Regulation of SETD2 stability is important for the fidelity of H3K36me3 deposition

Abstract

The SET domain-containing protein SETD2 is the sole methyltransferase in mammals that can trimethylate histone H3 at lysine 36. H3K36me3 is known to be involved in transcription elongation, pre-mRNA splicing, DNA methylation, and DNA damage repair. However, knowledge of the regulation of the SETD2 enzyme itself is limited. Here we show that the poorly characterized N-terminal region of SETD2 plays a determining role in regulating the stability of SETD2. This stretch of 1-1403 amino acid residues which contains disordered regions, is targeted for degradation by the proteasome. In addition, the SETD2 protein is aggregate-prone and forms insoluble inclusion bodies in nuclei especially upon proteasome inhibition. Removal of the N-terminal segment results in the stabilization of SETD2 and leads to a marked increase in global H3K36me3 which, uncharacteristically, can happen in an RNA Pol II-independent manner. The spurious H3K36me3 is deposited in a non-canonical distribution including reduced enrichment over gene bodies and exons. An increased SETD2 abundance leads to widespread changes in transcription and alternative splicing. Thus, the regulation of SETD2 levels through intrinsically disordered region-facilitated proteolysis is important to maintain the fidelity of transcription and splicing related processes. gene coding for the SETD2 histone methyltransferase is often deleted or mutated )(Su et al, 2017)(Li et al, 2016.In yeast, the SET domain-containing protein Set2 (ySet2) is the sole H3K36 methyltransferase (Strahl et al, 2002). ySet2 interacts with the large subunit of the RNA polymerase II, Rpb1, through its SRI domain, and co-transcriptionally deposits H3K36me3 (Xiao et al, 2003). The deletion of the SRI domain from ySet2 abolishes both the Set2-RNA Pol II interaction and H3K36me3 methylation in yeast (Suzuki et al, 2016). H3K36 methylation is a highly conserved histone mark and Set2 homologs are found in more complex eukaryotes (McDaniel & Strahl, 2017). These homologs share the conserved features like the AWS (associated with SET), SET [Su(var)3-9, Enhancer-of-zeste and Trithorax] and Post-SET domains that are required for the catalytic activity of the enzyme, and also, the protein-protein interaction regions such as the WW and SRI (Set2-Rpb1 Interaction) domains. However, there are differences in both the manner of H3K36me3 deposition in mammals as well as in the enzyme itself as compared to yeast. For instance, although SETD2 is the sole methyltransferase in mammals that can deposit the histone H3K36me3 mark, there are additional enzymes such as NSD1, NSD2, and ASH1L that can deposit H3K36me1 and me2 (Lucio-Eterovic et al, 2010)(Kuo et al, 2011)(Tanaka et al, 2007). Notably, SETD2 has a long N-terminal segment that is not present in ySet2. The function of this region has remained obscure (McDaniel & Strahl, 2017)(Hacker et al, 2016).Here we show that SETD2 is an inherently aggregate-prone protein and its N-terminal region regulates its half-life. This, in turn, is important for the fidelity of H3K36me3 ...

The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

“…Microscopy with the GFP-tagged version of the fragments revealed that both localized to the nucleus, which is consistent with our previous characterization of the SETD2 NLS (Fig. 2b) 36 . Next, Halo-SETD2 1-1692 and 1404-2564 (SETD2C) were affinity-purified from 293T extracts using Halo ligandconjugated magnetic resin.…”

“…Next, Halo-FLAG-SETD2C with and without RNase treatment, and also Halo-FLAG-SETD2CΔSRI (the SRI domain is known to mediate the Set2-Pol II interaction) from 293T extracts were affinity-purified. Previously we have shown that the SETD2-Pol II interaction is not affected by RNase treatment and is abolished upon deletion of the SRI domain from SETD2 36 . Notably, the interaction with hnRNP L persisted without the Pol II interaction domain and in fact, increased upon RNase treatment ( Fig.…”

“…1g, Supplementary Data 1). Previously, we and others have shown that the full-length SETD2 protein is robustly degraded by the ubiquitin-proteasome pathway 36,37 . Therefore, the low dNSAF values of SETD2 could be due to the low abundance of the endogenous SETD2 protein.…”

“…Earlier we showed that SETD2 is an aggregate-prone protein 36 . We co-expressed mCherry-hnRNP L and GFP-SETD2C in 293T cells.…”

“…Previously, we established that full-length SETD2 is robustly degraded by the proteasome and its smaller fragments are much better expressed 40 . Based on this knowledge, we opted to purify two overlapping fragments of SETD2: N + catalytic domains (1-1692) and C (1404-2564 (N3) both having at least one NLS and the catalytic domains, AWS, SET, and Post-SET ( Fig.…”