Abstract
Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and regulation of gene expression1–4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H35-11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help limit inappropriate H3K9me spreading12–15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me leading to aberrant gene silencing and epigenetic instability is not fully understood. Here we reveal an autoinhibited conformation in the conserved fission yeast S. pombe H3K9 methyltransferase Clr4/Suv39h that plays a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits its catalytic activity by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances Clr4 H3K9 methylation activity. Mutations predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains, and growth inhibition, demonstrating the importance of Clr4 intrinsic inhibition and auto-activation in regulating H3K9me deposition and preventing epigenetic instability. Conservation of the Clr4 autoinhibitory loop in other H3K9 methyltransferases, and automethylation of a corresponding lysine in the human SUV39H2 homolog16, suggest that the mechanism described here is broadly conserved.
While examining the in vitro methyltransferase activity of Clr4 (Extended Data Fig. 1a), we noticed that Clr4 methylated itself in the presence or absence of its canonical histone H3(1−20) substrate (Fig. 1a, Extended Data Fig. 1b). Whereas wild-type Clr4 readily methylated itself, it was unable to methylate a catalytically inactive GST-Clr4 (GST-Clr4dead) in trans, suggesting that Clr4 undergoes intra-molecular automethylation (Fig. 1b, Extended Data Fig. 1b). Moreover, Clr4 automethylation produced a more active enzyme, since pre-incubation of Clr4 with non-radioactive S-Adenosyl-L-methionine (SAM) to allow automethylation, prior to performing methylation reactions with 3H-SAM and histone H3(1−20), increased its activity on H3(1−20) (Fig. 1c, Extended Data Fig. 1c, d).
To identify the automethylated lysine(s) in Clr4, we incubated Clr4 with non-radioactive SAM for different time points followed by tandem mass spectrometry (LC-MS/MS) analysis. A purified catalytically dead Clr4 protein was also analyzed by quantitative mass spectrometry to rule out lysines methylated by E. coli enzymes. We consistently identified methylation of Clr4 K127 (me1 and me3) and K455 (me1, me2, and me3) in wild-type relative to catalytic dead Clr4, K127 (me2) in both wild-type and catalytically dead Clr4, and K464 (me1) in some preparations (Fig. 1d; Extended Data Fig. 1e, Extended Data Fig. 2). Among these lysines, only the substitution of K455 with arginine (K455R), a lysine mimic that cannot be methylated by SET domain methyltransferases such as Clr4, greatly diminished Clr4 automethylation while its combination with K464R substitution did not further decrease Clr4 automethylation (Fig. 1e; Extended Data Fig. 1f, g). Clr4-K455 was also the only lysine that showed increased time-dependent methylation upon Clr4 incubation with SAM (Extended Data Fig. 2d). Furthermore, in contrast to wild-type Clr4, pre-incubation of Clr4-K455R with non-radioactive SAM did not stimulate its ability to methylate H3(1−20) (Fig. 1f). Thus, Clr4-K455 is a primary target of automethylation and its substitution with arginine inhibits Clr4 autoactivation.
The previously solved crystal structure of Clr4 lacks cofactor, and K455 and surrounding residues point away from the active site17. We therefore solved the crystal structure of Clr4 (Clr4192−490) in complex with the methyl donor analog S-Adenosyl-L-homocysteine (SAH)(Fig. 1d). The 2.4 Å resolution Clr4192−490 structure displayed a similar overall conformation to the previously described Clr4192−490 structure, but contained additional density allowing us to model the post-SET domain and SAH (Fig. 1g; Extended Data Fig. 3a)17. Moreover, the catalytic pocket of Clr4192−490 was occluded by K455, whether our crystallization conditions included histone H3 peptide or not (Fig. 1h, Extended Data Fig. 3b). Clr4-K455 is located in a loop (aa 453 to aa 473) that connects the SET and post-SET domains, henceforth referred to as autoinhibitory (AI) loop (Fig. 1d; highlighted in red). Alignment of our Clr4192−490 structure with the published structure of DIM-5 in complex with a histone H3 peptide18 showed that the histone H3 peptide and the backbone of Clr4 AI loop occupied similar positions (root-mean-square deviation = 0.761 Å) but were stabilized through distinct contacts with the respective catalytic pockets (Extended Data Fig. 3c-e). More importantly, Clr4-K455 occupied a similar position to that of lysine 9 of the histone H3 substrate in DIM-5 near the sulfur of SAH and interacted almost identically with residues of the catalytic site (Fig. 1h, i; Extended Data Fig 3f). Thus, consistent with the biochemical and mass spectrometry identification of Clr4-K455 as a target of automethylation, it was located in Clr4 active site where it can block access to the histone substrate and could itself act as a methyl group acceptor. To directly test this hypothesis, Clr4-K455 was substituted with arginine, a lysine mimic which cannot be methylated by Clr4 or with alanine, a small non-polar side chain that cannot interact with the active site and should disfavor the autoinhibited conformation (Extended data Fig. 3g-i). Consistent with our hypothesis, while Clr4-K455R displayed reduced Clr4 activity on the H3 substrate relative to wild-type Clr4, Clr4-K455A increased histone H3(1−20) methylation (Fig. 1j).
To evaluate the consequences of automethylation on Clr4 conformation, we solved the crystal structure of automethylated Clr4192–490. The 2.8 Å structure revealed a dramatic conformational rearrangement in Clr4 (Fig. 2a). In contrast to the autoinhibited conformation (Fig. 2b, left), in the automethylated Clr4, K455 and C-terminal amino acids were disordered and therefore no longer occluded the catalytic pocket (Fig. 2b, right; Extended Data Fig. 4a-e). Conversely, the C terminal residues in the AI loop (residues 468 to 472), which were disordered in the autoinhibited structure (Fig. 2b, left), formed a helix that interacted with and stabilized the SET insertion (SET-I, residues 371 to 379) domain (Fig. 2b, right), which was also partially disordered in the autoinhibited structure (Fig. 2b, left). The SET-I domain has been proposed to play a conserved role in substrate recognition and cofactor binding and its conformation is also modulated by protein-protein interactions in other SET domain methyltransferases19,20. Clr4 therefore exists in an inhibited conformation, which is reversed by an automethylation-induced conformational switch.
Similar to wild-type Clr4, we detected stimulation of Clr4-K455A methyltransferase activity on histone H3(1−20) following preincubation with non-radioactive SAM (Fig. 1c; Fig. 3a). This observation suggested that, in addition to K455, methylation of at least one other lysine contributed to the relief of Clr4 autoinhibition. In addition to K455, Clr4 AI loop contains lysines at positions 464, which we already ruled out as a major target of automethylation (Fig. 1e), and 472. In vitro methyltransferase assays showed that Clr4-K472A had increased H3 substrate methylation activity, which was greatly enhanced in combination with K455A (Fig. 3b). In contrast, Clr4-K472R had slightly reduced automethylation activity and reduced H3 peptide substrate methylation activity, which was further reduced in combination with K455R (Fig. 3b). The addition of K472R mutation to the hyperactive Clr4-K455A protein also greatly reduced both Clr4-K455A automethylation and methyltransferase activity toward H3(1−20) (Extended Data Fig. 5a). Together these findings suggest that Clr4 full activation requires methylation of both K455 and K472. Consistently, Clr4-K472R H3(1−20) methylation activity was not stimulated after pre-incubation with non-radioactive SAM (Extended Data Fig. 5b) and kinetic analysis indicated faster and slower rates of histone H3(1−20) methylation for single and double Clr4-K455A or -K472A and Clr4-K455R or -K472R substitutions, relative to wild-type Clr4, respectively (Fig. 3c). Analysis of automethylated Clr4 by LC-MS/MS (Fig. 1d; Extended Data Fig. 1 and 2) did not uncover Clr4-K472 methylation probably because trypsin or LysC digestion generated K472 peptides that were not readily detectable by LC-MS/MS. However, the lysine corresponding to Clr4-K472 was recently reported to be automethylated in the human SUV39H2 (K392)16, which together with our findings supports an evolutionarily conserved role for this automethylation event. We note that in contrast to our findings, which reveal an activating role for automethylation of Clr4-K472, automethylation of the corresponding human SUV39H2-K392 was proposed to inhibit its methyltransferase activity16. This apparent contradiction is likely due to dilution of 3H-SAM by excess non-radioactive SAM, which was not removed prior to histone peptide methylation assays with SUV39H216.
To investigate effect of Clr4 autoregulation on heterochromatin formation and silencing, we constructed S. pombe cells expressing endogenous Clr4 mutant proteins with single or double substitutions at K455 and K472 (Extended Data Fig. 5c). We predicted that silencing would be weakened in cells expressing hypoactive Clr4 with lysine to arginine substitutions and strengthened in cells expressing hyperactive Clr4 with lysine to alanine substitutions (Fig. 3c, Extended Data Fig. 5d), or substitution with tryptophan, which is too bulky to fit into the active site and should destabilize the autoinhibited conformation. Gene silencing was examined using an ade6+ reporter transgene inserted within the heterochromatic mat locus (mat2P::ade6+; Fig. 3d, left). Silencing of ade6+ depends on heterochromatin spreading from surrounding regions and results in growth of red colonies on medium with limiting adenine. The lysine to arginine clr4 mutant cells displayed partial or complete loss of mat2P::ade6+ silencing relative to clr4+ cells, as they formed a greater percentage of white colonies, with the strongest effect observed in the double mutant clr4-K455R/K472R (Fig. 3d, middle). Conversely, substitutions of lysine to alanine or tryptophan (for K455) resulted in increased mat2P::ade6+ silencing as indicated by growth of a higher percentage of red colonies (Fig. 3d, right). Clr4 autoinhibition therefore plays a critical role in heterochromatin-dependent transgene silencing.
In order to gain insight into possible genome-wide changes in H3K9me in the Clr4 activity mutants, we performed chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq). In agreement with the silencing data, ChIP-seq analysis in hypoactive clr4-K455R and -K472R mutant cells, showed reduction or loss of H3K9me levels at rDNA, subtelomeric and pericentromeric DNA repeats (Fig. 3e; Extended Data Fig. 5e, f). More strikingly, several clones expressing hyperactive Clr4 enzymes (K to A or W substitutions) showed variable increase in H3K9me levels and spreading, beyond the restricted H3K9me domains in wild-type cells, at all heterochromatin regions, with dramatic increases in spreading at tel2R, spanning over 100 kb in some clones, and gained H3K9me3 at all meiotic genes and other euchromatic genes (Fig. 3e, f; Extended Data Fig. 5e, f, Supplementary Table 1 and 2), while other clones showed loss of reporter gene silencing and severely diminished or loss of H3K9me3 levels, particularly at the mat locus (Extended Data Fig. 6a, b). In addition, the hyperactive clr4 mutant cells grew slower than clr4+ or clr4Δ cells, suggesting that inappropriate heterochromatin formation in the hyperactive mutant cells inactivates genes that are required for growth (Extended Data Fig. 7a). Thus, disruption of Clr4 autoregulation results in illegitimate spreading of heterochromatin beyond its normal boundaries, ectopic gain of H3K9me at euchromatic loci, and growth inhibition.
We next asked whether Clr4 intrinsic autoinhibition acts in parallel with other anti-silencing pathways. We first tested whether a limitation in the cellular concentration of Clr4 contributes to anti-silencing by increasing the dosage of wild-type or mutant Clr4 enzymes. Relative to cells carrying one copy (1x), cells carrying two copies of clr4+ (2x) displayed stronger mat2P::ade6+ silencing, indicating that the dosage of clr4+ indeed influenced the strength of silencing at the mat locus (Extended Data Fig. 7b, first two rows). Moreover, increased dosage of hypoactive clr4 single and double mutants, partially suppressed their mat2P::ade6+ silencing defect (Extended Data Fig. 7b). In contrast, when we increased the dosage of hyperactive clr4-K455A or clr4-K455A/K472A mutants, we observed variable and decreased silencing as indicated by the appearance of pink and white colonies (Fig. 4a). Consistent with the hypothesis that a balance between Clr4 levels and activation potential regulates silencing in vivo, ChIP-seq analysis of H3K9me indicated that two independent clones carrying an extra copy of clr4+ displayed increased H3K9me spreading (e.g. tel2R) and gained de novo domains of H3K9me (Fig. 4b; Extended Data Fig. 7c, d; Supplementary Table 1). Furthermore, increased dosage of the hyperactive clr4 mutants exacerbated their growth defect, increased H3K9me spreading at rDNA, subtelomeric, and pericentromeric and resulted in the appearance of several de novo peaks of H3K9me, including at rpb1+, which encodes the largest subunit of RNA polymerase I, rik1+, and clr4+ (Fig. 4b, c; Extended Data Fig. 7c-e; Supplementary Table 1). The latter two genes encode subunits of the Clr4 methyltransferase complex, suggesting that epigenetic adaptation, previously observed in other mutant backgrounds that promote deleterious heterochromatin spreading21, may allow cells to overcome the toxicity of unchecked Clr4 hyperactivity.
We next investigated whether Clr4 intrinsic autoinhibition acts in parallel with Epe1, a putative H3K9 demethylase that limits heterochromatin spreading13–15. Consistent with previous studies, genome-wide analysis of H3K9me in epe1Δ cells revealed variations in the levels of H3K9me levels at native heterochromatic loci, increased H3K9me spreading at some heterochromatic loci, and higher levels of H3K9me at meiotic genes14,15 (Extended Data Fig. 8a, b; Supplementary Table 2). In addition, we observed ectopic peaks of H3K9me at several non-meiotic genes in epe1Δ cells (Extended Data Fig. 8a, b; Supplementary Table 2). Such ectopic H3K9me peaks became more prevalent in epe1Δ ago1Δ double mutant cells with some clones also displaying signatures of epigenetic adaptation, such as gain of H3K9me at clr4+ itself (Extended Data Fig. 8c, d), similar to Clr4 hyperactive mutants at increased dosage (Fig. 4c). The enhancement of the epe1Δ phenotype in the epe1Δ ago1Δ double mutant cells is likely to result from an increase in free Clr4 concentration due to its release from pericentromeric and telomeric DNA repeats, where heterochromatin formation is Ago1-dependent22. To more directly test the relationship between Epe1-mediated anti-silencing and Clr4 autoinhibition, we deleted epe1+ in clr4 hyperactive mutant cells. Surprisingly, and in contrast to epe1∆ clr4+ cells, epe1∆ clr4 hyperactive mutant cells displayed reduced, rather than increased, mat2P::ade6+ silencing phenotypes, which were more drastic in some epe1∆ clr4-K455A/K472A mutant clones (Fig. 4d; Extended Data Fig. 8e). ChIP-seq analysis of different clones with variable silencing defects showed that loss of mat2P::ade6+ silencing in a representative epe1∆ clr4-K455A/K472A clone was accompanied by loss of H3K9me at ade6+ and the mat locus (clone #5; Fig. 4e; Extended Data Fig. 8f). Remarkably, the loss or reduced silencing in epe1∆ clr4 hyperactive double mutants was often accompanied by gain of de novo peaks of H3K9me at genes that encode heterochromatin proteins, such as clr4+ itself, rik1+, or sir2+ (Fig. 4f; Extended Data Fig. 8f right; Supplementary Table 2), suggesting that the survival of cells lacking both intrinsic Clr4 autoinhibition and extrinsic H3K9me demethylation depends on epigenetic downregulation of factors required for heterochromatin formation. Thus, Clr4 intrinsic autoinhibition acts in parallel with extrinsic (Clr4 levels and Epe1-promoted demethylation) anti-silencing mechanisms to maintain accurate H3K9me domains and epigenetic stability. Our results also raise the possibility that Clr4 is a target of Epe1-mediated demethylation. However, since combining epe1∆ with Clr4 hyperactive mutants resulted in more severe growth and silencing phenotypes than that of the hyperactive mutants by themselves (Fig. 4), Epe1 is likely to have substrates beyond Clr4 K455 or K472.
Finally, combining mutations in the chromodomain of Clr4 (W31G/W41G), which disrupt its ability to recognize H3K9me, with Clr4 hyperactive mutants (K455A/K472A) suppressed the illegitimate heterochromatin formation phenotype of the hyperactive mutants (Extended Data Fig. 9a, b), indicating that autoregulation counteracts feedback-mediated methylation leading to aberrant heterochromatin formation (Extended Data Fig. 9c).
The intrinsic enzyme inhibition mechanism described here adds a new layer of control that complements extrinsic anti-silencing mechanisms involving Epe1-promoted H3K9 demethylation, boundary elements, and appropriate Clr4 levels (Extended Data Fig. 10a). Previous structural analysis of SET domain methyltransferases suggests that intrinsic inhibition is broadly conserved19,20,23,24. The autoinhibitory loop described here appears to be conserved in the mammalian Clr4 homologs23, although its physiological significance in mammals has not been addressed. In the crystal structure of human SUV39H2, K375, which corresponds to Clr4-K455 (Extended Data Fig. 10b), is located in a similar position between the SET and post-SET domains as Clr4-K455. But unlike Clr4-K455, SUV39H2-K375 is inserted only halfway into the catalytic pocket (Extended Data Fig. 10c)23. This lysine is also conserved in the mouse Suv39h2, and both the mouse and human SUV39H1 and SUV39H2 contain a lysine in analogous positions that may correspond to Clr4-K472, the second inhibitory lysine in the Clr4 AI loop, which was recently reported to be automethylated in human SUV39H2 (Extended Data Fig. 10b)16. The fission yeast Clr4 and mammalian SUV39H are components of positive feedback loops that can mediate sequence-independent spreading and epigenetic inheritance of histone modifications8,9,25,26. We propose that intrinsic enzyme inhibition, first described for cyclin-dependent kinases27, plays a critical role in preventing positive feedback-coupled chromatin-modifying enzymes from initiating illegitimate epigenetic inactivation of the genome.
Methods
Strains and plasmids construction
S. pombe strains used in this study and their genotypes are listed in Supplementary Table 3. Plasmids are listed in Supplementary Table 4. To generate pGEX-6P-1-Clr4 (pDM2113) and pGEX-6P-1-Clr4 192–490 (pDM1906), the coding sequence of Clr4 full length (amino acids 1–490) and Clr4 sequence encoding amino acids 192–490 were amplified from S. pombe genomic DNA and cloned into an EcoRI/XhoI and BamHI/XhoI digested pGEX-6P-1 plasmid backbone (GE Healthcare), respectively. pGEX-6P-1-Clr4-Y451N (GST-Clr4dead, pDM2142) contains Y451N amino acid substitutions in the catalytic SET domain28. Mutagenesis was performed by partial overlap PCR and plasmids sequences were verified by DNA sequencing.
All strains, except SPY7249-SPY7260, were constructed by transformation using a PCR-based targeting method and lithium acetate protocol29 and selected on yeast extract plus adenine (YEA) plates containing appropriate antibiotic. SPY7249-SPY7260 strains (ago1Δ epe1Δ strains #4–14 in Extended Data Figure 9) were constructed by crossing SPY2352 and SPY3216 followed by random spore analysis. Integrations were confirmed by colony PCR and clr4+ and mutant sequences were verified by DNA sequencing. All Flag-tagged Clr4 or mutant genes were expressed under the control of their endogenous promoters and terminators. SPY8363–5 strains were constructed by integrating full length 3xFlag-clr4-W31G/W41G/K455A/K472A mutants open reading frames in place of 3xFlag-clr4-K455A/K472A in SPY7361. SPY7363–4, SPY7374–6, SPY7384–5, SPY7366–7, SPY7368–9, and SPY7371–2 strains were constructed by integrating full length clr4+ or mutants open reading frames and the endogenous clr4+ promoter and terminator sequences about 450 bp upstream of the trp1+ gene in the SPY3 strain.
Silencing assays
Silencing of the ade6+ reporter gene was assessed by growth on YE plates at 32 °C followed by incubation at 4 °C 1–2 days to enhance the red pigmentation prior to imaging. The plates were incubated for 3–5 days at 32 °C and photographed. Ura4 silencing was assessed by growth on the drug FOA, which is toxic to cells expressing ura4. Cells were grown at 30 °C overnight in 5 ml of YEA medium. A concentration of 1 × 107 cells/ml was harvested, washed once with sterile water, suspended in 200 µl of sterile water, and then serially diluted 10 fold. Three µl of each dilution was spotted on the appropriate growth medium (YEA, EMM-Ura, YEA+FOA, containing 1 mg/ml of 5-fluoro-orotic acid, FOA).
Protein purification and methyltransferase assay
BL21(DE3) strains expressing GST-Clr4 and mutant proteins were grown at 37 °C to OD600 between 0.6–0.8. Protein expression was induced for 16 hrs at 18 °C with 0.2 mM of IPTG. GST-Clr4 and mutant fusion proteins were then purified on glutathione-Sepharose 4B beads (GE Healthcare) in the presence of 0.2% Triton X-100 as previously described30. GST fusion proteins were eluted either by addition of 15 mM glutathione or by addition of PreScission Protease.
Methyltransferase activity on Clr4 WT and mutant proteins was typically carried out with approximately 1.8 µM Clr4 with or without 18 µM H3 (1–20) peptide (Anaspec) in HMT buffer (50 mM Tris-HCl, pH 8, 20 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.02% Triton, 5% glycerol, 1 mM PMSF) in the presence of 0.42 µM 3H-S-Adenosylmethionine (3H-SAM, Perkin Elmer) and 70 µM cold SAM for 1 hr at 30 °C with mild agitation. Samples were boiled for 5 min, separated in two halves and loaded on 4–20% gradient SDS-PAGE (Biorad). Gels were either stained with Coomassie blue to visualize protein or transfered onto a PVDF membrane (Immobilon®-PSQ, Millipore), sprayed with EN3HANCE spray (Perkin Elmer) and exposed on Hyperfilm™ (Amersham) at −80 °C for 1 to several days for analysis of 3H incorporation. Kinetic analysis of Clr4 methyltransferase activity (Fig. 3c) was performed as described above except that 10.8 μM of biotinylated histone H3 (1–21) peptide (Anapspec, #61702) was used instead of H3 (1–20) peptide. Samples were taken at different time intervals and the reaction was stopped by the addition of 1x sample buffer. Samples were then spotted on a streptavidin matrix (SAM2® biotin capture membrane; Promega, #V2861), which was washed 3 times with 1M NaCl followed by two times with deionized water. After the membrane was fully dried, each membrane square was placed in a scintillation vial containing Ultima Gold scintillation cocktail (Perkin Elmer) and scintillation counting was performed on a Tri-Carb® 2910 TR scintillation counter (Perkin Elmer).
In vitro methyltransferase assays with unmethylated and automethylated Clr4 were performed by first incubating GST-Clr4 immobilized on beads in HMT buffer in the absence or presence of 250 µM cold SAM, respectively, for 1 hr at 30 °C with mild agitation. Unmethylated and automethylated GST-Clr4 beads were then washed three times in HMT buffer and used for methyltransferase assays with histone H3 (1–20) peptide as described above except that reactions were incubated for 10 minutes. Kinetic analysis of Clr4 methyltransferase activity following automethylation (Extended data Fig. 1d) was performed as described above except that unmethylated and automethylated GST-Clr4 beads were used for methyltransferase assays with 10.8 μM biotinylated histone H3 (1–21) peptide (Anapspec, #61702) in the presence of 70 μM cold SAM and 0.42 μM 3H-SAM. Samples were taken at different time intervals and the reaction was stopped by the addition of 1x sample buffer. Samples were then spotted on a streptavidin matrix (SAM2® biotin capture membrane; Promega, #V2861), which was washed 3 times with 1M NaCl followed by two washes with deionized water. After the membrane was fully dried, each membrane square was placed in a scintillation vial containing Ultima Gold scintillation cocktail (Perkin Elmer) and scintillation counting was performed on a Tri-Carb® 2910 TR scintillation counter (Perkin Elmer).
Mass spectrometry analysis for identification of Clr4 methylated lysines
For mass spectrometry analysis of Clr4 methylated lysines, a methyltransferase assay was performed as described above except that 2.4 μM recombinant Clr4 was incubated with 250 µM cold SAM for 4 hrs followed by trichloroacetic acid (TCA) precipitation and protein digestion. In brief, 100% TCA was added to each designated sample to achieve a final concentration of 12.5%. The sample was vortexed, incubated on ice for 1 hr, and centrifuged at 20,000 rpm for 30 min at 4°C. The supernatant was aspirated and the sample was washed once with 1 ml of acetone. The sample was centrifuged at 20,000 rpm for 10 min at 4°C. The supernatant was aspirated and the sample was washed once with 1 ml of methanol and allowed to air-dry.
Samples were digested with either trypsin or LysC at a 100:1 protein-to-protease ratio overnight at 37 °C. Samples were subsequently vacuum centrifuged to near dryness, acidified to 1% formic acid, desalted via StageTip, and dried via vacuum centrifugation. The desalted peptides were reconstituted in 5% acetonitrile, 5% formic acid for LC-MS/MS processing. Mass spectrometry data were collected using an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, San Jose, CA) coupled to a Proxeon EASY-nLC 1200 liquid chromatography (LC) pump (Thermo Fisher Scientific). Peptides were separated on a 100 μm inner diameter microcapillary column packed with 35 cm of Accucore C18 resin (2.6 μm, 150 Å, ThermoFisher). For each analysis, we loaded ~2 μg desalted peptides onto the column. Separation was in-line with the mass spectrometer and was performed using a 3 hr gradient of 5 to 22% acetonitrile in 0.125% formic acid at a flow rate of ∼450 nL/min. Each analysis used an SPS-MS3-based TMT method31,32, which has been shown to reduce ion interference compared to MS2 quantification33. The scan sequence began with an MS1 spectrum (Orbitrap analysis; resolution 120,000; mass range 400−1400 m/z; automatic gain control (AGC) target 5 × 105; maximum injection time 100 ms). Precursors for MS2/MS3 analysis were selected using a Top20 method. MS2 analysis consisted of collision-induced dissociation (CID); AGC 2.0 × 104; normalized collision energy (NCE) 35; maximum injection time 120 ms; and isolation window of 1.2 Da. Mass spectra were processed using a SEQUEST-based in-house software pipeline34. Spectra were converted to mzXML using a modified version of ReAdW.exe.
Database searching included only the Clr4 protein and the sequence coverage was up to 86%. This database was concatenated with one composed of all protein sequences in the reversed order. Searches were performed using a 50 ppm precursor ion tolerance for total protein level analysis. The product ion tolerance was set to 0.9 Da. These wide mass tolerance windows were chosen to maximize sensitivity in conjunction with Sequest searches and linear discriminant analysis34,35. We searched for the following modifications: Oxidized methionine (+15.9949), mono-methylated lysine (+14.0157), di-methylated lysine (+28.0313), and trimethylated lysine (+42.0470). Candidate peptides were manually triaged with mass errors limited to >−10 and <10 ppm, XCorr values >2, and with the requirement of trypsin specificity.
For quantitative mass spectrometry analysis of kinetics of Clr4 lysine methylation (Extended Data Fig. 2d), methyltransferase assays were performed twice in triplicate as described above except that 150 μg of recombinant Clr4 at a concentration of 6 μM was incubated with 640 µM cold SAM for 0, 20, 40, and 240 minutes followed by trichloroacetic acid (TCA) precipitation and protein digestion as described above. For quantitative mass spectrometry analysis of methylated lysines in wild-type versus catalytically dead Clr4 (Extended Data Fig. 1e), the experiment was performed twice in triplicate using 150 μg of E. coli purified recombinant wild-type and catalytically dead Clr4, which were precipitated using trichloroacetic acid (TCA) followed by protein digestion as described above. Tandem mass tag (TMT) reagents (0.8 mg) were dissolved in anhydrous acetonitrile (40 μL) of which 10 μL was added to the peptides (100 µg) with 30 μL of acetonitrile to achieve a final acetonitrile concentration of approximately 30% (v/v). Following incubation at room temperature for 1 h, the reaction was quenched with hydroxylamine to a final concentration of 0.3% (v/v). The TMT-labeled samples were pooled across all samples. The pooled sample was vacuum centrifuged to near dryness and subjected to C18 solid-phase extraction (SPE) (Sep-Pak, Waters).
All samples were analyzed on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a Proxeon EASY-nLC 1000 liquid chromatography (LC) pump (Thermo Fisher Scientific). Peptides were separated on a 100 μm inner diameter microcapillary column packed with 35 cm of Accucore C18 resin (2.6 μm, 150 Å, ThermoFisher). For each analysis, we loaded approximately 2 μg onto the column. Peptides were separated using a 150 min gradient of 3 to 25% acetonitrile in 0.125% formic acid with a flow rate of 450 nL/min. Each analysis used an MS3-based TMT method31, which has been shown to reduce ion interference compared to MS2 quantification36. Prior to starting the analysis, we perform two injections of trifluoroethanol (TFE) to elute any peptides that may be bound to the analytical column from prior injections to limit carry over. The scan sequence began with an MS1 spectrum (Orbitrap analysis, resolution 120,000, 350−1,400 Th, automatic gain control (AGC) target 5E5, maximum injection time 100 ms). The top ten precursors were then selected for MS2/MS3 analysis. MS2 analysis consisted of: collision-induced dissociation (CID), quadrupole ion trap analysis, automatic gain control (AGC) 2E4, NCE (normalized collision energy) 35, q-value 0.25, maximum injection time 120 ms), and isolation window at 0.7. Following acquisition of each MS2 spectrum, we collected an MS3 spectrum in which multiple MS2 fragment ions are captured in the MS3 precursor population using isolation waveforms with multiple frequency notches. MS3 precursors were fragmented by HCD and analyzed using the Orbitrap (NCE 65, AGC 1.5E5, maximum injection time 150 ms, resolution was 50,000 at 400 Th). For MS3 analysis, we used charge state-dependent isolation windows: For charge state z=2, the isolation window was set at 1.3 Th, for z=3 at 1 Th, for z=4 at 0.8 Th, and for z=5 at 0.7 Th. Mass spectra were processed using a Sequest-based pipeline34. Spectra were converted to mzXML using a modified version of ReAdW.exe. Database searching included only the Clr4 protein. This database was concatenated with one composed of the protein sequence in the reversed order. Searches were performed using a 3 Da precursor ion tolerance for total protein level analysis. The product ion tolerance was set to 0.9 Da. TMT tags on lysine residues and peptide N termini (+229.163 Da) and carbamidomethylation of cysteine residues (+57.021 Da) were set as static modifications, while oxidation of methionine residues (+15.995 Da) was set as a variable modification. Candidate peptides were manually triaged with mass errors limited to >−10 and <10 ppm, XCorr values >2, and with the requirement of trypsin specificity.
Chromatin immunoprecipitation and high throughput sequencing (ChIP-seq)
ChIP was performed essentially as described11. Briefly, 50 ml of logarithmically growing cells were fixed with 1% formaldehyde for 15 min at room temperature, quenched with 130 mM glycine for 5 min, harvested and washed twice with 1x TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). Cells were resuspended in 500 µl lysis buffer (50 mM Hepes-KOH, pH 7.5, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, and protease inhibitors) in 2 ml screw-cap tubes and lysed by bead beating with 1 ml acid-washed 0.5 mm glass beads with 6 cycles of 30 sec at 5000 rpm on a MagNA Lyser Instrument (Roche). Extracts were sonicated for 3×20 sec at 50% amplitude using a sonicator (Branson Digital Sonifier). For H3K9me2 ChIP, 2 µg of anti-H3K9me2 antibody (Abcam, ab1220) coupled to 30 µl Dynabeads Protein A (Invitrogen) was used for each immunoprecipitation. For H3K9me3 ChIP, 1 µg of anti-H3K9me3 antibody (Diagenode, C15500003) was first incubated with 30 µl Dynabeads M-280 Streptavidin beads (Invitrogen), followed by blocking with 5 µM biotin, according to the manufacturer’s instructions, prior to using for each immunoprecipitation. Half (250 µl for ChIP-seq) or one fifth (100 µl for ChIP-qPCR) of sheared chromatin lysate was added to the antibody-bead mixture and incubated for 2 hrs at 4 °C on a rotating device.
After reversing cross-links and DNA clean-up, libraries for Illumina sequencing were constructed as previously described11 following the manufacturer’s protocols, starting with 1 to 10 ng of immunoprecipitated DNA fragments. Briefly, each library was generated with custom-made adapters carrying unique barcode sequences at the ligating end37. Barcoded libraries were pooled and sequenced with Illumina HiSeq2000. Raw reads were separated according to their barcodes and mapped to the S. pombe genome using Bowtie’s default parameters. Reads that mapped to repeated regions were randomly assigned to the dg and dh repeats of each chromosome and therefore the reads at cenH in the mat locus are shared with those at the pericentromeric dg and dh repeats (with which cenH shares 98% sequence identity). Mapped reads were normalized to reads per million, tiled with igvtools, and visualized in IGV (http://www.broad.mit.edu/igv/). The raw and processed ChlP-seq data are publicly available at the NCBI Gene Expression Omnibus under accession number GSE102905.
Expression and purification of Clr4 SET domain for crystallization
The GST-Clr4 fusion protein containing amino acids 192 to 490 (Clr4192−490) was expressed in BL21-CodonPlus (Agilent technologies) cells. One liter cultures of Luria Burtani media supplemented with 100 μg/mL ampicillin were inoculated with overnight cultures and shaken at 37°C until they reached an OD600 of 0.6–1.0. Cultures were then cooled to 16 °C and ethanol, ZnSO4, and IPTG were added to a final concentration of 2%, 20 μM, and 0.2 mM, respectively. Cultures were shaken at 16 °C for an additional 16 hrs and then harvested by centrifugation. Cell pellets were resuspended in Buffer A (50 mM Tris-HCl, pH 8.0, and 500 mM NaCl) and 1 mg/mL DNAse, 1 mg/mL Lysozyme, and 1 mM MgCl2 and incubated at 4 °C with mixing for 1 hr. The suspension was subsequently sonicated and centrifuged at 32,000 × g for 20 min. The supernatant was nutated with Glutathione Sepharose 4B resin that had been preequilibrated in Buffer A for 1 hr at 4°C. The resin was washed with Buffer A and eluted with Buffer A containing 10 mM reduced glutathione. The eluted protein was dialysed overnight, with PreScission Protease (GE Healthcare) to cleave the GST tag, into Buffer B (5 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM DTT). The protein was again incubated with Glutathione Sepharose 4B resin equilibrated in Buffer B. The unbound fraction was further purified using a HiTrap Q (GE Healthcare) column and a High Load 16/60 Sephadex 200 pg column (GE Healthcare) equilibrated in Buffer B. Fractions containing Clr4192−490 were concentrated to 10 mg/mL for crystallization.
Crystallization
Clr4192−490 crystals were produced by the sitting drop vapor diffusion method at 25 °C. Clr4192−490 at a concentration of 10 mg/mL was preincubated with a threefold molar excess of SAH for 15 min on ice and mixed in an equal volume of reservoir solution containing 200 mM calcium acetate, 100 mM Imidazole, pH 7.5, 15% PEG 8,000. Clr4: SAH complex crystals were cryoprotected in reservoir solution containing 20 % glycerol. Automethylated Clr4192−490 was prepared for crystallization by incubating the protein at a concentration of 0.5 μM with 80 μM SAM at 25 °C. The buffer was exchanged extensively three times and replenished with fresh SAM after 8, 24, and 32 hours. The protein was concentrated to 10 mg/mL, supplemented with stoichiometric excess of SAM, and mixed with an equal volume of reservoir solution containing 0.1 M magnesium formate dihydrate and 20 % PEG 3350. Crystals of the automethylated Clr4 were cryoprotected in reservoir solution containing 30 % PEG 3,350. Another crystal was isolated from the same drop as the one used for data collection, washed three times in water, digested with Trypsin and LysC, and subjected to mass spectrometry analysis as described above (Supplementary Table 5).
Crystallographic data collection and structure determination
X-ray diffraction data was collected at The Northeastern Collaborative Access Team (NE-CAT) beamline 24-ID-E at The Advanced Photon Source (APS) at Argonne National Laboratory at a wavelength of 0.97918 Å38. All structural biology software was accessed through the SBGrid consortium. Data was processed using XDS39 and the CCP4 suite40. The data for the automethylated form of Clr4 was corrected for anisotropy using the Staraniso server (http://staraniso.globalphasing.org) developed by Global Phasing Ltd. Molecular replacement was performed with Phaser41 and coordinates from PDB entry 1MVH. Phenix was used for refinement and generating omit maps42 and model building was carried out using COOT43. Ramachandran statistics for the autoinhibited and automethylated Clr4 structures were 95.98% favored, 3.82% allowed, and 0.19% outliers and 90.86% favored, 8.96% allowed, 0.19% outliers, respectively. All structure alignments and figures were generated using PyMOL Molecular Graphics System, Version 1.8.0.3, Schrödinger, LLC. Structure coordinates were submitted to the Protein Data Base (PDB) and crystallographic data collection and refinement statistics are presented in Supplementary Table 6.
Extended Data
Supplementary Material
Acknowledgments
We are grateful for assistance from the staff at NE-CAT at Argonne National Laboratory, the SBGrid consortium at Harvard Medical School, and Simon Jenni for useful discussions, Haejin Yoon for help with the scintillation counter, Zarmik Moqtaderi and Ruby Yu for Python scripts, Gergana Shipkovenska, Antonis Tatarakis, Xiaoyi Wang, Andy Yuan, and Haining Zhou for comments on the manuscript, and members of the Moazed lab for discussion. This work used NE-CAT beamlines (GM103403), a Pilatus detector (RR029205), a Eiger detector (OD021527) and APS Synchrotron source (DE-AC02–06CH11357). This work was supported by an EMBO long-term fellowship and a Swiss National Science Foundation postdoctoral fellowship (N.I.), K01 DK098285 (J.A.P.), NIH P50 GM107618 (M.K., S.P.G.), and NIH RO1 GM072805 (D.M.). D.M. is a Howard Hughes Medical Institute Investigator.
Footnotes
The authors declare no competing interests.
Code availability
The Python script used in this study is available upon request.
Data availability
Genome-wide datasets are deposited in the Gene Expression Omnibus (GEO) under the accession number GSE102905. Structure coordinates were deposited in the Protein Data Base (PDB ID 6BOX and 6BP4).
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