antibody was used for immunoprecipitation of endogenous H3K36me3-bound DNA. ChIP PCR primers were optimized for active genes, such as actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a “gene desert” on chromosome 12 (negative control). ChIP libraries were then generated from 3 paired uninvolved kidney and RCC and 2 RCC cell lines. In order to identify H3K36Me3 upregulated regions in uninvolved kidney and RCC, reads from the ChIP sequencing were mapped to the human genome using Burrows-Wheeler Aligner and SICER algorithms. Results: Using ChIP PCR, we found that active genomic regions were enriched 15-30 fold over the negative controls indicating that the quality and yield of immunoprecipitated DNA/chromatin complexes from frozen tissue was sufficient for ChIP sequencing. A preliminary ChIP sequencing analysis of RCC cell lines and frozen ccRCC tissue indicates that H3K36me3 enriched DNA sequences were mapped to exons (31.3%) compared to introns (13.5%, p<0.001), consistent with the role of H3K36me3 in transcription. Conclusions: Genomic regions enriched for H3K36Me3 binding were identified from patient-derived tissue and RCC cell lines. Current efforts are focused on comparing the H3K36me3 profiles between matched tumor and uninvolved kidney ChIP libraries to generate a genome wide map of dysregulated H3K36me3 modifications.