Article Oligomerization and Auto-methylation of the Human Lysine Methyltransferase SETD6 Lital Estrella Weil 1,2, Yulia Shmidov 3, Margarita Kublanovsky 1,2, David Morgenstern 5, Michal Feldman 1,2, Ronit Bitton 3,4 and Dan Levy 1,2 1 - The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, P.O.B. 653, Be'er-Sheva 84105, Israel 2 - The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O.B. 653, Be'er-Sheva 84105, Israel 3 - Department of Chemical Engineering, Ben-Gurion University of the Negev, P.O.B. 653, Be'er-Sheva 84105, Israel 4 - Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P.O.B. 653, Be'er-Sheva 84105, Israel 5 - The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 7610001, Israel Correspondence to Dan Levy: The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Be'er-Sheva 84105, Israel. [email protected] https://doi.org/10.1016/j.jmb.2018.08.028 Edited by Igor Stagljar Abstract Signaling via lysine methylation by protein lysine methyltransferases (PKMTs), has been linked to diverse biological and disease processes. The mono-methyltransferase SETD6 (SET-domain-containing protein 6) is a member of the PKMT family and was previously shown to regulate essential cellular processes such as the NF-κB, WNT and the oxidative stress pathways. However, on the biochemical level, little is known about the enzymatic mode of action of SETD6. Here we provide evidence that SETD6 forms high-molecular-weight structures. Specifically, we demonstrate that SETD6 monomeric, dimeric and trimeric forms are stabilized by the methyl donor, S-adenosyl-L-methionine. We then show that SETD6 has auto-methylation activity at K39 and K179, which serves as the major auto-methylation sites with a moderate auto-methylation activity toward K372. A point mutation at K179 but not at K39 and K372, located at the SET domain of SETD6, impaired SETD6 ability to form a trimer, strongly implying a link between the auto-methylation and the oligomerization state. Finally, by radioactive in vitro methylation experiments and biochemical kinetics analysis, we show that the auto-methylation at K39 and K179 increases the catalytic rate of SETD6. Collectively, our data support a model by which SETD6 auto-methylation and self-interaction positively regulate its enzymatic activity in vitro and may suggest that other PKMTs are regulated in the same manner. © 2018 Elsevier Ltd. All rights reserved. Introduction methyl-donor for the methyl-transferase reaction, which gives rise to the methylated lysine and an Protein lysine methylation is a common and versatile S-adenosyl-L-homocysteine (SAH) product [4]. post-translational modification that has been shown to Self-modification and self-interaction are basic be involved in the regulation of many signaling mechanisms that are shared among many kinases. pathways [1]. Lysine methylation is catalyzed by These biochemical properties are important for the protein lysine methyltransferases (PKMTs). There enzymes stability, catalytic activity and their ability to are over 60 candidate members of this enzyme family, transduce down-stream signals [5–8]. While homo- the vast majority of which contain a conserved catalytic dimerization of PKMTs was not reported yet, there are SETdomainthatcatalyzestheadditionofmethyl several examples showing a link between auto- groups (mono, di or tri) to histone and non-histone methylation and PKMT enzymatic activity. The H3K9 proteins [2,3]. S-adenosyl-L-methionine (SAM) is the PKMT G9a activity was shown to be regulated by auto- 0022-2836/© 2018 Elsevier Ltd. All rights reserved. J Mol Biol (2018) 430, 4359–4368 4360 SETD6 Oligomerization and Auto-methylation methylation at K239. The auto-methylation of G9a is an sulfosuccinimidyl-suberate (BS3), which covalently effective mediator for HP1 and G9a interaction similar crosslinks proteins 1.14 nm apart, via lysine residues to H3K9me3 binding [9]. A conformational switch [22]. Under untreated conditions, a single monomeric mediated by the auto-methylation of the yeast methyl- SETD6 band was observed. After 30 min of incubation, transferase Clr4 (Suv39h) at multiple lysines (K455 we could detect complexes of the expected dimers and and K472) was shown to enhance H3K9 methylation trimers sizes, reaching a saturation after 2 h. His- activity [10]. In addition, a recent study demonstrated SUMO was used as a negative control for the BS3 that the auto-methylation of SUV39H2 at K392 crosslinking assay (Fig. S1). reduces its enzymatic activity by blocking its interaction To examine SETD6 oligomerization state under with the substrates [11], whereas PRMT8 auto- native conditions, recombinant His-SETD6 was load- methylation reduces its interaction with SAM [8]. ed on a Superdex200 Increase size exclusion column While PKMTs are considered to be attractive thera- (SEC) (Fig. 1b). Consistent with the crosslinking peutic targets [12–14], the regulatory mechanisms of experiments, we observed higher amount of SETD6 their activity and specifically how their auto- monomers relative to the dimer–trimer forms (Fig. 1b, methylation regulates their activity are still unclear. upper panel), which was validated by Coomassie and The SET-domain-containing protein 6 (SETD6) is a Western blot analysis with anti-SETD6 antibody member of the PKMT family. SETD6 is located on the (Fig. 1b, bottom panel). To test whether constitutive long arm of chromosome 16 (16q21). The gene spans equilibrium between the monomeric, dimeric and 5049 bases with eight coding exons. Human SETD6 trimeric forms of SETD6 exists, the trimeric/dimeric has two splice variants: the long, 473-residue “isoform eluted fractions (fractions 13 and 14 from panel 1B), a” and the short, 449-residue “isoform b,” which lacks were loaded again on the SEC, resulting in a similar an in-frame segment (residues 40–63) [15].SETD6is distribution of the SETD6 native complexes (Fig. 1c). linked to the regulation of various major cellular This result suggests the existence of such equilibrium processes such as mono-methylation of the NF-κB between the different oligomerization states. Better subunit RelA at Lys 310, to suppress the activation of separation and enrichment of the dimer–trimer NF-κB target genes [16] and to promote RelA populations were obtained by loading crosslinked transcriptional activity in bladder cancer [17].In recombinant SETD6 on SEC (Fig. 1d). To test addition, SETD6 was shown to play a role in the SETD6 oligomerization state in cells, over-expressed NRF2 oxidative stress response by interacting with Flag-SETD6 was immunoprecipitated from HEK293T DJ1 [18], the WNT signaling pathway by methylation cells, eluted with Flag-peptides followed by separation of PAK4 [19], nuclear hormone receptor signaling [20] on native gel and Western analysis with anti-SETD6 and embryonic stem cell differentiation via methylation antibody (Fig. 1e). The result indicates that SETD6 of H2AZ [21]. forms dimeric and trimeric structures in a pattern and Despite the emerging importance of SETD6 in the ratio similar to recombinant His-SETD6. Taken togeth- regulation of diverse cellular processes, little is known er, our results suggest that SETD6 forms monomeric, about the biochemical properties which regulate its dimeric and trimeric structures in vitro andincells. activity. In this study, we show that SETD6 forms monomers, dimers and trimers and that these SAM stabilizes SETD6 monomeric, dimeric and structures are stabilized by SAM. We discovered trimeric structures that SETD6 is capable of auto-methylation and mapped the auto-methylation sites to K39, K179 and As SAM serves as the methyl donor during the K372. A point mutation at K179 impaired its ability to methylation reaction [1], we hypothesized that SAM form a trimer, implying a link between the auto- stabilizes the monomeric, dimeric and trimeric com- methylation and oligomerization properties. Finally, by plexes of SETD6. To address this hypothesis, biochemical kinetics analysis, we show that the auto- recombinant SETD6 was incubated in the absence methylation at K39 and K179 increases SETD6's or presence of SAM under methylation reaction affinity to its methylated substrate. conditions. Native gel electrophoresis, followed by Coomassie stain or Western blot analysis, was used to detect changes in the presence of SETD6 Results monomeric, dimeric, and trimeric as well as higher- molecular-weight structures (Fig. 2aandb).The addition of SAM resulted in stabilization of the SETD6 forms monomers, dimers and trimers as monomeric, dimeric and trimeric forms of SETD6 well as higher-molecular-weight structures and a significant reduction in the high-molecular- weight bands. However, in the absence of SAM, we In order to test if SETD6 forms strutures with could detect bands with significantly higher size molecular weight higher than monomers, we performed than monomers, dimers and trimers. Importantly, the a crosslinking experiment (Fig. 1a) with recombinant high-molecular-weight bands were not reduced after His-SETD6 in the presence of 68 μMbis- the addition of equivalent amount of SAH, which SETD6 Oligomerization and Auto-methylation Fig. 1. SETD6 forms monomers, dimers and trimers as well as high-molecular-weight structures. (a) Recombinant His-SETD6 was treated with 68 μMBS3 for the indicated times followed by Coomassie stain. (b) SEC profile of His-SETD6. Eluted fractions (11–18) were loaded on SDS gel followed by Coomassie and Western blot analysis. Standard curve obtained with proteins of known masses is shown on the right. The position of the monomer, dimer and trimer on the curve is indicated with an arrow. (c) The dimer/trimer eluted fractions (13–14) were re-loaded again resulting in a similar distribution of the dimer/trimer versus monomer high-molecular-weight structures. (d) Recombinant His-SETD6 was treated with 68 μMBS3 for 30 min followed by SEC. (e) Flag-SETD6 was over-expressed in HEK293T followed by immunoprecipitation with Flag antibody.