Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation.

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 tumor-suppressive. 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.

Hacker KE ，Fahey CC ，Shinsky SA ，Chiang YJ ，DiFiore JV ，Jha DK ，Vo AH ，Shavit JA ，Davis IJ ，Strahl BD ，Rathmell WK ... -  《-》

Set2 methyltransferase facilitates cell cycle progression by maintaining transcriptional fidelity.

Dronamraju R ，Jha DK ，Eser U ，Adams AT ，Dominguez D ，Choudhury R ，Chiang YC ，Rathmell WK ，Emanuele MJ ，Churchman LS ，Strahl BD ... -  《-》

The Benzene Hematotoxic and Reactive Metabolite 1,4-Benzoquinone Impairs the Activity of the Histone Methyltransferase SET Domain Containing 2 (SETD2) and Causes Aberrant Histone H3 Lysine 36 Trimethylation (H3K36me3).

Human SETD2 is the unique histone methyltransferase that generates H3K36 trimethylation (H3K36me3), an epigenetic mark that plays a key role in normal hematopoiesis. Interestingly, recurrent inactivating mutations of SETD2 and aberrant H3K36me3 are increasingly reported to be involved in hematopoietic malignancies. Benzene (BZ) is a ubiquitous environmental pollutant and carcinogen that causes leukemia. The leukemogenic properties of BZ depend on its biotransformation in the bone marrow into oxidative metabolites, in particular 1,4-benzoquinone (BQ). This hematotoxic metabolite can form DNA and protein adducts that result in the damage and the alteration of cellular processes. Recent studies suggest that BZ-dependent leukemogenesis could depend on epigenetic perturbations, notably aberrant histone methylation. We investigated whether H3K36 trimethylation by SETD2 could be impacted by BZ and its hematotoxic metabolites. Herein, we show that BQ, the major leukemogenic metabolite of BZ, inhibits irreversibly the human histone methyltransferase SETD2, resulting in decreased H3K36me3. Our mechanistic studies further indicate that the BQ-dependent inactivation of SETD2 is due to covalent binding of BQ to reactive Zn-finger cysteines within the catalytic domain of the enzyme. The formation of these quinoprotein adducts results in loss of enzyme activity and protein crosslinks/oligomers. Experiments conducted in hematopoietic cells confirm that exposure to BQ results in the formation of SETD2 crosslinks/oligomers and concomitant loss of H3K36me3 in cells. Taken together, our data indicate that BQ, a major hematotoxic metabolite of BZ, could contribute to BZ-dependent leukemogenesis by perturbing the functions of SETD2, a histone lysine methyltransferase of hematopoietic relevance. SIGNIFICANCE STATEMENT: Benzoquinone is a major leukemogenic metabolite of benzene. Dysregulation of histone methyltransferase is involved in hematopoietic malignancies. This study found that benzoquinone irreversibly impairs SET domain containing 2, a histone H3K36 methyltransferase that plays a key role in hematopoiesis. Benzoquinone forms covalent adducts on Zn-finger cysteines within the catalytic site, leading to loss of activity, protein crosslinks/oligomers, and concomitant decrease of H3K36me3 histone mark. These data provide evidence that a leukemogenic metabolite of benzene can impair a key epigenetic enzyme.

Berthelet J ，Michail C ，Bui LC ，Le Coadou L ，Sirri V ，Wang L ，Dulphy N ，Dupret JM ，Chomienne C ，Guidez F ，Rodrigues-Lima F ... -  《-》

Depletion of H3K36me2 recapitulates epigenomic and phenotypic changes induced by the H3.3K36M oncohistone mutation.

Rajagopalan KN ，Chen X ，Weinberg DN ，Chen H ，Majewski J ，Allis CD ，Lu C ... -  《-》

Histone H3 K36 methylation is associated with transcription elongation in Schizosaccharomyces pombe.

Morris SA ，Shibata Y ，Noma K ，Tsukamoto Y ，Warren E ，Temple B ，Grewal SI ，Strahl BD ... -  《-》