The Effect of Cancer-Associated SETD2 Mutations on Transcription and Chromatin Organization Public Deposited

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  • March 19, 2019
Creator
  • Fahey, Catherine
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • Clear cell renal cell carcinoma is characterized by mutations in chromatin modifying enzymes. Among these is SETD2, a non-redundant histone H3 lysine 36 methyltransferase. Mutations in SETD2 in ccRCC are either early-inactivating, occur in the catalytic SET domain, or are found in the Set2 Rpb1-interacting domain. We inactivated SETD2 in ccRCC cells lines and reintroduced a truncated but functional wildtype SETD2 (tSETD2), as well as three ccRCC-associated point mutations in order to examine the effect on DNA damage repair, chromatin organization, transcription, and H3K36 trimethylation. We found that SETD2 loss results in complete loss of H3K36me3. SETD2Δ cells do not resolve γH2A.X foci after DNA damage and show altered chromatin accessibility. Transcription is largely unaffected by SETD2 loss. tSETD2 restores H3K36me3 to loci marked by H3K36me3 in wildtype cells, indicating that the N-terminus of SETD2 in not required for H3K36me3 placement. The first examined point mutant, R1625C, occurs in the SET domain and disrupts catalytic activity of SETD2 by reducing the interaction between SETD2 and histone H3, which results in decreased protein stability in the cell. The second examined point mutation, R2510H, occurs in the SRI domain. This SRI domain mutant is catalytically active. The third mutation, T2457*, deletes the SRI domain from tSETD2, and is also catalytically active. Both the R2510H and the T2457* mutations disrupt the interaction between SETD2 and RNA polymerase II. Surprisingly, both of these mutants also restore H3K36me3 to loci marked by wildtype SETD2. These mutants also show increased H3K36me3 levels near the TSS relative to wildtype. This suggests that the SRI domain is necessary for interaction with RNAPII, is not required for normal H3K36me3 placement across the genome, and may be involved at the TSS for H3K36me3 marking. The separation of function for the SET and SRI domains is of important consideration when developing therapeutics which target SETD2 mutation in ccRCC.
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  • In Copyright
Advisor
  • Baldwin, Albert
  • Rathmell, W. Kimryn
  • Kim, William
  • Strahl, Brian
  • Davis, Ian
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017
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