Variation in chromatin accessibility in human kidney cancer links H3K36 methyltransferase loss with widespread RNA processing defects.

Large-scale cancer sequencing studies continue to identify mutations in genes encoding chromatin regulatory proteins in a wide variety of human cancers. The downstream molecular consequences of these mutations, however, remain unknown. Clear cell renal cell carcinoma (ccRCC) is a particularly relevant model for the study of chromatin regulation in cancer for several reasons. First, relative to mutations in other classes of genes, ccRCCs are marked by frequent mutation of chromatin regulators (Dalgliesh et al. 2010; Varela et al. 2011; Pena-Llopis et al. 2012; Ryan and Bernstein 2012). Three of the more commonly mutated genes in ccRCC include chromatin modifiers SETD2, PBRM1, and BAP1 (Dalgliesh et al. 2010; Varela et al. 2011; Pena-Llopis et al. 2012; Kapur et al. 2013), suggesting that alterations at the level of chromatin may play a prominent role in the development of ccRCC (Dalgliesh et al. 2010; Varela et al. 2011). Mutation-associated changes in chromatin organization may promote oncogenesis in novel ways, and it has been suggested that specific chromatin regulator mutations may confer differences in patient survival or associate with more advanced disease (Hakimi et al. 2012). However, the downstream effect of these mutations on tumor chromatin biology remains unknown. Second, this cancer is tightly associated with a distinct transcriptional program resulting from the inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene (Kim and Kaelin 2004; Bratslavsky et al. 2007; Nickerson et al. 2008; Jonasch et al. 2012). The loss of VHL results in the stabilization of hypoxia inducible factors (HIFs), transcription factors that activate a complex program of downstream targets, including vascular endothelial growth factor (VEGF) and other genes (Gordan et al. 2008; Gore and Larkin 2011; Jonasch et al. 2012). Third, besides VHL and chromatin regulators, mutations in other cancer-associated pathways are generally absent from ccRCC tumors. Elucidating the functional consequences of mutations in genes encoding chromatin regulatory proteins on chromatin organization and transcription in human tumor specimens requires the application of techniques developed for cultured cells to primary human tissues. Formaldehyde-assisted isolation of regulatory elements (FAIRE) interrogates chromatin accessibility by isolating nucleosome-depleted regions of DNA (Nagy et al. 2003; Hogan et al. 2006; Giresi et al. 2007; Giresi and Lieb 2009; Simon et al. 2012). These regions harbor regulatory elements such as active transcriptional start sites, transcriptional enhancers, insulators, silencers, and locus control regions (Hogan et al. 2006; Giresi et al. 2007; Giresi and Lieb 2009; Gaulton et al. 2010; Song et al. 2011; Simon et al. 2012). As a component of the ENCODE Project, FAIRE has been used to identify regulatory elements across a wide range of cell lines (Song et al. 2011; Thurman et al. 2012). However, the application of FAIRE to primary human tissue or to explore the association between chromatin and genetic alterations in cancer has yet to be evaluated. We modified FAIRE for use on primary human clinical samples to define the chromatin landscape in a large cohort of ccRCC tumors and matched normal tissues. We identified tumor- and normal-kidney-specific classes of chromatin accessibility changes, as well as those associated with chromatin modifier mutations. We focused our study on SETD2, which trimethylates lysine-36 on histone H3 (H3K36me3) (Rayasam et al. 2003; Sun et al. 2005; Brown et al. 2006; Edmunds et al. 2008; Yoh et al. 2008; Duns et al. 2010). Associated with the RNA polymerase II complex, SETD2-dependent methylation tends to occur toward the 3′ ends of genes and over nucleosomes located at exons (Edmunds et al. 2008; Kolasinska-Zwierz et al. 2009; Schwartz et al. 2009). SETD2 and H3K36me3 seem to play a role in cotranscriptional RNA processing. In cell-culture-based studies, silencing of SETD2 or readers of H3K36me3 has been associated with differential exon inclusion for individual genes (Luco et al. 2010; Pradeepa et al. 2012) and alternative transcription start site utilization (Carvalho et al. 2013). However, the consequence of SETD2 deficiency on chromatin organization and RNA processing remains to be explored on a genome-wide scale and in a disease-relevant model. SETD2 is mutated in ∼12% of primary human ccRCC tumors and results in H3K36me3 deficiency (Gerlinger et al. 2012). A similar rate of SETD2 mutation has also been observed in high-grade gliomas (Fontebasso et al. 2013). A recent study of intratumor heterogeneity in ccRCC identified distinct SETD2 mutations in all subsections of the same tumor suggesting the importance of disrupting SETD2 function for a subset of tumors (Gerlinger et al. 2012). We found that SETD2 mutation was associated with chromatin accessibility differences preferentially in gene bodies, and these genes frequently exhibited RNA processing defects. Nearly 25% of all expressed genes demonstrated aberrancies in splicing, including exon skipping, intron retention, and alternative transcription start and termination sites. We observed that misspliced exons were marked by a striking increase in chromatin accessibility immediately upstream of the aberrant splice and a loss of nucleosome occupancy directly over the exon. This study represents the first investigation of chromatin organization in human tumors to identify the impact of chromatin modifier mutations on the genomic landscape. Understanding chromatin dysregulation in cancer may ultimately inform the application of emerging classes of chromatin-targeted small molecules in renal cancer.