Investigation into the Role of the Set2 Auto‐Inhibitory Domain in H3K36 Methyltransferase Activity

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Set2 is nucleosomal‐specific methyltransferase that is responsible for mediating histone H3 lysine 36 (H3K36) mono‐, di‐, or trimethylation in Saccharomyces cerevisiae. The human homolog of Set2, SETD2, mediates only H3K36me3. H3K36 methylation has been linked to the prevention of inappropriate or “cryptic” transcription, and SETD2 functions as a tumor repressor through its regulation of the cell cycle and mitotic function. While Set2 catalyzes H3K36 methylation via its SET methyltransferase domain located at its N‐terminus, Set2 contains a Set2‐Rpb1 interaction (SRI) domain at its C‐terminus that binds RNA polymerase II and couples H3K36 methylation with transcriptional elongation. In addition to the SET and SRI domains, a third domain termed the auto inhibitory domain (AID) was identified within Set2 and functions to control the extent of methylation and substrate specificity of Set2's H3K36 methyltransferase activity (1). Additionally, a new study revealed key residues within the AID that are required for the autoinhibitory function of this domain (2). Here, we used an in vitro biochemical approach to further examine the role of the AID in Set2 methyltransferase activity. We designed and expressed full‐length (FL) Set2 containing a recently characterized mutation of the AID (i.e., H336Y) (2) as well as the AID in isolation to test the possibility that this domain directly interacts with the SET domain of Set2 to regulate its H3K36 methylation. Consistent with previous reports, we found that the H336Y AID mutation in Set2 resulted in significantly higher H3K36 methylation activity on nucleosomal and core histone substrates in vitro. Importantly, the normal preference of Set2 for nucleosomes over core histones was lost in the H366Y AID mutation; i.e., the H366Y AID mutation in Set2 enhanced core histone methylation activity to those found with oligonucleosomes. Examination of the AID's ability to interact with Set2's SET domain to regulate its H3K36 methyltransferase activity was next examined. Contrary to our predictions, we found that the addition of AID in Set2 methyltransferase assays did not significantly alter Set2's methyltransferase activity on either oligonucleosomes or core histones. Further preliminary co‐immunoprecipitation analyses did not support a direct interaction of the AID with Set2. These data suggest that the AID's repressive action on Set2 may be regulated through a unique AID structure that exists adjacently to the SET domain which enforces substrate preference and activity rather than by a scenario where the AID interacts directly with, and occludes, the SET domain. Taken together, our data extends our understanding of the autoinhibition functions found in chromatin regulators that direct the precise transcriptional control between chromatin accessibility and repression.Support or Funding InformationNIH to BDSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.