H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly
2019; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês
10.15252/embr.201948111
ISSN1469-3178
AutoresEriko Oya, Reiko Nakagawa, Yuriko Yoshimura, Mayo Tanaka, Gohei Nishibuchi, Shinichi Machida, A Shirai, Karl Ekwall, Hitoshi Kurumizaka, Hideaki Tagami, Jun‐ichi Nakayama,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle29 August 2019free access Transparent process H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly Eriko Oya Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Reiko Nakagawa orcid.org/0000-0002-6178-2945 Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan Search for more papers by this author Yuriko Yoshimura Division of Chromatin Regulation, National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Mayo Tanaka Division of Chromatin Regulation, National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Gohei Nishibuchi Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Search for more papers by this author Shinichi Machida Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Atsuko Shirai Cellular Memory Laboratory, RIKEN, Wako, Japan Search for more papers by this author Karl Ekwall orcid.org/0000-0002-3029-4041 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Hitoshi Kurumizaka orcid.org/0000-0001-7412-3722 Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Hideaki Tagami Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Search for more papers by this author Jun-ichi Nakayama Corresponding Author [email protected] orcid.org/0000-0002-5597-8239 Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Division of Chromatin Regulation, National Institute for Basic Biology, Okazaki, Japan Department of Basic Biology, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, Japan Search for more papers by this author Eriko Oya Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Reiko Nakagawa orcid.org/0000-0002-6178-2945 Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan Search for more papers by this author Yuriko Yoshimura Division of Chromatin Regulation, National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Mayo Tanaka Division of Chromatin Regulation, National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Gohei Nishibuchi Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Search for more papers by this author Shinichi Machida Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Atsuko Shirai Cellular Memory Laboratory, RIKEN, Wako, Japan Search for more papers by this author Karl Ekwall orcid.org/0000-0002-3029-4041 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Hitoshi Kurumizaka orcid.org/0000-0001-7412-3722 Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Hideaki Tagami Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Search for more papers by this author Jun-ichi Nakayama Corresponding Author [email protected] orcid.org/0000-0002-5597-8239 Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Division of Chromatin Regulation, National Institute for Basic Biology, Okazaki, Japan Department of Basic Biology, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, Japan Search for more papers by this author Author Information Eriko Oya1,2,†, Reiko Nakagawa3, Yuriko Yoshimura4, Mayo Tanaka4, Gohei Nishibuchi1,†, Shinichi Machida5,†, Atsuko Shirai6, Karl Ekwall2, Hitoshi Kurumizaka5,7, Hideaki Tagami1 and Jun-ichi Nakayama *,1,4,8 1Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan 2Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden 3Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan 4Division of Chromatin Regulation, National Institute for Basic Biology, Okazaki, Japan 5Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan 6Cellular Memory Laboratory, RIKEN, Wako, Japan 7Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 8Department of Basic Biology, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, Japan †Present address: Faculty of Science and Engineering, Chuo University, Bunkyo-ku, Tokyo, Japan †Present address: Graduate School of Science, Osaka University, Toyonaka, Japan †Present address: Institute of Human Genetics, CNRS UMR 9002, Montpellier, France *Corresponding author. Tel: +81 564 55 7680; E-mail: [email protected] EMBO Rep (2019)20:e48111https://doi.org/10.15252/embr.201948111 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The methylation of histone H3 at lysine 9 (H3K9me), performed by the methyltransferase Clr4/SUV39H, is a key event in heterochromatin assembly. In fission yeast, Clr4, together with the ubiquitin E3 ligase Cul4, forms the Clr4 methyltransferase complex (CLRC), whose physiological targets and biological role are currently unclear. Here, we show that CLRC-dependent H3 ubiquitylation regulates Clr4's methyltransferase activity. Affinity-purified CLRC ubiquitylates histone H3, and mass spectrometric and mutation analyses reveal that H3 lysine 14 (H3K14) is the preferred target of the complex. Chromatin immunoprecipitation analysis shows that H3K14 ubiquitylation (H3K14ub) is closely associated with H3K9me-enriched chromatin. Notably, the CLRC-mediated H3 ubiquitylation promotes H3K9me by Clr4, suggesting that H3 ubiquitylation is intimately linked to the establishment and/or maintenance of H3K9me. These findings demonstrate a cross-talk mechanism between histone ubiquitylation and methylation that is involved in heterochromatin assembly. Synopsis The Clr4 methyltransferase complex in fission yeast preferentially ubiquitylates H3K14. H3K14ub is linked to H3K9me-enriched heterochromatin and promotes Clr4 methyltransferase activity. The Clr4 methyltransferase complex (CLRC) preferentially ubiquitylates H3K14. K14-ubiquitylated histone H3 is enriched in heterochromatin. CLRC-mediated H3 ubiquitylation promotes Clr4 methyltransferase activity. Introduction In eukaryotic cells, the formation of higher-order chromatin structure, known as heterochromatin, plays an important role in diverse chromosomal processes. Heterochromatin assembly is intimately associated with changes in post-translational histone-tail modifications 1-3. Histone H3 lysine 9 methylation (H3K9me), a hallmark of heterochromatin structure, is catalyzed by SUV39H-family histone methyltransferases (HMTases) and functions as a binding site for recruiting heterochromatin protein 1 (HP1) family proteins 4-6. Heterochromatic structures are epigenetically inherited through cell division in a metastable manner 7, 8. In the fission yeast Schizosaccharomyces pombe (S. pombe), heterochromatin plays critical roles in the assembly of functional chromosomal domains such as centromeres, telomeres, and the mating-type loci 3, 9. Clr4, a homolog of mammalian SUV39H, is the sole H3K9 methyltransferase expressed in S. pombe and plays a central role in heterochromatin assembly 4, 6, 10. H3K9me creates binding sites for the chromodomain (CD) proteins Swi6, Chp1, and Chp2, and these proteins further recruit a variety of chromatin proteins to form repressive higher-order chromatin 11-14. Clr4 also possesses a CD that can bind H3K9me, and it has been suggested that the ability of Clr4 and other HMTases to "write" and "read" H3K9me facilitates heterochromatin spreading and the maintenance of H3K9me during cell division 15. In fission yeast, the assembly and maintenance of pericentromeric heterochromatin is directly linked to the RNA interference (RNAi) pathway 3, 16-18. Pericentromeric repeats are transcribed by RNA polymerase II, and the nascent RNAs are converted to small interfering RNAs (siRNAs) through the actions of the RNA-dependent RNA polymerase complex (RDRC) and Dicer (Dcr1) ribonuclease. The siRNAs are then loaded onto Argonaute (Ago1), the catalytic component of the RNA-induced transcriptional silencing (RITS) complex, which targets pericentromeric repeats through base-pairing interaction between the siRNAs and nascent transcripts. In addition to Ago1, the RITS complex contains the GW motif protein Tas3 and the chromodomain protein Chp1, and the association of the RITS complex with chromatin is facilitated by the Chp1-CD's H3K9me-binding and nucleic acid-binding activities 19. The deletion of RNAi pathway components leads to a loss of silencing and reduced H3K9me at the pericentromeric regions, indicating that the RNAi pathway and recruitment of the siRNA-bound RITS complex to chromatin are coupled with the targeting of Clr4. While RNAi also targets the silent mating-type loci and telomeres, alternative pathways act redundantly with the RNAi pathway to recruit the Clr4 HMTase activity 20, 21. Clr4 forms a multi-protein complex called the Clr4 methyltransferase complex (CLRC). The CLRC consists of the cullin scaffold protein Cul4, the β-propeller protein Rik1, the WD-40 protein Raf1 (Dos1/Cmc1/Clr8), the replication foci targeting sequence (RFTS)-like domain-containing protein Raf2 (Dos2/Cmc2/Clr7), and the RING-box protein Rbx1 (Pip1) 22-26. All of these CLRC components except Rbx1 have been shown to be required for heterochromatic silencing. Cul4, Rik1, and Raf1 in fission yeast show a strong structural resemblance to the conserved CUL4-DDB1-DDB2 E3 ubiquitin ligase (CRL4DDB1) 27, 28. While Raf2 has no analogous component in the CRL4DDB1 complex, it interacts with Cul4, Rik1, and Raf1, and thus is proposed to be a hub for the core components in the CLRC 28, 29. Deleting genes encoding CLRC components results in the loss of both H3K9 methylation and siRNA 24, 30, indicating that a physical interaction between CLRC and RITS couples the siRNA production with H3K9 methylation. Stc1, a tandem zinc finger domain-containing protein, was not identified among the CLRC components, but was shown to mediate the interaction between the CLRC and RITS, presumably through interactions with Raf2 and Ago1 31, 32. On the other hand, studies using cul4 mutant cells suggested that Rik1 is loaded onto heterochromatic repeats in an RNAi-dependent manner and targets other CLRC components to heterochromatic loci 15, 25. Intriguingly, analyses of cells expressing mutant histone H3 (H3K9R) or CLRC components demonstrated that the CLRC complex promotes siRNA production, independently of H3K9 methylation 27, 29, 33, although the details of the mechanism remain unknown. As predicted from its structural similarity to CRL4DDB1, the CLRC exhibits ubiquitin ligase activity in vitro 22. In addition, heterochromatin defects in Cul4 mutants cannot be rescued by expressing a Cul4 protein lacking Nedd8 modification, which is essential for its ubiquitin ligase activity 25, 34. These data show that the CLRC is an active ubiquitin ligase whose ligase activity is required for heterochromatin formation. Structural and functional studies also suggested that Raf1 plays a critical role in target recognition by the CLRC 27, 28. However, whether the CLRC acts as an E3 ubiquitin ligase in vivo and how ubiquitylation modulates Clr4's HMTase activity remain unclear. Here, we demonstrated that affinity-purified CLRC preferentially ubiquitylates lysine 14 on histone H3 (H3K14) in vitro, and the H3K14 ubiquitylation is tightly associated with H3K9me-enriched heterochromatin in vivo. Importantly, the K14-ubiquitylated H3 promotes H3K9's methylation by Clr4. This study demonstrates a cross-talk mechanism between histone methylation and ubiquitylation for heterochromatin assembly. Results Histone H3 is ubiquitylated in vitro by the CLRC To identify the physiological substrate(s) ubiquitylated by CLRC, we first affinity-purified the CLRC and characterized it. For this purpose, an S. pombe strain expressing C-terminally TAP-tagged Rik1 (Rik1-TAP) was constructed, and Rik1-TAP and its associated proteins were affinity-purified. Analysis of the purified fraction revealed protein bands that were specific to the Rik1-TAP preparation but were absent from a control preparation using an untagged strain (mock) (Fig 1A). Mass spectrometry analysis of the purified fraction identified a number of specific proteins, including Cul4, Nedd8, Raf2, Raf1, and Clr4, in agreement with previous studies (Fig 1A) 22, 24, 25, 27. In addition, a limited number of histone H2B or H4 peptides were detected in the purified fraction, as previously observed 22, 24. Rik1 is reported to form a protein complex with Mms19 and Cdc20 35, but these two proteins were not identified in our Rik1-TAP preparations. Figure 1. Histone H3 is ubiquitylated in vitro by the CLRC Purified TAP-tagged Rik1-containing complexes analyzed by SDS–PAGE and silver staining. mock: a mock purification from an untagged strain. Proteins identified by LC-MS/MS are indicated at right, with the number of identified unique peptides in parentheses. In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting (WB) using the indicated antibodies. Asterisks indicate ubiquitylated histone H3 species. Ubiquitylation assays using recombinant histone H3, the N-terminal tail of histone H3 (residues 1-36) fused with GST (H3N-GST), or GST alone as the substrate. Both fission yeast (sp) and human (hs) full-length H3 proteins and H3N-GST fusion proteins were examined. The proteins were analyzed by SDS–PAGE followed by either silver staining or Western blotting with an anti-biotin antibody. Asterisks indicate ubiquitylated histone H3 species. Download figure Download PowerPoint Given that histones have been identified in several independent Rik1-TAP purifications 22, 24 and that histone ubiquitylation has a direct effect on other histone-modifying enzymes 36, 37, we chose histones as candidate CLRC substrates and examined whether CLRC ubiquitylates them. We performed an in vitro ubiquitylation assay using recombinant E1, E2 (UbcH5b), biotinylated ubiquitin, and histones. Incubation of the Rik1-TAP preparation with these components in the absence of histone substrate resulted in a ladder of biotinylated ubiquitin (Fig 1B, lane 3, and Fig EV1A–C). The ladder probably represented the self-ligation of polyubiquitin in the absence of substrate 38, 39, and its appearance suggested that the purified CLRC possessed ubiquitin ligase activity. When histone H3 was added as a substrate, several additional bands appeared, whose molecular weights corresponded to histone H3 modified with mono (~8.6 kDa)- or di-ubiquitin (~17 kDa) (Fig 1B, lane 4, indicated by asterisks). This activity appeared to be specific for the Rik1-TAP preparation, since we did not detect it in the control assay using a mock preparation obtained from the Rik1-untagged strain (Fig 1B, lane 5, mock). Although a weak band (~25 kDa) was also detected in the control assay without E3 (Fig 1B, lane 2), it migrated slightly more slowly than the H3 species modified with mono-ubiquitin, and appeared to be a background product. When histone H2A, H2B, or H4 was used in the assay, a very weak activity was detected for H2A (Fig EV1A, indicated by asterisks), and no obvious change was seen for H2B or H4 (Fig EV1B and C). Although H2A might also serve as a substrate of CLRC, the purified CLRC exhibited a stronger activity for H3, and thus, in this study, we focused on H3's ubiquitylation and its roles in heterochromatin assembly. Click here to expand this figure. Figure EV1. In vitro ubiquitylation assay using affinity-purified CLRC A–C. In vitro ubiquitylation assays, in which purified CLRC was incubated with biotinylated ubiquitin and recombinant histone H2A (A), H2B (B), or H4 (C). The proteins were resolved by SDS–PAGE and analyzed by Western blotting using the indicated antibodies. The single and double asterisks indicate mono- and di-ubiquitylated histone species, respectively, and the triple asterisk indicates an unrelated cross-reactive band for the anti-H2A antibody. Download figure Download PowerPoint Characterization of CLRC's H3 ubiquitylation activity Cul4 functions as scaffold to form E3 ubiquitin ligase and is modified by the ubiquitin-like protein Nedd8, which induces the E3 ubiquitin ligase activity 34. To determine whether Cul4's activity is involved in the CLRC's H3 ubiquitylation, we first created a cul4 mutant allele in which Lys680 (the site of Nedd8 conjugation) was mutated to arginine (cul4K680R) as previously reported 25. However, the cul4 K680R mutant showed a severe growth defect (Fig EV2B), which made it difficult to assess Cul4's involvement in CLRC's H3 ubiquitylation. Instead, we isolated a milder cul4 mutant allele (cul4-1), which did not exhibit a noticeable growth defect (Fig EV2B), but resulted in loss of the silencing of ade6+ or ura4+ marker genes inserted at the centromeric repeats (otr1R::ade6+) and the mating-type locus (kint2::ura4+), respectively (Fig EV2A and B). In this cul4 mutant, its original 3′-untranslated region was replaced with TEF terminator and a hygromycin-resistant gene without altering cul4 coding region, which presumably led to a change in cul4 expression or mRNA stability. This mutation changed the profile of the Rik1-TAP preparation with a clear reduction in the co-purified Cul4 (Fig EV2C) and markedly reduced the H3 ubiquitylation activity (Fig EV2D). This result suggested that Cul4 plays a critical role in CLRC's H3 ubiquitylation activity. Click here to expand this figure. Figure EV2. Mutation of cul4 affects CLRC's ubiquitylation activity Diagram of the mating-type loci and part of centromere 1 in S. pombe. The positions of the silencing reporter genes (Kint2::ura4+ and otr1R::ade6+) are shown. Heterochromatic silencing assays of wild-type and cul4 mutant strains. Silencing at the mating-type Kint2::ura4+ and otr1R::ade6+ was evaluated. Ten-fold serial dilutions of the indicated strains were spotted onto non-selective medium (N/S), medium lacking uracil (-Ura), medium containing 5-FOA (5-FOA), and medium containing low adenine (Low ade). Purified TAP-tagged Rik1-containing complexes analyzed by SDS–PAGE and silver staining. mock: a mock purification from an untagged strain. Proteins identified by LC-MS/MS are indicated at right. In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting using the indicated antibodies. The single and double asterisks indicate mono- and di-ubiquitylated histone H3 species, respectively. Recombinant histone H3 protein and core histones in the reconstituted nucleosomes (Nuc.) were analyzed by SDS–PAGE and visualized by CBB staining. In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 or reconstituted nucleosomes (Nuc.) as the substrate. Proteins were analyzed by Western blotting using the indicated antibodies. The single and double asterisks indicate mono- and di-ubiquitylated histone H3 species, respectively. Download figure Download PowerPoint To characterize the CLRC's ubiquitylation activity further, we prepared a fusion protein of the N-terminal tail of H3 (residues A1-K36) and GST (H3N-GST). Since the human H3 histone-tail sequence is slightly different from that of S. pombe, we prepared both human and S. pombe H3N-GST fusion proteins, and examined them in the ubiquitylation assay. While both the human and S. pombe full-length H3 histones were ubiquitylated by CLRC (Fig 1C, lanes 2 and 4), the H3N-GSTs were more efficiently ubiquitylated than the full-length histones (Fig 1C, lanes 6 and 8). These results suggested that the CLRC preferentially ubiquitylates the N-terminal tail of H3 and that the C-terminal region of H3 may have an inhibitory effect on the CLRC's activity for the H3 N-terminal tail. We also noticed that CLRC did not efficiently ubiquitylate H3 in reconstituted nucleosomes (Fig EV2E and F), suggesting that nucleosomal DNA may also have an inhibitory effect on the CLRC's activity and additional factor(s) may help to promote it in vivo. CLRC preferentially ubiquitylates histone H3 lysine 14 We next sought to identify the ubiquitylated lysine residues of the H3 N-terminal tail. For this investigation, we performed mass spectrometry (LC-MS/MS) analysis of the mono- and di-ubiquitylated H3N-GST species recovered from the CLRC-mediated in vitro ubiquitylation assay (Figs 2A and EV3A). Because trypsin cleaves the C-terminal Arg-Gly-Gly of ubiquitin, ubiquitylated lysine residues could be identified by the presence of a characteristic diGly remnant (Fig 2B) 40. In this analysis, four residues, K14, K18, K23, and K27, were identified as candidate H3 ubiquitylation sites (Fig EV3B). The number of identified peptides containing each of the candidate ubiquitylation sites suggested that K14 and K18 are the predominant H3 ubiquitylation sites. Figure 2. Histone H3 lysine 14 is ubiquitylated in vitro by CLRC Ubiquitylation assay using H3N-GST. The components included in the assay and the ubiquitylated H3N-GST species are indicated at right. MS/MS spectrum of the histone H3 peptide corresponding to residues 10–17. The observed y and b ions and fragment map are shown. Ubiquitylation assay using biotinylated ubiquitin and recombinant wild-type H3N-GST (WT) and arginine-substituted H3N-GST mutants as substrates. Proteins were analyzed by Western blotting and silver staining. mock: reaction without substrate. Asterisks indicate ubiquitylated H3N-GST proteins. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Analysis of the histone H3 ubiquitylation states Ubiquitylation assay using recombinant H3N-GST. The proteins were analyzed by SDS–PAGE and silver staining. The components included in the assay are indicated at right, and the ubiquitylated H3N-GST species are indicated by asterisks. The proteins subjected to LC-MS/MS analysis are indicated by boxes. Summary of the ubiquitylated peptides identified by LC-MS/MS. Recombinant S. pombe histone H3 (wild type; WT) and mutant histone H3 with K14A substitution (H3K14A) used in (D) were analyzed by SDS–PAGE and visualized by CBB staining. In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant wild-type (WT) or mutant (K14A) histone H3 as the substrate. Proteins were analyzed by Western blotting using the indicated antibodies. The single and double asterisks indicate mono- and di-ubiquitylated histone H3 species, respectively. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to conventional ChIP analysis, and the levels of act1+ and pericentromeric dg loci relative to the input were determined. Error bars indicate standard errors from three technical replicates. Abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines. Download figure Download PowerPoint To confirm the results obtained by LC-MS/MS analysis, we prepared H3N-GST fusion proteins containing amino acid substitutions for each of the candidate lysine residues (K4R, K9R, K14R, K18R, K23R, K27R, and K36R) and assessed their effect on H3's ubiquitylation. In agreement with the LC-MS/MS analysis (Fig EV3B), the K14R substitution resulted in a marked reduction in the CLRC-mediated H3 ubiquitylation (Fig 2C, indicated by asterisks). Although the K18R, K23R, or K27R substitution appeared to slightly reduce the H3 ubiquitylation, the effect was much weaker than that of the K14R substitution. The other lysine substitutions (K4R, K9R, and K36R) had little or no impact on the CLRC-mediated H3 ubiquitylation (Fig 2C). We also confirmed that K14A substitution also markedly reduced the CLRC-mediated ubiquitylation of full-length histone H3 (Fig EV3C and D). These results suggested that CLRC possesses an E3 ubiquitin ligase activity that preferentially targets histone H3K14, and that K14-mono-ubiquitylated H3 is a major product of CLRC's activity. At this point, we could not distinguish whether the di-ubiquitylated H3 species contained two mono-ubiquitins at different lysine residues or one di-ubiquitin at a single lysine residue. In either case, our findings indicated that H3K14 ubiquitylation (H3K14ub) may play a role in promoting additional ubiquitylation. Heterochromatin is enriched in H3K14ub in vivo Since our in vitro assays suggested that the CLRC preferentially ubiquitylates histone H3K14, we next sought to assess the presence of H3K14ub in vivo. We speculated that H3K14ub would be concentrated at heterochromatic regions where the CLRC is predominantly localized 15. To examine this possibility, we carried out chromatin immunoprecipitation (ChIP) assays using antibodies against heterochromatic H3K9me2 or euchromatic H3K4me2 41, and performed LC-MS/MS analyses to identify the histone modifications associated with these regions (Figs 3 and EV3E and F, and Appendix Table S1, Appendix Figs S1–S8). We confirmed that H3K9me2 was enriched in centromeric dg repeats, whereas H3K4me2 was preferentially enriched in the actively transcribed act1+ gene (Fig EV3E). Subsequent LC-MS/MS analysis revealed that K14-ubiquitylated H3 peptides were detected exclusively in the H3K9me2-associated chromatin (Figs 3B–D and EV3F). It was also notable that nearly all of the tryptic K14-ubiquitylated H3 peptides contained H3K9me2 or H3K9me3 (Fig 3C). These results demonstrated that H3K14ub is concentrated in H3K9me-associated heterochromatin, where CLRC is predominantly localized, in vivo. Our LC/MS/MS analysis also revealed that K56- or K79-ubiquitylated H3 peptides were preferentially concentrated in the H3K4me2-associated chromatin (Figs 3C and D, and EV3F), although their biological functions and potential involvement in transcription are unknown. Figure 3. H3K14 ubiquitylation is associated with H3K9me-enriched chromatin Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS–PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis. MS/MS spectrum of the histone H3 peptide corresponding to residues 9–17. The observed y and b ions and fragment map are shown. Summary of the ubiquitylated peptides identified by LC-MS/MS. Relative abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to LC-MS/MS analysis, and the abundance of ubiquitylated lysine residues in each fraction is shown (see also Fig EV3F). Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines. Download figure Download PowerPoint H3K14 is critical for heterochromatin assembly in vivo We next asked whether H3K14 is essential for heterochromatin assembly in fission yeast. Schizosaccharomyces pombe encodes three copies of genes that encode histone H3 (H3.1, H3.2, and H3.3). We introduced an alanine substitution mutation at individual lysine residues within the H3.1 N-terminal tail and assessed the effect on the silencing of kint2::ura4+ (Fig 4A). Introduction of the K9A mutation clearly abolished the kint2::ura4+ silencing (Fig 4B), and ChIP analysis showed that the level of H3K9me2 at heterochromatic regions was reduced to half or less that of wild type (Fig 4C). Notably, the K14A or K14R mutation also led to defective silencing and decreased H3K9me2 levels, comparable to that of the K9A mutation (Figs 4B and C, and EV4A). The status of silencing defect caused by amino acid substitution for K9 or K14 was also confirmed by quantitative reverse-transcriptase PCR (RT–qPCR) (Fig EV4B and C). Alanine substitution of the other lysine residues in the H3.1 N-terminal tail had no effect on gene silencing (Fig 4B)
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