Residues that are conserved across species are indicated in green. The arrow indicates Arg-2510, the residue mutated for study. Residues mutated in ccRCC are in red and marked with an asterisk. D, SRI domain sequence alignment across multiple species. The black box indicates residues previously shown to be an important catalytic site. The arrow indicates Arg-1625, the residue mutated for study. Residues mutated in ccRCC are shown in red and marked with an asterisk. Amino acids 1612–1673 of the human SET domain (amino acids 1550–1667) are shown. C, partial SET domain sequence alignment across multiple species. The N terminus is marked in green, the C terminus is marked in pink, and residues mutated are shown as sticks. B, alignment of human SET domain crystal structure ( blue) with I-TASSER protein structure prediction for yeast SET domain ( yellow). Annotated domains include AWS, SET, PS, CC, LCR, WW, and SRI. The percentage of conserved residues within the BLAST-aligned domain sequence is indicated. A, comparison of SETD2 and yeast Set2 ( ySet2) annotated protein structure. SETD2 and yeast Set2 show high sequence and structural conservation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. H3K36 SETD2 Set2 cancer chromatin enzyme histone methylation mutant. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer. 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. 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. This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. 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). 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. 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. 7 From the Department of Genetics, Curriculum in Genetics and Molecular Biology, the Lineberger Comprehensive Cancer Center, and the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, 8 From the Department of Genetics, Curriculum in Genetics and Molecular Biology, the Lineberger Comprehensive Cancer Center, and the Division of Hematology and Oncology, Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232 yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair.7 From the Department of Genetics, Curriculum in Genetics and Molecular Biology, the Lineberger Comprehensive Cancer Center, and the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, 8 From the Department of Genetics, Curriculum in Genetics and Molecular Biology, the Lineberger Comprehensive Cancer Center, and the Division of Hematology and Oncology, Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232 PMC article.6 From the Department of Genetics, Curriculum in Genetics and Molecular Biology, the Lineberger Comprehensive Cancer Center, and the Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27514, and.5 the Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan 48109.4 From the Department of Genetics, Curriculum in Genetics and Molecular Biology, the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599. 3 the Lineberger Comprehensive Cancer Center, and.2 the Lineberger Comprehensive Cancer Center, and the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.1 From the Department of Genetics, Curriculum in Genetics and Molecular Biology, the Lineberger Comprehensive Cancer Center, and.
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