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The SUV39H1 protein lysine methyltransferase methylates chromatin proteins involved in heterochromatin formation and VDJ recombination Srikanth Kudithipudi, Maren Kirstin Schuhmacher, Adam Fiseha Kebede, and Albert Jeltsch ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01076 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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ACS Chemical Biology

The SUV39H1 protein lysine methyltransferase methylates chromatin proteins involved in heterochromatin formation and VDJ recombination Srikanth Kudithipudi1, Maren Kirstin Schuhmacher1, Adam Fiseha Kebede2, & Albert Jeltsch1,* 1) Institute of Biochemistry, Stuttgart University, Pfaffenwaldring 55, D-70569 Stuttgart, Germany 2) School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany, present address: Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67404 Illkirch, France *Corresponding author: Prof. Albert Jeltsch phone: +49-711-685-64390 fax: +49-711-685-64392 [email protected] Running title: Role of SUV39H1 in methylation of chromatin proteins Abstract SUV39H1 is an H3K9 methyltransferase involved in the formation of heterochromatin. We investigated its substrate specificity profile and show recognition of H3 residues between K4 and G12 with highly specific readout of R8. The specificity profile of SUV39H1 is distinct from its paralog SUV39H2, indicating that they can have different additional substrates. Using the specificity profile, several novel SUV39H1 candidate substrates were identified. We observed methylation of 19 novel substrates at the peptide level and for 6 of them at the protein level. Methylation of RAG2, SET8 and DOT1L was confirmed in cells, which all have important roles in chromatin regulation. Methylation of SET8 allosterically stimulates its H4K20 monomethylation activity connecting SUV39H1 to the generation of increased H4K20me3 levels, another heterochromatic modification. Methylation of RAG2 alters its subnuclear localization indicating that SUV39H1 might regulate VDJ recombination. Taken together, our results indicate that beyond the generation of H3K9me3, SUV39H1 has additional roles in chromatin biology by direct stimulation of the establishment of H4K20me3 and the regulation of chromatin binding of RAG2. Key words: Protein lysine methylation; enzyme specificity; peptide array; SUV39H1; SET8; RAG2; DOT1L

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Histone proteins are subject to several post-translational modifications, which are essential for the regulation of chromatin states.1-3 H3K9me3 is a hallmark of facultative and constitutive heterochromatin in almost all eukaryotes,4-7 and it is also enriched in silenced genes.8 Suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A) was the first identified human protein lysine methyltransferase (PKMT).9 It introduces H3K9me3 and is one human homolog of the Drosophila Su(var)3-9 enzyme, a second human paralog is called SUV39H2 (KMT1B). Deletion of both SUV39H genes results in the loss of pericentric H3K9 trimethylation leading to chromosomal instabilities.10, 11 SUV39H1 exists in multimeric complexes with the other H3K9 PKMTs such as G9a, GLP and SETDB1 and the deletion of SUV39H1 destabilizes the corresponding proteins and leads to a decrease in the H3K9 methylation signal at the global level.12 SUV39H1 mediated H3K9 trimethylation regulates the expression of several genes, and dysregulation of SUV39H1 is observed in different cancers.13, 14 Trimethylation of H3K9 by SUV39H and other enzymes creates a binding site for reading domains, for example the chromodomain of heterochromatic protein 1 (HP1).15, 16 HP1 proteins further recruit SUV4-20H enzymes to heterochromatic regions, which generate H4K20me3 by using H4K20me1 generated by SET8 as substrate. By this mechanism, H3K9me3 introduced by SUV39H enzymes indirectly stimulates generation of H4K20me3, another characteristic heterochromatic histone tail modification.17, 18 SUV39H1 consists of two conserved chromatin domains, a catalytic SET domain and a chromodomain. The SET domain of SUV39H1 introduces methyl groups on the H3 substrate in a non-processive manner19 and the chromodomain binds to the H3K9 trimethylated histones and regulates the catalytic activity of the SET domain.20-22 The catalytic activity of SUV39H1 is influenced by other modifications of the H3 tail, for example trimethylation of K4 has been shown to reduce the activity of SUV39H123, 24 in agreement with the observation in Drosophila that H3K9 methylation dependent heterochromatin formation is initiated through active removal of H3K4 methylation by an LSD1 homolog.25 During last years, several PKMTs that were initially characterized as histone lysine methyltransferases have been shown to methylate various non-histone proteins.26-28 However, till now no additional targets were reported for SUV39H1, although it was the first identified histone PKMT. In contrast, CLR4, the yeast homolog of SUV39H1, was shown to methylate the Mlo3 protein and Mlo3 methylation is required to produce centromeric siRNA and to suppresses the antisense RNA.29 Here we determined the specificity profile of SUV39H1 using peptide arrays. We show that the specificity profile of SUV39H1 differs from SUV39H2 suggesting that these paralogs have non-redundant functions. Based on the specificity profile, we identified possible substrate sequences in proteins known to interact with SUV39H1 either directly or indirectly and in addition we also screened data bases to identify proteins known to be methylated in human cells that fit the SUV39H1 substrate sequence motif. We showed methylation of 19 peptide substrates and confirmed methylation of 6 novel substrates at the protein level. Cellular methylation by SUV39H1 was confirmed for RAG2, SET8 and DOT1L, which all have important roles in gene regulation and chromatin biology. We investigated the biological role of the methylation of these proteins and show that methylation of RAG2 alters its sub2 Environment ACS Paragon Plus

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nuclear localization and methylation of SET8 increases its activity. We conclude that beyond the introduction of H3K9me3 SUV39H1 has additional roles in the chromatin regulation. Results Specificity analysis of SUV39H1 The murine SUV39H1 SET domain (amino acids 82-412) was expressed as GST fusion protein in E. coli BL21 cells and purified using affinity glutathione sepharose beads (Suppl. Fig. 1A). As an initial activity test, the transfer of radioactively labeled methyl groups from [methyl-3H]-S-adenosyl-L-methionine (AdoMet) to recombinant H3 was detected by autoradiography (Suppl. Fig. 1A). To determine the substrate specificity profile of SUV39H1, peptide arrays were synthesized on cellulose membranes by employing the SPOT synthesis method. Since SUV39H1 methylates lysine 9 of histone H3, we used the H3 (1-20) sequence as a template to prepare the arrays. To investigate the influence of all amino acids of the H3 (1-20) sequence on SUV39H1 activity, mutant peptides were prepared in which each amino acid was individually exchanged with the 19 other amino acids. A total of 420 peptides were synthesized on a single membrane including one control peptide in each row. The peptide arrays were incubated with the enzyme in the presence of radioactively labeled AdoMet and the transfer of methyl groups to the immobilized peptides was detected by autoradiography (Fig. 1A). The experiment was repeated three times and the results of each experiment were normalized and averaged (Fig. 1B). The standard deviations (SD) calculated for the relative methylation activities observed with the individual peptides showed that the results are highly reproducible, 83% of the peptides had an SD smaller than ±30% (Suppl. Fig. 1B). To visualize these data, we have calculated the discrimination factor as described previously,30 which summarizes the preference of SUV39H1 for each amino acid at each position over the other tested amino acids (Fig. 1C). The results show that SUV39H1 has a clear specificity profile distinct from its homolog SUV39H231 and also from other H3K9 PKMTs like G9a32 or Dim5.32 Similarly as G9a and SUV39H2, SUV39H1 shows high specificity for an arginine at the -1 position (R8) (using K9 as reference position), replacing this R by any other amino acid completely abolished the activity of SUV39H1. Apart from this, residues from the -5 to +3 positions were recognized with variable stringency (Table 1). At the -5 site (K4), lysine and more weakly arginine are preferred. At the -3 position (T6), the enzyme prefers T, S, A and Y. At the -2 position, SUV39H1 accepts several residues including polar (N, Q), small (A) and hydrophobic (L, P, W) ones. At the +1 position, positively charged K and R are equally accepted as the native S10. At the +2 position, SUV39H1 tolerates only small amino acids like A, G, S, in addition to the native amino acid T11 and at the +3 site, G, K and Q are preferred. Interestingly, the substrate specificity profiles of the two SUV39H enzymes differ from each other (Table 1). SUV39H1 has strong preferences for residues N-terminal to the target lysine, whereas SUV39H2 is more specific for residues C-terminal to the target lysine. One clear difference between SUV39H1 and SUV39H2 is the preference of SUV39H1 for R and K at the +1 site, where SUV39H2 prefers S (and T). Overall, SUV39H2 is more specific than SUV39H1, because it displays a high preference for the native H3 tail residues at 6 sequence positions (R8-G12), while SUV39H1 shows only stringent readout of R8. Overall, these 3 Environment ACS Paragon Plus


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differences indicate that the same methylation site is recognized in a different manner and both enzymes could have different non-histone substrate proteins. Identification of novel non-histone substrate peptides of SUV39H1 Next, we were interested in investigating if SUV39H1 methylates any of its known interacting proteins. Though, the substrate sequence motif of SUV39H1 is long, we have initially focused the search on the RK motif not to exclude any potential target substrate at this stage. We retrieved the known interaction partners of SUV39H1 from the Human Protein Reference Database (http://www.hprd.org/). Currently, about 40 interacting partners including DNMTs, HDACs and many more chromatin-associated proteins have been reported for SUV39H1. Furthermore, we also included proteins which indirectly interact with the SUV39H1 via another protein. Altogether, we identified 276 target proteins containing RK sites, some proteins containing more than one. To investigate the methylation of these putative substrates, we synthesized an array of 407 peptides of 20 amino acid length also including the H3 (1-20) and the H3K9A peptide in which the target lysine is exchanged to alanine as controls, and tested for methylation by SUV39H1 (Suppl. Fig. 2A). Out of the 407 potential target peptides, 13 peptides were methylated with comparable intensities as the H3 tail and 27 additional peptides were methylated as well, but more weakly than the H3 (1-20) peptide. The PhosphoSite Plus data base contains all known lysine methylation sites in mouse and human proteins identified by various experimental methods.33 We retrieved all the lysine trimethylated human proteins in 2013 and screened for proteins containing the substrate sequence motif of SUV39H1 (Table 1). 35 proteins were identified, one of which is SET8, which was also identified in the first screening of SUV39H1 interactors. To test them at the peptide level, 15 amino acid length peptides were synthesized including H3 (2-16) and the methylated peptides identified in the first screening (RAG2 and SET8) as positive controls and H3K9A as a negative control (Suppl. Fig. 2B). Upon methylation with SUV39H1, 8 nonhistone peptides were found to be strongly methylated and another 6 peptides were weakly methylated. In agreement with the specificity profile, a comparison of the sequences of methylated peptides (Fig. 1D) show a striking enrichment of K and R at the +1 position and an overrepresentation of K and R at the -5 site. Moreover, the validated substrate sequences show an enrichment of K or R at the K14 position (+5) as well, which was not apparent from the peptide scans. Methylation of non-histone protein substrates After confirming the methylation of 19 potential new substrates at the peptide level, we proceeded to investigate their methylation at the protein level. We identified the domain boundaries of the respective proteins (http://bioinf.cs.ucl.ac.uk/dompred) and cloned the corresponding domains containing the target lysine as GST fusions. Some proteins failed at different stages of cloning, protein expression and purification; we eventually succeeded in purifying 10 of the protein domains (Fig. 2A, Suppl. Fig. 3A, Suppl. Table 4). Methylation at the protein level was analyzed by incubating the purified target protein domains with SUV39H1 in methylation buffer containing radioactively labeled AdoMet. The transfer of radioactively labeled methyl groups to the target proteins was analyzed by separating the proteins on SDS-PAGE and then subjecting the gels to autoradiography (Fig. 2A, Suppl. Fig. 4 Environment ACS Paragon Plus

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3A). Altogether, 7 out of the 10 protein domains were methylated indicating that the peptide based pre-screening was very successful. Two of the proteins, RAG2 and SET8, were strongly methylated, while 5 more showed moderate to weak methylation signals (DOT1L, JARID2, SCML2, MLL1 and SIN3B). To confirm that the identified non-histone target proteins were methylated at the predicted lysine residue, the target lysine was altered to arginine by site directed mutagenesis and the purified protein mutants were tested for methylation (Fig. 2B). The methylation signal was completely lost for 6 of the target lysine mutant proteins confirming that the predicted lysine was the site of modification. Since we had problems to express and purify the SIN3B K268R mutant at sufficient amount and quality, we could not confirm the methylation of the target lysine for this protein. Next, we aimed to determine the degree of methylation of the strongly methylated nonhistone targets RAG2 and SET8. SUV39H1 is known to trimethylate H3K9. RAG2 and SET8 were methylated in vitro with SUV39H1 using unlabeled AdoMet and the methylation level was analyzed using a commercial antibody offered as a pan-methyllysine antibody, which had been characterized by us to be specific for trimethyllysine in an RK context (Suppl. Fig. 4). The results showed that the antibody specifically recognized methylated RAG2 and SET8 while no signal was detected with the unmethylated proteins (Fig. 3A). In an independent experiment by mass spectrometry, we also showed that in vitro methylated RAG2 protein is di- and mostly tri-methylated at the target lysine by SUV39H1 (Fig. 3B). We could not detect methylation of SET8 by mass spectrometry, because the peptides containing the target lysine were missing both in the methylated and unmethylated samples. Collectively results from the two experiments showed that the SUV39H1 is adding up to three methyl groups to the target lysine of the newly identified substrates RAG2 and SET8. Details of the SUV39H1 substrate recognition As described above, the sequence preferences of SUV39H231 are distinct from SUV39H1. We, therefore, tested if SUV39H2 methylates any of the novel SUV39H1 substrates. For this purpose, the 6 substrates that were methylated by SUV39H1 at the protein level were incubated with SUV39H2 in the presence of radioactively labeled AdoMet. In contrast to the results obtained with SUV39H1 and in agreement with the SUV39H2 specificity profile, none of the SUV39H1 substrates was methylated by SUV39H2 (Suppl. Fig. 3B). This indicates that the SUV39H2 enzyme is more specific for the H3 substrate than SUV39H1, as no substrates were even identified with the SUV39H2 substrate specificity profile31 and one previously reported substrate could not be validated.34 As described above, our specificity profile shows that the lysine at the -5 position (K4 in H3) is an important contact for SUV39H1. In agreement with literature findings,23, 24 we observed that trimethylation of this residue reduces the activity of SUV39H1 (Suppl. Fig. 5A). Upon inspection of our novel SUV39H1 substrates, we observed that a lysine is present at -6 to -4 position in all methylated peptide substrates. Therefore, we tested the recognition of the additional lysine residues at the -4 and -5 position in RAG2 and SET8 as well and found that they are important for SUV39H1 activity (Suppl. Fig. 5B and C). Interestingly, the exact position of this additional lysine appears to be flexible within the -4 and -6 site. 5 Environment ACS Paragon Plus


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Cellular methylation of SUV39H1 substrate proteins After confirming the methylation of SUV39H1 non-histone targets in vitro, we investigated their methylation in cells. For this, we focused on the three target proteins (RAG2, SET8 and DOT1L) that were strongly methylated in vitro. For cellular experiments, we used the full length SET8 protein and domains of RAG2 and DOT1L. For RAG2, the C-terminal part (residues 311-520) was used which contains the PHD domain and a C-terminal nuclear localization signal, which is sufficient for its nuclear localization and activity.35 For DOT1L, the N-terminal part (1-472) was used, which contains the methyltransferase and nucleosome binding regions.36 To assess if these proteins are methylated in vivo, we ectopically expressed GFP-tagged target proteins in HEK293 cells in the presence and absence of SUV39H1. Two days after transfection, the target proteins were purified using GFP-Trap followed by western blot analysis using the pan-trimethyllysine antibody. The results show that the antibody only recognized RAG2, SET8 and DOT1L proteins that were co-expressed with SUV39H1, but not the proteins isolated from the cells without ectopic SUV39H1 expression (Fig. 3C). These results indicate that RAG2, SET8 and DOT1L proteins are methylated in cells by SUV39H1, but the endogenous levels of SUV39H1 in the HEK293 cells were insufficient to methylate the overexpressed substrates. Lysine methylation changes the sub-nuclear localization of RAG2 To explore potential biological effects of SUV39H1 methylation of RAG2, we investigated if K507 is relevant for the sub-nuclear localization of RAG2. NIH3T3 cells were transfected with RAG2 and RAG2 K507R expressing constructs fused to YFP and CFP. The results demonstrate that the wild type RAG2 protein is localized in the nucleus with a speckled distribution (Fig. 4A) as shown previously by others.37 In contrast to wild type RAG2, the RAG2 K507R mutant showed a uniform distribution in the nucleus (Fig. 4B). To explore the effect of SUV39H1 methylation of K507 on RAG2 localization, we co-expressed SUV39H1 and RAG2 wild type in NIH3T3 cells. Interestingly, after co-transfection of RAG2 with SUV39H1, the wild type RAG2 protein also showed a homogenous distribution in the nucleus (Fig. 4C and Suppl. Fig. 6A), while SUV39H1 showed the expected binding to DAPI stained heterochromatic foci in mouse cells.38 In control experiments, transfection of RAG2 K507R with SUV39H1 showed no changes in the localization pattern of RAG2 K507R (Suppl. Fig. 6B) and co-transfection of the RAG2 wild type protein with an inactive variant of SUV39H1 containing an H324L mutation39 did not disrupt the speckled sub-nuclear pattern of RAG2 (Fig. 4D). Collectively these results show that the unmethylated K507 is critical for the subnuclear localization of RAG2 and its methylation or mutation severely impaired the localization of RAG2 protein at nuclear spots. K210 methylation stimulates the activity of SET8 SET8 is an H4K20 monomethyltransferase40 that is essential for the subsequent generation of heterochromatic H4K20me2/3 by SUV4-20H enzymes.41, 42 Methylation of SET8 occurs at K210 in a protein part preceding the SET domain. Since allosteric regulation of PKMTs by regions preceding the SET domain has been observed,22 we aimed to investigate if SUV39H1 methylation of SET8 influences its activity. To this end, HEK293 cells were transfected with plasmids expressing YFP-fused SET8 in combination with either an empty vector or with the CFP-fused SUV39H1. After 72 hours of transfection, SET8 was purified using GFP-Trap and 6 Environment ACS Paragon Plus

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equal amounts of the SET8 protein purified from the cells with and without co-expression of SUV39H1 were used for in vitro methylation assays with recombinant H4 to determine the activity of SET8. The results showed an approximately 2-fold higher methylation of H4 with the SET8 purified after co-transfection with SUV39H1 (Fig. 5A). Control experiments with the SET8 K210R mutant did not show stimulation of activity after co-expression with SUV39H1 (Suppl. Fig. 7). We further validated the stimulation of SET8 activity by SUV39H1 methylation using in vitro methylated SET8. YFP fused SET8 protein was purified from HEK293 cells by GFP-Trap and the beads with bound SET8 were incubated with SUV39H1 in methylation buffer containing unlabeled AdoMet. Control reactions were prepared without addition of SUV39H1. The beads were washed to remove the AdoMet and SUV39H1 and the methylation activity of SET8 was investigated using recombinant H4 and radioactively labeled AdoMet (Fig. 5B). The results confirmed that the methylated SET8 exhibited an about 2-fold higher H4 methylation activity than the unmethylated SET8. Taken together these data indicate that the methylation of SET8 stimulates its H4 methyltransferase activity (Fig. 5C). Discussion The SUV39H1 PKMT generates H3K9me3 and is a key player in heterochromatin formation. By setting H3K9me3 it recruits reading proteins, for example HP1 family members, which then attract other factors, such as the SUV4-20H enzymes that generate H4K20me3. Despite the fact that it was the first PKMT discovered in mammals,9 so far no additional substrates of SUV39H1 have been described. In this study, we have determined the substrate specificity profile of SUV39H1 by employing peptide arrays and found that it differs from its paralog SUV39H2. This is an interesting observation, because in mammals many chromatin modifying enzymes are present in two or more paralogs, like SUV39H1/H2, MLL1/MLL2, MLL3/MLL4, G9A/GLP, SETDB1/2, and SUV4-20H1/H2. Our data indicate that one reason for the presence of different paralogs could be that they diverged in their substrate recognition, resulting in a situation in which both still interact with their histone substrate, but they have non-overlapping additional substrates, as demonstrated here for SUV39H1 and SUV39H2. A similar observation was recently made for the SUV4-20H1 and SUV4-20H2 enzymes,43 suggesting that this mechanism may also apply to paralogs of other PKMTs. Based on the specificity profile of SUV39H1, for the first time we were able to show that SUV39H1 also methylates proteins other than histones. We have identified several candidate substrates from the proteins that interact with SUV39H1 and showed methylation of 19 novel substrates at the peptide level and 6 at the protein level. By using a validated antibody, we showed that the RAG2, SET8 and DOT1L are trimethylated by SUV39H1 in cells at the target lysine residue. We studied the biological roles of these methylation events as discussed in the following paragraphs. RAG2 together with RAG1 forms the recombination machinery catalyzing the V (variable) D (diverse) J (joining) recombination of different immunoglobulin and T cell receptor genes in developing lymphocytes.44-46 We show here that the SUV39H1 methylates K507 of RAG2, which is located on the C-terminal tail of RAG2 next to its PHD domain and methylation of K507 leads to changes in the sub-nuclear localization pattern of RAG2. Our finding suggests 7 Environment ACS Paragon Plus


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that SUV39H1 could have a direct influence on VDJ recombination catalyzed by RAG2. This conclusion is in agreement with previous data showing that SUV39H1 regulates class switch recombination in B cells47 and H3K9me3 is associated with this process.48 Our data indicate that in addition to the generation of H3K9me3, SUV39H1 also has other roles in chromatin regulation. Loss of the SUV39H enzymes leads to decreased levels of heterochromatic H4K20me3,17 which so far was attributed to the recruitment of SUV4-20H enzymes by HP1 binding to H3K9me3. One of the new substrates of SUV39H1 is SET8, a PKMT which generates monomethylated H4K20.23, 40, 49 It has been shown that H4K20me1 produced by SET8 is the substrate used by the SUV4-20H enzymes for the generation of H4K20me3.41, 42 We show here that SUV39H1 methylation of SET8 increases its catalytic activity. This adds a second dimension to the control of SUV39H1 on heterochromatic H4K20 trimethylation, because SUV39H1 is not only involved in H4K20me3 generation by the indirect recruitment of the SUV4-20H enzymes via HP1 but also by stimulating SET8 to generate more H4K20me1 which is used by SUV4-20H as substrate (Fig. 5C). DOT1L, the third novel SUV39H1 substrate described here, is an evolutionarily conserved histone PKMT specific for lysine 79 of H3 (H3K79) which has important roles in development and cancer.50 DOT1L-deficient mouse embryos show reduced levels of heterochromatic H3K9me3 and H4K20me3 marks at centromeres and telomeres indicating that DOT1L plays an important role in heterochromatin formation as well.51 Our data indicate that there exists a reciprocal connection between H3K9 methylation and DOT1L, because SUV39H1 can methylate DOT1L, although the biological effects of this methylation event needs further investigation. Conclusions Protein lysine methylation is emerging as a general post-translational modification with essential functions regulating protein stability, activity, and protein-protein interactions. One of the outstanding challenges in this field is to identify the full substrate spectrum of protein lysine methyltransferases (PKMTs) which is a prerequisite to understand their cellular role. This paper investigates the SUV39H1 protein lysine methyltransferase, which introduces heterochromatic H3K9me3 and was the first PKMT identified in human cells back in 2000. Nevertheless, so far neither a detailed specificity analysis of SUV39H1, nor systematic search for additional substrates has been conducted, and no additional substrates have been identified. We determined the specificity profile of SUV39H1 showing that several residues on both sides of the methylation target K9 are very important for target site interaction. The specificity profile of SUV39H1 is distinct from its paralog SUV39H2 suggesting that they have non-redundant functions. Based on the specificity profile we describe several novel SUV39H1 substrates and confirmed cellular methylation of RAG2, SET8 and DOT1L, which are all functionally connected to SUV39H1. DOT1L has a role in the generation of H3K79me3, SET8 is involved in the formation of H4K20me3, another important heterochromatic modification, and RAG2 catalyzes VDJ recombination, a process known to be influenced by H3K9me3 and SUV39H1. Our data show that SUV39H1 methylation of SET8 can stimulate the formation of H4K20me3, and SUV39H1 methylation of RAG2 8 Environment ACS Paragon Plus

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regulates its chromatin interaction, suggesting that SUV39H1 has other roles in chromatin biology beyond the generation of H3K9me3. Experimental procedures Cloning, expression and purification of proteins The GST fused murine SUV39H1 SET domain (82-412) cloned into pGEX-2T vector was obtained from Dr. Xiaodong Cheng. Potential substrate protein domains or full length proteins (domain boundaries and protein accession numbers are described in Suppl. Table 3) were amplified by PCR using cDNA prepared from HEK293 cells as a template. The obtained sequences were cloned into pGEX-6P2 for bacterial expression and into pEYFP-C1 or pECFP-C1 for mammalian expression. The target lysine residues in the wild type protein were mutagenized by PCR mega primer method.52 GST fused DOT1L (1-472) constructs were obtained from Dr. Yongcheng Song53 and Dr. Yi Zhang.36 The proteins were overexpressed in BL21(DE3) cells by the addition of 1 mM IPTG after the cells reached an OD (measured at 600 nm) between 0.6 to 0.8. The cells were further grown at 20 °C for 12 hours and harvested by centrifuging at 4500 g for 15 minutes. The purification of GST fused proteins was performed as described before.54 SUV39H2 was prepared as described.31 Synthesis of peptide arrays and peptide array methylation Peptide array synthesis using the SPOT method55 and analysis were performed as described in our previous publication30 using an Autospot peptide array synthesizer (Intavis). All peptide arrays were prepared with free N-termini. The membranes containing the peptides were preincubated in methylation buffer (50 mM Tris/HCl pH 9.0, 5 mM MgCl2, 4 mM DTT) for 5 minutes. Then, the peptide arrays were incubated with 50 nM SUV39H1-SET in methylation buffer supplemented with 0.76 µM radioactively labeled methyl-[3H]-AdoMet (Perkin Elmer) for one hour at room temperature. Afterwards, the membranes were washed with washing buffer (100 mM ammonium bicarbonate and 1% SDS) for 5 times. Finally, membranes were incubated for 5 minutes in amplify NAMP100V (GE Healthcare) and then exposed to HyperfilmTM high performance autoradiography film (GE Healthcare) in dark at -800C for 1 to 2 days. Imaging and analysis was performed as described before.30 CelluSpots peptide binding experiments CelluSpots assays to determine the specificity of modified histone tail antibodies were carried out using MODified Histone Peptide Array (Active Motif) basically as described.56, 57 Annotations of all spots are given in Suppl. Table 5. In vitro protein methylation assay Protein methylation reactions were performed by incubating the substrate proteins in methylation buffer (50 mM Tris/HCl pH 9.0, 5 mM MgCl2, 4 mM DTT) supplemented with 1.4 µM SUV39H1 and 0.76 µM radioactively labeled AdoMet for 3 h at 25 °C. The reaction was halted by incubating the samples in the SDS loading buffer at 95 °C for 5 minutes. Proteins were then separated on 16% SDS-PAGE and the transfer of radioactively labeled methyl groups to the substrate proteins was detected by autoradiography or the protein methylation was detected using the methyl specific antibody by western blot. 9 Environment ACS Paragon Plus


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Mass Spectrometry After in vitro protein methylation, samples were separated on SDS-PAGE and stained with Coomassie brilliant blue. Protein bands were excised from the gel and washed 3 times using a 1:1 ratio of 50 mM ammonium bicarbonate buffer and 100% acetonitrile to remove the Coomassie stain. The gel pieces were then incubated in 100% acetonitrile for 10 minutes and lyophilized. Then, the dried gel pieces were subjected to trypsin digestion by incubating with digestion buffer (40 mM ammonium bicarbonate and 10% acetonitrile) supplemented with 5 ng/µl trypsin (Promega) overnight at 37 °C. Peptides were extracted with extraction buffer (25% acetonitrile in 0.1% TFA), dried by vacuum centrifugation and dissolved in 0.1% TFA for the analysis. Sample spotting on an anchor chip plate and collecting the spectra was performed as described31 using an Autoflex Speed system (Bruker Daltonics). Data were analyzed with the Biotools 2.0 software (Bruker Daltonics). Cell culture, transfection and immunoprecipitation Cells were grown in Dulbecco´s Modified Eagle´s Medium (Sigma) supplemented with either 5% fetal bovine serum (HEK293 cells) or 10% fetal bovine serum (NIH3T3 cells), along with penicillin, streptomycin and L-glutamine (Sigma). For immunoprecipitation experiments, the target protein domains or full length constructs were transfected with or without SUV39H1 expression construct in HEK293 cells using polyethylenamine (Promega), according to the manufacturer´s instructions. After 48-72 h of transfection, the cells were washed with PBS buffer and harvested by centrifuging at 525 g for 5 min. The YFP fused proteins were immunoprecipitated from mammalian cell extract using GFP-Trap A beads (Chromotek) following the manufacturer’s instructions. SET8 methylation assays YFP-tagged SET8 were purified after expression in HEK293 cells as described in the last section. For in vitro SET8 methylation, the SET8-YFP (bound to the beads) was incubated with 7 µM SUV39H1 in methylation buffer (50 mM Tris/HCl pH 9.0, 5 mM MgCl2, 4 mM DTT) containing 0.6 mM unlabeled AdoMet overnight at 25 °C on a shaker. Then, the beads were washed three times with lysis buffer (10 mM Tris/HCl pH 7.5, 300 mM NaCl, 0.5 mM EDTA, 0.5 % NP40) to remove the SUV39H1 and the unlabeled AdoMet. As control, an identically treated sample without SUV39H1 was prepared. For SET8 activity assay, the beads were resuspended in SET8 methylation buffer (20 mM HEPES pH 8.0, 50 mM NaCl, 5 mM DTT) and 60 µg/ml recombinant H4 and 0.76 µM radioactively labeled AdoMet were added. The methylation was performed for 3 h at 25 °C on a shaker. The reaction was halted by heating the samples at 95 °C for 5 minutes in the SDS loading buffer. The samples were separated on a 16% SDS-PAGE and the transfer of the methyl groups was detected by autoradiography. Imaging of fixed cells NIH3T3 cells were cultured on glass coverslips until they attain 70 to 80% confluence, the cells were then transfected with RAG2-YFP or RAG2-CFP either with or without SUV39H1 (YFP or CFP) expression construct using Fugene HD (Promega). After 24 h of transfection, cells were washed with PBS buffer and fixed using 3.7% paraformaldehyde for 10 minutes at room temperature. The cells were finally washed with PBS buffer and the coverslips were 10 Environment ACS Paragon Plus

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mounted on a glass slide using Mowiol. Images were taken using an LSM 710 confocal microscope (Carl Zeiss). Acknowledgements This work has been supported by the DFG JE 252/7-2. We thank Dr. S. Yongcheng and Dr. Y. Zhang for the DOT1L bacterial expression constructs and Dr. X. Cheng for the SUV39H1 bacterial expression construct.

Author contributions SK and AJ designed the experiments. SK and MKS performed the experiments with help of AFK. All authors contributed to data analysis and interpretation. SK and AJ wrote the manuscript draft. All authors contributed to the editing of the manuscript and approved its final version.

Supporting Information Available: This material is available free of charge via the Internet.



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References

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Tables Position H3 sequence -5 -4 -3 -2 -1 0 +1 +2 +3 +4

K4 Q5 T6 A7 R8 K9 S10 T11 G12 G13

SUV39H1 SUV39H2 preference preference K > R>>N X not hydrophobic X ASTY X ANLPW > QH X R R K K RKS > T S>T AGST T > S,G,A QGK > X G >> R X G >> X

Table 1: Specificity profile comparison of SUV39H paralogs. The SUV39H1 profile has been determined in this work. The SUV39H2 profile is adopted from Schuhmacher et al, 2015.31


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Figure legends Fig. 1: Substrate specificity analysis of SUV39H1. A) Peptide arrays were synthesized using the H3 (1-20) sequence as a template and each amino acid was individually exchanged with all the naturally available amino acids. The peptide array membrane was then incubated with SUV39H1 in the presence of radioactively labeled AdoMet and the transfer of methyl groups was detected by autoradiography. B) The results were compiled from three experiments and the data were averaged after normalizing the activity (see also Suppl. Fig. 1B). Activity is represented in a greyscale. C) Discrimination factors were determined for the recognition of amino acids at each position of the H3 (1-15) sequence. D) Alignment of the sequences of 19 newly discovered SUV39H1 peptide substrates with the sequence of H3K9 (Histone H3). Fig. 2: In vitro methylation of non-histone target proteins. A) Purified GST fused protein domains were analyzed on SDS-PAGE and stained with Coomassie as loading control. GSTfused protein domains were incubated with SUV39H1 in the presence of radioactively labeled AdoMet and separated on SDS-PAGE. The transfer of methyl groups was detected by autoradiography. Asterisks indicate the target protein size. B) Confirmation of methylation of the predicted lysine residue. Purified wild type proteins and the target lysine mutants were incubated with the SUV39H1 in the presence of radioactively labeled AdoMet and the transfer of methyl groups were detected by autoradiography. The Coomassie stain images show the equal loading of the wild type and target lysine mutant proteins. Fig. 3: Methylation studies with three novel SUV39H1 substrates. A) Methylation level analysis of SUV39H1 methylated SET8 and RAG2 using a pan-trimethyllysine antibody (Suppl. Fig. 4). Methylated RAG2 and SET8 proteins (M) were generated by incubation with SUV39H1 in the presence of unlabeled AdoMet. Unmethylated RAG2 and SET8 proteins (UM) were used as controls. Western blots were probed with the pan-trimethyllysine antibody. The Coomassie stain represents the loading control of proteins used in the assay. B) Methylation level analysis of SUV39H1 methylated RAG2 by mass spectrometry. In vitro methylated and unmethylated RAG2 protein domains were subjected to in-gel digestion with trypsin and the extracted peptides were analyzed by mass spectrometry. Peptide peaks corresponding to the fragments containing the un- (604.0 Da), di- (632.4 Da) and trimethylated (646.4 Da) target lysine are annotated. C) Cellular methylation of novel targets by SUV39H1. YFP fused substrate proteins were transiently expressed in the HEK293 cells in the presence and absence of ectopically expressed SUV39H1. YFP fused substrate proteins were purified by GFP-Trap and then subjected to western blot analysis using the same antibody as in panel A). Methylation signal was detected with the RAG2, SET8 and DOT1L proteins purified from the HEK293 cells with the ectopically expressed SUV39H1. GFP-Trap purified proteins were probed with GFP antibody as loading control. Fig. 4: Effects of the methylation of RAG2 (311-520). A) Sub-nuclear localization of RAG2 wild type in NIH3T3 cells. RAG2-YFP (yellow) showed a spotty sub-nuclear pattern. B) In contrast to wild type RAG2, the CFP fused RAG2 K507 mutant (blue) showed a diffused appearance in the nucleus of NIH3T3 cells. C) After co-transfection of RAG2-YFP (yellow) and SUV39H1-CFP (blue), RAG2 lost the speckled nuclear localization and changed to a 17 Environment ACS Paragon Plus


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diffuse nuclear localization. SUV39H1 showed a regular binding to heterochromatic spots. D) RAG2-YFP shows the “normal” speckled appearance in the nucleus after co-expression with the SUV39H1 H324L inactive mutant. See Suppl. Fig. 6 for additional controls. Fig. 5: SET8 methylation increases its H4 methylation activity. A) YFP-fused full length human SET8 was purified from HEK293 cells with and without co-expression of SUV39H1. The purified SET8 protein was incubated with recombinant H4 in the presence of radioactively labeled AdoMet. The samples were separated on SDS-PAGE and the methylation signal was detected by autoradiography. The bar diagram shows the averaged SET8 activities observed in three independent biological experiments, the error bar represents the standard error of the mean. B) Stimulation of the SET8 protein methyltransferase activity after in vitro methylation with SUV39H1. YFP fused SET8 protein bound to GFP-Trap beads was methylated by SUV39H1 using unlabeled AdoMet. Control reactions were prepared without SUV39H1 and treated identically. Afterwards, AdoMet and SUV39H1 were removed by washing the beads. The unmethylated and methylated SET8 was then incubated with recombinant H4 in the presence of radioactively labeled AdoMet and the H4 methylation was detected by autoradiography. The bar diagram represents the average H4 methylation signals obtained from two independent experiments, the error bar represents the standard error of the mean. C) Model of the role of SUV39H1 methylation of SET8 in the generation of H4K20me3. For details refer to the text.


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Fig. 1: Substrate specificity analysis of SUV39H1. A) Peptide arrays were synthesized using the H3 (1-20) sequence as a template and each amino acid was individually exchanged with all the naturally available amino acids. The peptide array membrane was then incubated with SUV39H1 in the presence of radioactively labeled AdoMet and the transfer of methyl groups was detected by autoradiography. B) The results were compiled from three experiments and the data were averaged after normalizing the activity (see also Suppl. Fig. 1B). Activity is represented in a greyscale. C) Discrimination factors were determined for the recognition of amino acids at each position of the H3 (1-15) sequence. D) Alignment of the sequences of 19 newly discovered SUV39H1 peptide substrates with the sequence of H3K9 (Histone H3). Fig. 1 186x151mm (300 x 300 DPI)

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Fig. 2: In vitro methylation of non-histone target proteins. (A) Purified GST fused protein domains were analyzed on SDS-PAGE and stained with Coomassie as loading control. Asterisks indicate the target protein size. GST-fused protein domains were incubated with SUV39H1 in the presence of radioactively labeled AdoMet and separated on SDS-PAGE. The transfer of methyl groups was detected by autoradiography. Asterisks indicate the target protein size. (B) Confirmation of methylation of the predicted lysine residue. Purified wild type proteins and the target lysine mutants were incubated with the SUV39H1 in the presence of radioactively labeled AdoMet and the transfer of methyl groups were detected by autoradiography. The Coomassie stain images show the equal loading of the wild type and target lysine mutant proteins. Fig. 2 190x142mm (300 x 300 DPI)


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Fig. 3: Methylation studies with three novel SUV39H1 substrates. (A) Methylation level analysis of SUV39H1 methylated SET8 and RAG2 using a pan-trimethyllysine antibody (Suppl. Fig. 4). Methylated RAG2 and SET8 proteins were generated by incubation with SUV39H1 in the presence of unlabeled AdoMet. Unmethylated RAG2 and SET8 proteins were used as a control. Western blots were probed with the pan-trimethyllysine antibody that specifically recognizes trimethylated proteins. The Coomassie stain represents the loading control of proteins used in the assay. (B) Methylation level analysis of SUV39H1 methylated RAG2 by mass spectrometry. In vitro methylated and unmethylated RAG2 protein domains were subjected to in-gel digestion with trypsin and the extracted peptides were analyzed by mass spectrometry. Peptide peaks corresponding to the fragments containing the un- (604.0 Da), di- (632.4 Da) and trimethylated (646.4 Da) target lysine are annotated. (C) Cellular methylation of novel targets by SUV39H1. YFP fused substrate proteins were transiently expressed in the HEK293 cells in the presence and absence of ectopically expressed SUV39H1. YFP fused substrate proteins were purified by GFP-Trap and then subjected to western blot analysis using the same antibody as in panel A). Methylation signal was detected with the RAG2, SET8 and DOT1L proteins purified from the HEK293 cells with the ectopically expressed SUV39H1. GFP-Trap purified proteins were probed with GFP antibody as loading control. Fig. 3 140x79mm (300 x 300 DPI)



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Fig. 4: Effects of the methylation of RAG2 (311-520). A) Sub-nuclear localization of RAG2 wild type in NIH3T3 cells. RAG2-YFP (yellow) showed a spotty sub-nuclear pattern. B) In contrast to wild type RAG2, the CFP fused RAG2 K507 mutant (blue) showed a diffused appearance in the nucleus of NIH3T3 cells. C) After co-transfection of RAG2-YFP (yellow) and SUV39H1-CFP (blue), RAG2 lost the speckled nuclear localization and changed to a diffuse nuclear localization. SUV39H1 showed a regular binding to heterochromatic spots. D) RAG2-YFP shows the “normal” speckled appearance in the nucleus after co-expression with the SUV39H1 H324L inactive mutant. See Suppl. Fig. 6 for additional controls. Fig. 4 115x52mm (300 x 300 DPI)


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Fig. 5: SET8 methylation increases its H4 methylation activity. (A) YFP-fused full length human SET8 was purified from HEK293 cells with and without co-expression of SUV39H1. The purified SET8 protein was incubated with recombinant H4 in the presence of radioactively labeled AdoMet. The samples were separated on SDS-PAGE and the methylation signal was detected by autoradiography. The bar diagram shows the average SET8 activities observed in three independent biological experiments, the error bar represents the standard error of the mean. (B) Stimulation of the SET8 protein methyltransferase activity after in vitro methylation with SUV39H1. YFP fused SET8 protein bound to GFP-Trap beads was methylated by SUV39H1 using unlabeled AdoMet. Control reactions were prepared without SUV39H1 and treated identically. Afterwards, AdoMet and SUV39H1 were removed by washing the beads. The unmethylated and methylated SET8 was then incubated with recombinant H4 in the presence of radioactively labeled AdoMet and the H4 methylation was detected by autoradiography. The bar diagram represents the average H4 methylation signals obtained from two independent experiments, the error bar represents the standard error of the mean. (C) Model of the functional role of SUV39H1 methylation of SET8. For details refer to the text.



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Fig. 5 190x258mm (300 x 300 DPI)


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Table of content image 148x123mm (300 x 300 DPI)


The SUV39H1 Protein Lysine Methyltransferase ... - ACS Publications

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