Investigating the role of SETD2 mutations and H3K36me3 loss in clear cell renal cell carcinoma

Hacker, Kathryn Esther

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March 20, 2019

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Hacker, Kathryn Esther
- Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology

Abstract

Comprehensive sequencing of human cancers has identified frequent mutations in genes encoding chromatin regulatory proteins, highlighting the importance of chromatin maintenance in tumor suppression. Specifically, the only human histone H3K36 trimethyltransferase, SETD2, is mutated in up to 15% of clear cell renal cell carcinoma (ccRCC). To understand how SETD2 mutations promote cancer growth or development, we analyzed the molecular and phenotypic effects of SETD2 inactivation and H3K36me3 loss in primary human ccRCC tumors and human renal cell lines. First, we undertook an innovative and comprehensive genomic analysis of ccRCC by performing Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE-seq) across a large cohort of primary tumors and matched uninvolved kidney. FAIRE-seq interrogates chromatin structure by enriching for nucleosome-depleted regions of DNA, which are associated with gene regulatory activity in normal cells. Using this approach, we linked SETD2 mutations with nucleosome loss at genes normally marked by H3K36 tri-methylation, which is globally eliminated in SETD2 mutant tumors. To determine the consequences of this altered chromatin, we investigated how H3K36me3 deficiency affects the ccRCC transcriptome and observed RNA processing defects, including intron retention and alternative splicing, within pre-mRNA transcripts that persisted into the mature mRNA pool. Remarkably, these defects affected approximately 25% of all expressed genes, suggesting that RNA processing defects severely deregulate a wide variety of important cellular pathways. To further investigate the consequences of SETD2 mutations on tumor growth, we used TAL-Effector Nucleases to inactivate SETD2 in renal cell lines and observed global H3K36me3 loss and recapitulation of RNA processing defects observed in human tumors. Additionally, SETD2 loss significantly enhanced tumor-promoting properties, including increased cell proliferation, anchorage-independent growth, and migration. Finally, modeling SETD2 mutations observed in ccRCC and re-introducing them into SETD2-inactivated cells provides insight into H3K36me3 function and how these mutations promote ccRCC. This mechanistic link between defects in chromatin organization, aberrant RNA processing, and tumor-promoting phenotypes provides critical evidence for how recurrent mutations in chromatin regulatory proteins promote cancer and identifies novel targets with therapeutic potential to improve patient care.

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