Summary We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) based on multidimensional and comprehensive characterization, including mitochondrial DNA (mtDNA) and whole genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared to other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT up-regulation in cancer distinct from previously-observed amplifications and point mutations.

SUMMARY Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a “tubulin code”. PTMs of histones comprise an analogous “histone code”, although the “readers, writers and erasers” of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules, and that the histone methytransferase, SETD2, which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis, and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei and polyploidy. These data now identify SETD2 as a dual function methyltransferase for both chromatin and the cytoskeleton, and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes.

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumorsuppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.Cancer is increasingly characterized by alterations in chromatin-modifying enzymes (1). SETD2, a non-redundant histone H3 lysine 36 (H3K36) 4 methyltransferase (2), has been found to be mutated in a growing list of tumor types, most notably in clear cell renal cell carcinoma (ccRCC) (1, 3, 4), but also in high grade gliomas (5), breast cancer (6), bladder cancer (7), and acute lymphoblastic leukemia (8 -10). Recent studies exploring intratumor heterogeneity in ccRCC identified distinct mutations in SETD2 from spatially distinct subsections of an individual tumor, suggesting that mutation of SETD2 is a critical and selected event in ccRCC cancer progression (11). Mutations in SETD2 are predominantly inactivating, such as early nonsense or frameshift mutations, which lead to nonfunctional protein and global loss of H3K36 trimethylation (H3K36me3) (4,11,12). Missense mutations tend to cluster in two domains (1,4,12,13): the SET domain, which catalyzes H3K36me3 (14), and the Set2 Rpb1 interaction (SRI) domain, which mediates the interaction between SETD2 and the hyperphosphorylated form of RNA polymerase II (RNAPII) (13).SETD2 and its yeast counterpart, Set2, both associate with RNAPII in a co-transcriptional manner (13,15,16). In yeast, Set2 mediates all H3K36 methylation states (H3K36me1/me2/ me3) (17) and regulates the recruitment of chromatin-remodeling enzymes (Isw1b) and a histone deacetylase (Rpd3) (18) that functions to keep gene bodies deacetylated, thereby maintaining a more compact chromatin structure (19,20) that is more resistant to inappropr...

The H3 lysine 36 histone methyltransferase is mutated across a range of human cancers. Although other enzymes can mediate mono- and dimethylation, SETD2 is the exclusive trimethylase. SETD2 associates with the phosphorylated carboxy-terminal domain of RNA polymerase and modifies histones at actively transcribed genes. The functions associated with SETD2 are mediated through multiple effector proteins that bind trimethylated H3K36. These effectors directly mediate multiple chromatin-regulated processes, including RNA splicing, DNA damage repair, and DNA methylation. Although alterations in each of these processes have been associated with SETD2 loss, the relative role of each in the development of cancer is not fully understood. Critical vulnerabilities resulting from SETD2 loss may offer a strategy for potential therapeutics.

Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase , which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic,-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In -inactivated human kidney cells, rescue with a pathogenic mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability. Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. .