Clr4 (Cryptic loci regulator 4, Kmt1) is the sole histone H3 lysine 9 (H3K9) methyltransferase of the fission yeast Schizosaccharomyces pombe and the ortholog of metazoan SUV39H1/2 and Drosophila Su(var)3-9. It is a SET-domain protein lysine methyltransferase that, using S-adenosyl-L-methionine, deposits H3K9 mono-, di- and trimethylation, the defining histone mark of heterochromatin. Clr4 has a bipartite architecture: an N-terminal chromodomain that reads pre-existing H3K9me and a C-terminal pre-SET/SET/post-SET catalytic module that writes the mark, with a triangular zinc cluster in the pre-SET region and an autoregulatory (autoinhibitory) loop whose automethylation switches the enzyme to its active conformation. Clr4 is the catalytic subunit of the CLRC complex (Clr4, Rik1, Cul4/Pcu4, Raf1/Dos1, Raf2/Dos2 and the RING-box protein Pip1), a CRL4-type cullin-RING E3 ubiquitin ligase whose preferred substrate is histone H3 lysine 14 (H3K14ub); H3K14 ubiquitylation in turn strongly stimulates Clr4 H3K9 methyltransferase activity. H3K9me deposited by Clr4 recruits HP1-family proteins (Swi6, Chp2) and the chromodomain protein Chp1 of the RITS complex, establishing transcriptional gene silencing at centromeres, telomeres, the silent mating-type region and ribosomal DNA repeats. Through its read-write feedback loop and its coupling to the nuclear RNAi machinery, Clr4 mediates nucleation, spreading and epigenetic maintenance of heterochromatin, and it can also methylate non-histone substrates such as Mlo3. It localizes to the nucleus and is enriched at heterochromatic chromatin domains.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Generic DNA binding from an ARBA machine-learning model (electronic). GO:0003677 does not imply sequence specificity, and the Clr4 chromodomain has experimentally demonstrated sequence-independent nucleic-acid (dsDNA/ssDNA) binding (PMID:22727667 IDA), so this generic parent term is corroborated rather than spurious. It is redundant with the more specific dsDNA/ssDNA-binding IDAs and is a non-core, ancillary property; Clr4 is not a sequence-specific DNA-binding factor and its locus targeting is mediated by H3K9me reading and CLRC/RNAi recruitment. Reason: Generic DNA-binding term corroborated by the chromodomain nucleic-acid-binding IDA; kept as a redundant, non-core property rather than removed. (An earlier REMOVE call conflated GO:0003677 with sequence-specific DNA binding.) Supporting Evidence: PMID:22727667 Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for heterochromatic gene silencing. |
| GO:0003727 single-stranded RNA binding | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Generic ssRNA binding from an ARBA model (electronic). This is the redundant electronic counterpart of the experimental ssRNA-binding IDA (PMID:22727667), which assays the Clr4 chromodomain and shows it binds nucleic acid. The electronic term is therefore corroborated rather than unsupported, but it is redundant with the IDA and reflects an ancillary, non-core property of the chromodomain. Reason: Electronic ssRNA-binding term corroborated by the experimental IDA (Clr4 chromodomain nucleic-acid binding, Ishida et al. 2012); kept as a redundant, non-core property rather than removed. (An earlier REMOVE call claiming the experimental source describes Chp1 not Clr4 was incorrect.) Supporting Evidence: PMID:22727667 Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for heterochromatic gene silencing. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Nucleus is the correct compartment for Clr4, which acts on chromatin. This electronic call is corroborated by experimental localization. A more specific heterochromatin localization is captured by other annotations. Reason: Nuclear localization is well established experimentally and by SubCell mapping. Supporting Evidence: PMID:18345014 ClrC components are distributed throughout heterochromatic domains. |
| GO:0005694 chromosome | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Chromosome localization is consistent with Clr4 acting on chromatin and is supported by experimental ChIP localization across heterochromatic domains. A more informative heterochromatin term is preferable but this is correct. Reason: Correct but general; more specific heterochromatin location terms are also annotated. Supporting Evidence: PMID:18345014 ClrC components are distributed throughout heterochromatic domains. |
| GO:0005721 pericentric heterochromatin | IEA GO_REF:0000117 | ACCEPT | Summary: Pericentric heterochromatin is a bona fide site of Clr4 action; the same localization is supported experimentally (IDA, PMID:18345014). This electronic call is correct. Reason: Correct localization, also supported by experimental IDA annotations. Supporting Evidence: PMID:18345014 ClrC components are distributed throughout heterochromatic domains. |
| GO:0005816 spindle pole body | IEA GO_REF:0000044 | MARK AS OVER ANNOTATED | Summary: Spindle pole body localization derives from SubCell mapping seeded by a single genome-wide GFP localization screen (PMID:16823372). Clr4 function is in nuclear chromatin; an SPB pool is not supported by any functional study and is likely an artefact or minor mislocalization. Should not be treated as a functional location. Reason: Based solely on a high-throughput localization mapping; no functional evidence links Clr4 to the spindle pole body, whereas all characterized functions are chromatin-associated. Supporting Evidence: PMID:16823372 ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. |
| GO:0008270 zinc ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Clr4 binds zinc through cysteine-rich pre-SET (triangular three-zinc cluster) and post-SET regions that are structurally required for catalysis. This is a genuine structural property documented in crystal structures, but it is a supporting cofactor-binding role rather than a core function. The Clr4 KMT (catalytic) domain crystallized by Du et al. 2025 (PMID:40446033) is explicitly composed of the pre-SET, SET catalytic core and post-SET subdomains, the cysteine-rich modules that coordinate the structural zinc. Reason: Structurally verified zinc binding required for SET-domain catalysis; supporting rather than core molecular function. Supporting Evidence: PMID:10949293 We mapped the catalytic motif to the evolutionarily conserved SET domain, which requires adjacent cysteine-rich regions to confer histone methyltransferase activity. PMID:40446033 we cocrystallized the catalytic KMT domain [composed of the N-terminal subdomain (NT), pre-SET, SET catalytic core, and post-SET subdomains] with the H3K14 ubiquitinated or unmodified H3 N-terminal tail peptide in the presence of S-adenosylmethionine (SAM). |
| GO:0016279 protein-lysine N-methyltransferase activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: Protein-lysine N-methyltransferase activity reflects Clr4's ability to methylate non-histone protein lysines (e.g. Mlo3) and to automethylate. It is a valid but broader sibling of the H3K9-specific activity; the histone-specific terms better capture the core function. Reason: Correct general activity, supported experimentally; the H3K9-specific methyltransferase term is the core function. Supporting Evidence: PMID:28143796 later it was also found to methylate the Mlo3 protein, which has a role in heterochromatin formation as well |
| GO:0030466 silent mating-type cassette heterochromatin formation | IEA GO_REF:0000117 | ACCEPT | Summary: Heterochromatin formation at the silent mating-type region is a core biological role of Clr4, supported by classic genetics (clr4 is one of the cryptic-loci-regulator genes) and by experimental IMP annotations. Reason: Core silencing function corroborated by experimental IMP evidence (PMID:8001791, PMID:11283354). Supporting Evidence: PMID:8001791 the transcription and recombination blocks require three newly defined trans-acting loci, clr2, clr3 and clr4, in addition to the previously identified clr1, rik1 and swi6 loci. |
| GO:0031509 subtelomeric heterochromatin formation | IEA GO_REF:0000117 | ACCEPT | Summary: Subtelomeric heterochromatin formation is a core role of Clr4 and is also supported by experimental IMP evidence (PMID:24240238). Reason: Core heterochromatin role; corroborated by experimental annotation. Supporting Evidence: PMID:24240238 deletion of telomere shelterin components restores pericentric heterochromatin and its functions in RNAi mutants |
| GO:0031934 mating-type region heterochromatin | IEA GO_REF:0000117 | ACCEPT | Summary: Localization to mating-type region heterochromatin is correct and is also supported experimentally (is_active_in IDA, PMID:18345014). Reason: Correct site of action, also supported by experimental IDA. Supporting Evidence: PMID:18345014 ClrC components are distributed throughout heterochromatic domains. |
| GO:0031981 nuclear lumen | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Nuclear lumen is a generic compartment term derived from an ARBA model. The more specific nucleus and heterochromatin terms better describe Clr4's location. Reason: Correct but over-general compartment; subsumed by more specific nuclear/heterochromatin annotations. Supporting Evidence: PMID:18345014 ClrC components are distributed throughout heterochromatic domains. |
| GO:0042054 histone methyltransferase activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Histone methyltransferase activity is correct but is the over-general parent of the H3K9-specific activity that is the true core function. The specific H3K9 methyltransferase term should be used. Reason: Over-general parent of the well-supported H3K9-specific methyltransferase activity. Proposed replacements: histone H3K9 methyltransferase activity Supporting Evidence: PMID:11283354 lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. |
| GO:0046974 histone H3K9 methyltransferase activity | IEA GO_REF:0000002 | ACCEPT | Summary: This is the central catalytic function of Clr4: it is the sole H3K9 methyltransferase of fission yeast. The electronic call is strongly corroborated by extensive experimental evidence (IDA/IMP). Crystal structures of the Clr4 KMT (SET) domain bound to H3 peptide with the SAM cofactor (Du et al. 2025, PMID:40446033) directly visualize the SAM-dependent H3K9 methyltransferase active site, with the H3 K9 side chain inserted into the catalytic pocket positioned to attack the S-methyl group of SAM, and explain how proximal H3K14 ubiquitination stimulates this activity. Reason: Core molecular function, supported by numerous direct assays. Supporting Evidence: PMID:11283354 lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. PMID:40446033 Clr4 is the sole known methyltransferase catalyzing H3K9 methylation in Schizosaccharomyces pombe. PMID:40446033 The SAM cofactor is located on the larger side of the ovoid structure, embedded in the SAM pocket of the SET domain, with the post-SET subdomain covering above as a lid. |
| GO:0062072 histone H3K9me2/3 reader activity | IEA GO_REF:0000117 | ACCEPT | Summary: The Clr4 chromodomain reads pre-existing H3K9me2/3, providing the read half of its read-write feedback loop. Supported experimentally (PMID:18345014, PMID:31165882). Reason: Genuine reader function of the chromodomain, also experimentally supported. Supporting Evidence: PMID:18345014 the chromodomain of Clr4 binds specifically to H3K9me that is essential for the spreading of heterochromatin. |
| GO:0140720 subtelomeric heterochromatin | IEA GO_REF:0000117 | ACCEPT | Summary: Localization to subtelomeric heterochromatin is correct and is also supported by experimental IDA (PMID:18345014, PMID:32269268). Reason: Correct site of action, also supported experimentally. Supporting Evidence: PMID:32269268 Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin. |
| GO:0140727 siRNA-mediated pericentric heterochromatin formation | IEA GO_REF:0000117 | ACCEPT | Summary: siRNA-mediated pericentric heterochromatin formation is a core role: Clr4 is recruited via the RNAi/RITS pathway to nucleate H3K9me at centromeric repeats. Also supported by experimental EXP evidence (PMID:15615848). Reason: Core RNAi-directed heterochromatin function, supported experimentally. Supporting Evidence: PMID:15615848 its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins |
| GO:0140947 histone H3K9me2 methyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Clr4 catalyzes H3K9 dimethylation; this Rhea/EC-derived specific activity is correct and supported by experimental EXP evidence (PMID:30051891). Reason: Specific catalytic activity within the core H3K9 methyltransferase function; experimentally supported. Supporting Evidence: PMID:30051891 automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. |
| GO:0140948 histone H3K9 monomethyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Clr4 catalyzes H3K9 monomethylation; this specific activity is correct and supported by experimental EXP evidence (PMID:10949293, PMID:11283354). Reason: Specific catalytic activity within the core function; experimentally supported. Supporting Evidence: PMID:11283354 lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein |
| GO:0140949 histone H3K9 trimethyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Clr4 catalyzes H3K9 trimethylation (the heterochromatic H3K9me3 mark); this specific activity is correct and supported by experimental EXP evidence (PMID:11283354). Reason: Specific catalytic activity within the core function; experimentally supported. Supporting Evidence: PMID:11283354 Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. |
| GO:0005515 protein binding | IPI PMID:16024659 A Rik1-associated, cullin-dependent E3 ubiquitin ligase is e... | REMOVE | Summary: This IPI captures the interaction of Clr4 with Rik1 (UniProtKB:Q10426) within the CLRC complex. The bare 'protein binding' term is uninformative; the biologically meaningful annotation is membership in the CLRC complex, already captured by GO:0043494. Reason: Uninformative bare protein-binding term; the underlying Clr4-Rik1 interaction is better represented by the CLRC complex membership annotation. Supporting Evidence: PMID:16024659 subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1 and Clr4 |
| GO:0005515 protein binding | IPI PMID:16127433 Ubiquitin ligase component Cul4 associates with Clr4 histone... | REMOVE | Summary: This IPI captures Clr4 binding to Cul4/Pcu4 (UniProtKB:O14122) and Rik1. The bare term is uninformative; the direct Clr4-Pcu4 interaction underlies CLRC assembly, captured by the CLRC complex annotation. Reason: Uninformative bare protein-binding term; the Clr4-Cul4/Pcu4 interaction is better represented by CLRC complex membership. Supporting Evidence: PMID:16127433 Clr4 associates with Cul4, a cullin family protein that serves as a scaffold for assembling ubiquitin ligases. |
| GO:0005515 protein binding | IPI PMID:20211136 Stc1: a critical link between RNAi and chromatin modificatio... | REMOVE | Summary: This IPI captures interaction with Stc1 (UniProtKB:O94276), the LIM-domain protein bridging the RNAi effector Ago1 to the CLRC complex. The bare term is uninformative; the functional significance is recruitment of CLRC by the RNAi machinery. Reason: Uninformative bare protein-binding term; the Clr4/CLRC-Stc1 interaction is better represented within the RNAi-directed heterochromatin process terms. Supporting Evidence: PMID:20211136 This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. |
| GO:0046974 histone H3K9 methyltransferase activity | IDA PMID:40923761 Intrinsically disordered region of Clr4/Suv39 regulates its ... | ACCEPT | Summary: Direct demonstration of Clr4 H3K9 methyltransferase activity; the study shows the intrinsically disordered region regulates this enzymatic activity and heterochromatin spreading. Confirms the core function. Reason: Direct assay of the core catalytic function. Supporting Evidence: PMID:40923761 Intrinsically disordered region of Clr4/Suv39 regulates its enzymatic activity and ensures heterochromatin spreading. |
| GO:0005634 nucleus | EXP PMID:18345014 Roles of the Clr4 methyltransferase complex in nucleation, s... | ACCEPT | Summary: Experimentally supported nuclear localization, consistent with Clr4's chromatin function. Reason: Experimentally supported correct compartment. Supporting Evidence: PMID:18345014 ClrC components are distributed throughout heterochromatic domains. |
| GO:0005694 chromosome | EXP PMID:18345014 Roles of the Clr4 methyltransferase complex in nucleation, s... | KEEP AS NON CORE | Summary: Experimentally supported chromosome localization (ChIP across heterochromatic domains). Correct but general relative to the specific heterochromatin terms. Reason: Correct but general; more specific heterochromatin location terms preferred. Supporting Evidence: PMID:18345014 ClrC components are distributed throughout heterochromatic domains. |
| GO:0016279 protein-lysine N-methyltransferase activity | EXP PMID:28143796 Clr4 specificity and catalytic activity beyond H3K9 methylat... | KEEP AS NON CORE | Summary: Experimentally demonstrated protein-lysine methyltransferase activity on non-histone substrates (Mlo3 and additional targets). A genuine but broader activity than the core H3K9-specific function. Reason: Real non-histone methyltransferase activity; supporting/secondary to the H3K9 core function. Supporting Evidence: PMID:28143796 Peptide methylation was observed on Mlo3 and 7 novel target sites |
| GO:0140947 histone H3K9me2 methyltransferase activity | EXP PMID:30051891 Automethylation-induced conformational switch in Clr4 (Suv39... | ACCEPT | Summary: Experimentally supported H3K9 dimethyltransferase activity; the automethyl conformational switch enhances Clr4 H3K9me activity. Part of the core catalytic repertoire. Crystal structures of the Clr4 KMT domain bound to H3K14-ubiquitinated H3 peptide and SAM (Du et al. 2025, PMID:40446033) show how proximal H3K14 ubiquitination promotes H3K9me2/3 deposition. Reason: Experimentally supported specific catalytic activity within the core function. Supporting Evidence: PMID:30051891 automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. PMID:40446033 The side chain of histone H3 K9Nle inserts into a hydrophobic tunnel and attacks the S-methyl group of SAM PMID:40446033 The ubiquitin constrained by the isopeptide bond linkage at the H3K14 position forms multivalent interactions with the Clr4 catalytic domain, stabilizing substrate binding and rapidly promoting H3K9me2/3 deposition. |
| GO:0140948 histone H3K9 monomethyltransferase activity | EXP PMID:10949293 Regulation of chromatin structure by site-specific histone H... | ACCEPT | Summary: Experimentally supported H3K9 monomethyltransferase activity; the founding study mapping SUV39/Clr4 SET-domain H3K9 methylation. The SAM-dependent nature of this catalysis is directly visualized in the Clr4 KMT-domain crystal structures with SAM bound (Du et al. 2025, PMID:40446033), where the SAM cofactor occupies the SAM pocket of the SET domain and the H3 K9 side chain attacks its S-methyl group. Reason: Experimentally supported specific catalytic activity within the core function. Supporting Evidence: PMID:10949293 encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro PMID:40446033 The SAM cofactor is located on the larger side of the ovoid structure, embedded in the SAM pocket of the SET domain, with the post-SET subdomain covering above as a lid. PMID:40446033 K9 was replaced with norleucine (Nle), which leads to SAM-dependent high affinity to Clr4 catalytic pocket and was widely used in MTase structural studies |
| GO:0140948 histone H3K9 monomethyltransferase activity | EXP PMID:11283354 Role of histone H3 lysine 9 methylation in epigenetic contro... | ACCEPT | Summary: Experimentally supported H3K9 monomethyltransferase activity, with catalytic mutagenesis (R320H, G378S, G486D) abolishing activity. Reason: Experimentally supported specific catalytic activity within the core function. Supporting Evidence: PMID:11283354 lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein |
| GO:0140949 histone H3K9 trimethyltransferase activity | EXP PMID:11283354 Role of histone H3 lysine 9 methylation in epigenetic contro... | ACCEPT | Summary: Experimentally supported H3K9 trimethyltransferase activity producing the hallmark H3K9me3 heterochromatic mark. Reason: Experimentally supported specific catalytic activity within the core function. Supporting Evidence: PMID:11283354 Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. |
| GO:0031491 nucleosome binding | EXP PMID:31165882 Disordered region of H3K9 methyltransferase Clr4 binds the n... | ACCEPT | Summary: Clr4 binds the nucleosome core through both its chromodomain and the disordered linker; this binding contributes to H3K9 methylation in vitro and in vivo. A genuine, mechanistically important binding activity that supports substrate engagement. Reason: Directly demonstrated nucleosome binding contributing to catalysis and de novo H3K9me deposition. Supporting Evidence: PMID:31165882 the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. |
| GO:0031491 nucleosome binding | IPI PMID:31165882 Disordered region of H3K9 methyltransferase Clr4 binds the n... | ACCEPT | Summary: IPI annotation of nucleosome (histone) binding from the same study, supported by direct binding assays with reconstituted nucleosomes. Reason: Direct nucleosome-binding interaction evidence; same well-supported function. Supporting Evidence: PMID:31165882 both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. |
| GO:0046974 histone H3K9 methyltransferase activity | IMP PMID:31165882 Disordered region of H3K9 methyltransferase Clr4 binds the n... | ACCEPT | Summary: IMP support for H3K9 methyltransferase activity: disordered-region mutants reduce nucleosome binding and H3K9 methylation in vivo. Confirms the core function. Reason: Mutational support for the core catalytic function. Supporting Evidence: PMID:31165882 interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. |
| GO:0005721 pericentric heterochromatin | NAS PMID:24449894 CRL4-like Clr4 complex in Schizosaccharomyces pombe depends ... | ACCEPT | Summary: Pericentric heterochromatin localization (NAS, ComplexPortal). Correct and also supported by experimental IDA; pericentric heterochromatin is a principal site of CLRC/Clr4 action. Reason: Correct localization, corroborated by experimental IDA evidence. Supporting Evidence: PMID:24449894 Repressive histone H3 lysine 9 methylation (H3K9me) and its recognition by HP1 proteins are necessary for pericentromeric heterochromatin formation. |
| GO:0043494 CLRC complex | NAS PMID:16024659 A Rik1-associated, cullin-dependent E3 ubiquitin ligase is e... | ACCEPT | Summary: Clr4 is the catalytic subunit of the CLRC (Clr4 methyltransferase) complex, a CRL4-type cullin-RING E3 ligase containing Rik1, Cul4/Pcu4, Raf1/Dos1, Raf2/Dos2 and Pip1. This is a core, well-established complex membership. Reason: Core complex membership; the central organizing fact of Clr4 biology. Supporting Evidence: PMID:16024659 subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1 and Clr4 |
| GO:0140727 siRNA-mediated pericentric heterochromatin formation | NAS PMID:24449894 CRL4-like Clr4 complex in Schizosaccharomyces pombe depends ... | ACCEPT | Summary: Core RNAi-directed pericentric heterochromatin formation; H3K9me deposition at pericentromeres depends on the RNAi pathway recruiting CLRC. Also supported by experimental EXP evidence (PMID:15615848). Reason: Core RNAi-directed heterochromatin process, corroborated experimentally. Supporting Evidence: PMID:24449894 H3K9me deposition depends on the RNAi pathway. |
| GO:0005721 pericentric heterochromatin | IDA PMID:18345014 Roles of the Clr4 methyltransferase complex in nucleation, s... | ACCEPT | Summary: Direct evidence that Clr4/ClrC is active in pericentric heterochromatin, where it nucleates and spreads H3K9me. Core site of action. Reason: Direct experimental evidence of Clr4 activity at pericentric heterochromatin. Supporting Evidence: PMID:18345014 The Clr4 methyltransferase complex (ClrC) is responsible for nucleation and spreading of heterochromatin |
| GO:0031934 mating-type region heterochromatin | IDA PMID:18345014 Roles of the Clr4 methyltransferase complex in nucleation, s... | ACCEPT | Summary: Direct evidence that Clr4 is active in mating-type region heterochromatin, a principal silencing domain. Core site of action. Reason: Direct experimental evidence of Clr4 activity at the mating-type heterochromatin. Supporting Evidence: PMID:18345014 The Clr4 methyltransferase complex (ClrC) is responsible for nucleation and spreading of heterochromatin |
| GO:0062072 histone H3K9me2/3 reader activity | EXP PMID:18345014 Roles of the Clr4 methyltransferase complex in nucleation, s... | ACCEPT | Summary: Experimentally supported reader activity: the Clr4 chromodomain binds H3K9me2/3, enabling the read-write feedback that spreads and maintains heterochromatin. Core function. Reason: Experimentally supported chromodomain reader function central to spreading/maintenance. Supporting Evidence: PMID:18345014 the ability of Clr4 to both 'write' and 'read' H3K9me facilitates heterochromatin maintenance through successive cell divisions. |
| GO:0140720 subtelomeric heterochromatin | IDA PMID:18345014 Roles of the Clr4 methyltransferase complex in nucleation, s... | ACCEPT | Summary: Direct evidence that Clr4 is active in subtelomeric heterochromatin, one of the major H3K9me domains. Core site of action. Reason: Direct experimental evidence of Clr4 activity at subtelomeric heterochromatin. Supporting Evidence: PMID:18345014 The Clr4 methyltransferase complex (ClrC) is responsible for nucleation and spreading of heterochromatin |
| GO:0031508 pericentric heterochromatin formation | IMP PMID:19136623 Phosphorylation of Swi6/HP1 regulates transcriptional gene s... | ACCEPT | Summary: Pericentric heterochromatin formation is a core role of Clr4. This IMP cites a study primarily on Swi6 phosphorylation/TGS; the broader requirement of Clr4 for pericentric H3K9me/heterochromatin is firmly established. Reason: Core heterochromatin process, well established across the literature. Supporting Evidence: PMID:11283354 Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation. |
| GO:0031509 subtelomeric heterochromatin formation | IMP PMID:24240238 Elimination of shelterin components bypasses RNAi for perice... | ACCEPT | Summary: IMP support for Clr4 in subtelomeric heterochromatin formation; the study dissects telomeric/subtelomeric heterochromatin and its interplay with pericentric assembly via Swi6 redistribution. Core role. Reason: Core subtelomeric heterochromatin function, experimentally supported. Supporting Evidence: PMID:24240238 deletion of telomere shelterin components restores pericentric heterochromatin and its functions in RNAi mutants |
| GO:1902794 siRNA-independent facultative heterochromatin formation | IMP PMID:22144463 RNA elimination machinery targeting meiotic mRNAs promotes f... | ACCEPT | Summary: Clr4 forms facultative heterochromatin islands at meiotic genes via the RNA elimination machinery (Mmi1/Red1), independently of the canonical siRNA pathway. A genuine, distinct biological role. Reason: Experimentally supported facultative heterochromatin role via RNA elimination machinery. Supporting Evidence: PMID:22144463 RNA elimination machinery is enriched at meiotic loci and interacts with Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly. |
| GO:0140720 subtelomeric heterochromatin | IDA PMID:32269268 Abo1 is required for the H3K9me2 to H3K9me3 transition in he... | ACCEPT | Summary: Direct evidence of Clr4 activity at subtelomeric heterochromatin in the context of the H3K9me2-to-me3 transition. Core site of action. Reason: Direct experimental localization/activity at subtelomeric heterochromatin. Supporting Evidence: PMID:32269268 Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin. |
| GO:0033562 co-transcriptional gene silencing by RNA interference machinery | IMP PMID:17512405 RNAi-dependent and -independent RNA turnover mechanisms cont... | ACCEPT | Summary: Clr4-deposited H3K9me underpins RNAi-coupled co-transcriptional silencing of heterochromatic repeats, working with RNA turnover pathways. A genuine process role of Clr4 within the RNAi-directed heterochromatin system. Reason: Experimentally supported role in RNAi-coupled co-transcriptional gene silencing. Supporting Evidence: PMID:17512405 the RNAi pathway is required for heterochromatin-dependent silencing of transgene insertions at centromeric repeats |
| GO:0005515 protein binding | IPI PMID:21436456 Clr4/Suv39 and RNA quality control factors cooperate to trig... | REMOVE | Summary: This IPI captures the interaction of Clr4 with Mlo3 (PomBase:SPBC1D7.04), an RNA quality-control/export-related protein and a non-histone methylation substrate of Clr4. The bare term is uninformative; the functionally meaningful aspects (Mlo3 methylation and RNAi/antisense processing) are captured elsewhere. Reason: Uninformative bare protein-binding term; the Clr4-Mlo3 interaction is better represented by the protein-lysine methyltransferase activity and process annotations. Supporting Evidence: PMID:21436456 Clr4 and the RNAi effector RITS (RNA-induced transcriptional silencing) interact with Mlo3, a protein related to mRNA quality control and export factors. |
| GO:0016279 protein-lysine N-methyltransferase activity | IDA PMID:21436456 Clr4/Suv39 and RNA quality control factors cooperate to trig... | KEEP AS NON CORE | Summary: Direct support for Clr4 methylating the non-histone protein Mlo3, consistent with its protein-lysine methyltransferase activity. Genuine but secondary to the core H3K9 function. Reason: Real non-histone methyltransferase activity; supporting/secondary to the H3K9 core function. Supporting Evidence: PMID:21436456 Clr4 and the RNAi effector RITS (RNA-induced transcriptional silencing) interact with Mlo3, a protein related to mRNA quality control and export factors. |
| GO:0140727 siRNA-mediated pericentric heterochromatin formation | EXP PMID:15615848 RNA-dependent RNA polymerase is an essential component of a ... | ACCEPT | Summary: Experimentally supported role in the RNAi self-enforcing loop coupling siRNA production to pericentric heterochromatin assembly. Core RNAi-directed heterochromatin function. Reason: Experimentally supported core RNAi-directed pericentric heterochromatin role. Supporting Evidence: PMID:15615848 its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins |
| GO:0043130 ubiquitin binding | EXP PMID:34524082 SUV39 SET domains mediate crosstalk of heterochromatic histo... | ACCEPT | Summary: The Clr4 catalytic (SET/KMT) domain harbors a ubiquitin-binding region (UBR) that specifically binds the ubiquitin moiety of H3K14ub, stimulating H3K9 methylation by over 250-fold. Genuine, mechanistically important binding activity. Reason: Experimentally demonstrated ubiquitin binding via the catalytic-domain UBR. Supporting Evidence: PMID:34524082 the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. |
| GO:0046974 histone H3K9 methyltransferase activity | EXP PMID:34524082 SUV39 SET domains mediate crosstalk of heterochromatic histo... | ACCEPT | Summary: Experimentally supported H3K9me2/3 methyltransferase activity; the study characterizes how H3K14ub stimulates this core activity and that disrupting it abolishes heterochromatin silencing similar to clr4 deletion. Reason: Experimentally supported core catalytic function. Supporting Evidence: PMID:34524082 Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe |
| GO:0140006 histone H3 reader activity | EXP PMID:34524082 SUV39 SET domains mediate crosstalk of heterochromatic histo... | ACCEPT | Summary: Clr4 reads the histone H3 tail (recognizing H3K14ub via its UBR and H3K9me via its chromodomain). This reader activity licenses and propagates H3K9 methylation. Genuine molecular function. Reason: Experimentally supported histone H3 reading (H3K14ub sensing) coupled to catalysis. Supporting Evidence: PMID:34524082 the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation. |
| GO:0061649 ubiquitin-modified histone reader activity | IPI PMID:34010645 The histone H3K9M mutation synergizes with H3K14 ubiquitylat... | ACCEPT | Summary: Clr4 reads ubiquitin-modified histone (H3K14ub), which together with the H3K9M mutation selectively sequesters Clr4 at heterochromatin. Genuine reader function consistent with the UBR characterized in PMID:34524082. Reason: Experimentally supported reading of H3K14-ubiquitylated histone via the catalytic-domain UBR. Supporting Evidence: PMID:34010645 The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin. |
| GO:0046974 histone H3K9 methyltransferase activity | IDA PMID:31468675 H3K14 ubiquitylation promotes H3K9 methylation for heterochr... | ACCEPT | Summary: Direct demonstration of Clr4 H3K9 methyltransferase activity stimulated by CLRC-mediated H3K14 ubiquitylation. Confirms the core catalytic function and its regulation. Reason: Direct assay of the core catalytic function in the context of H3K14ub regulation. Supporting Evidence: PMID:31468675 H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly. |
| GO:0030466 silent mating-type cassette heterochromatin formation | IMP PMID:11283354 Role of histone H3 lysine 9 methylation in epigenetic contro... | ACCEPT | Summary: IMP support that Clr4 (and its chromo+SET domains) is required for silencing at the mating-type region via H3K9 methylation and Swi6 recruitment. Core role. Reason: Experimentally supported core mating-type heterochromatin function. Supporting Evidence: PMID:11283354 Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. |
| GO:0031508 pericentric heterochromatin formation | IMP PMID:11283354 Role of histone H3 lysine 9 methylation in epigenetic contro... | ACCEPT | Summary: IMP support that Clr4 is required for pericentric heterochromatin formation; H3K9 methylation by Clr4 is necessary for Swi6 localization at heterochromatic regions. Core role. Reason: Experimentally supported core pericentric heterochromatin function. Supporting Evidence: PMID:11283354 Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation. |
| GO:0046974 histone H3K9 methyltransferase activity | IDA PMID:11283354 Role of histone H3 lysine 9 methylation in epigenetic contro... | ACCEPT | Summary: Direct in vivo demonstration that Clr4 methylates H3K9 at heterochromatic regions; chromo- and SET-domain dependent. Core catalytic function. Reason: Direct experimental evidence for the core catalytic function. Supporting Evidence: PMID:11283354 lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. |
| GO:0046974 histone H3K9 methyltransferase activity | IMP PMID:11283354 Role of histone H3 lysine 9 methylation in epigenetic contro... | ACCEPT | Summary: IMP support for H3K9 methyltransferase activity, from catalytic mutants (R320H, G378S, G486D) that abolish methylation and silencing. Core function. Reason: Mutational evidence supporting the core catalytic function. Supporting Evidence: PMID:11283354 Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. |
| GO:0003690 double-stranded DNA binding | IDA PMID:22727667 Intrinsic nucleic acid-binding activity of Chp1 chromodomain... | KEEP AS NON CORE | Summary: PomBase experimental (IDA) annotation from Ishida et al. 2012, which assayed the nucleic-acid-binding activity of fission-yeast chromodomain proteins. The paper's title and our cached abstract foreground the Chp1 chromodomain, but the full text also assays the Clr4 chromodomain and reports that Clr4 (the SUV39H homolog) binds nucleic acid via its chromodomain. The dsDNA-binding IDA to clr4 is therefore a valid experimental observation; it reflects an ancillary biochemical property of the chromodomain rather than Clr4's core H3K9 methyltransferase function. Reason: Valid PomBase IDA: Ishida et al. 2012 assays the Clr4 chromodomain and shows it binds nucleic acid, so dsDNA binding is retained as a real but non-core property of the chromodomain. (An earlier assessment that this paper concerned only Chp1 was based on the abstract-only cache and was incorrect; the full text reports Clr4 chromodomain nucleic-acid binding.) Supporting Evidence: PMID:22727667 Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for heterochromatic gene silencing. |
| GO:0003697 single-stranded DNA binding | IDA PMID:22727667 Intrinsic nucleic acid-binding activity of Chp1 chromodomain... | KEEP AS NON CORE | Summary: As with the dsDNA-binding annotation, this ssDNA-binding IDA comes from Ishida et al. 2012 (PomBase). The cached abstract foregrounds the Chp1 chromodomain, but the full text also assays the Clr4 chromodomain and shows Clr4 binds nucleic acid via its chromodomain. The IDA is valid but reflects an ancillary chromodomain property, not Clr4's core methyltransferase function. Reason: Valid PomBase IDA (Clr4 chromodomain assayed in Ishida et al. 2012); retained as a non-core property. (Earlier "concerns Chp1, not Clr4" assessment was based on the abstract-only cache and was incorrect.) Supporting Evidence: PMID:22727667 Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for heterochromatic gene silencing. |
| GO:0003727 single-stranded RNA binding | IDA PMID:22727667 Intrinsic nucleic acid-binding activity of Chp1 chromodomain... | KEEP AS NON CORE | Summary: This ssRNA-binding IDA also comes from Ishida et al. 2012 (PomBase). The cached abstract foregrounds Chp1, but the full text assays the Clr4 chromodomain and reports Clr4 binds nucleic acid (RNA/DNA) via its chromodomain. The IDA is valid but is an ancillary chromodomain property rather than Clr4's core methyltransferase function. Note the paper itself flags that the role of Clr4-chromodomain nucleic- acid binding in heterochromatin assembly was unclear at that time. Reason: Valid PomBase IDA (Clr4 chromodomain assayed in Ishida et al. 2012); retained as a non-core property. (Earlier "concerns Chp1, not Clr4" assessment was based on the abstract-only cache and was incorrect.) Supporting Evidence: PMID:22727667 Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for heterochromatic gene silencing. |
| GO:0030466 silent mating-type cassette heterochromatin formation | IMP PMID:8001791 Three additional linkage groups that repress transcription a... | ACCEPT | Summary: Classic genetic IMP defining clr4 as a trans-acting silencing gene required for transcriptional and recombinational repression at the mat2-mat3 region. Core mating-type heterochromatin function. Reason: Foundational genetic evidence for the core mating-type silencing role. Supporting Evidence: PMID:8001791 the transcription and recombination blocks require three newly defined trans-acting loci, clr2, clr3 and clr4, in addition to the previously identified clr1, rik1 and swi6 loci. |
| GO:0005634 nucleus | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: High-throughput GFP localization placing Clr4 in the nucleus, consistent with its chromatin function. Correct compartment. Reason: Correct nuclear localization, consistent with all functional data. Supporting Evidence: PMID:16823372 ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. |
| GO:0043494 CLRC complex | IDA PMID:16127433 Ubiquitin ligase component Cul4 associates with Clr4 histone... | ACCEPT | Summary: Direct evidence (co-purification, direct Clr4-Cul4/Pcu4 interaction) for Clr4 as a subunit of the CLRC complex. Core complex membership. Reason: Direct experimental evidence for the core CLRC complex membership. Supporting Evidence: PMID:16127433 Clr4 associates with Cul4, a cullin family protein that serves as a scaffold for assembling ubiquitin ligases. |
| GO:0046974 histone H3K9 methyltransferase activity | IDA PMID:10949293 Regulation of chromatin structure by site-specific histone H... | ACCEPT | Summary: Direct in vitro demonstration that the SUV39/Clr4 family selectively methylates H3K9. Founding evidence for the core catalytic function. Reason: Direct experimental evidence for the core catalytic function. Supporting Evidence: PMID:10949293 encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro |
| GO:0008168 methyltransferase activity | IDA PMID:10949293 Regulation of chromatin structure by site-specific histone H... | MARK AS OVER ANNOTATED | Summary: Methyltransferase activity is correct but is the over-general grandparent of the H3K9-specific activity that is the core function. The specific histone H3K9 methyltransferase term should be used. Reason: Over-general term; the specific H3K9 methyltransferase activity is the well-supported core function. Proposed replacements: histone H3K9 methyltransferase activity Supporting Evidence: PMID:10949293 encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro |
Loading supporting content…
Download this section (compressed HTML)Q: How is the balance between Clr4 intrinsic autoinhibition (automethylation of the AI loop) and extrinsic anti-silencing factors (Epe1, boundary elements, Clr4 dosage) quantitatively tuned to define heterochromatin domain boundaries in vivo?
Q: What is the full in vivo repertoire of non-histone Clr4 substrates (beyond Mlo3 and automethylation), and what are their biological consequences?
Q: To what extent is the H3K14ub-to-H3K9me crosstalk a general feature of SUV39 enzymes across eukaryotes, and how does it intersect with the read-write spreading mechanism?
Experiment: Quantitative genome-wide ChIP-seq of H3K9me1/2/3 and Clr4 occupancy in panels of catalytic, chromodomain, AI-loop (K455/K472) and UBR mutants to dissect the relative contributions of writing, reading, autoregulation and H3K14ub-sensing to nucleation versus spreading.
Experiment: Reconstitution of CLRC on nucleosomal arrays to measure how H3K14 ubiquitylation, Clr4 automethylation and chromodomain engagement cooperatively control processive H3K9 methylation and spreading kinetics.
Experiment: Proteome-wide identification of Clr4 lysine-methylation targets (e.g. SILAC/quantitative MS of clr4 mutants) to map the non-histone methylome and test its role in RNA surveillance and heterochromatin.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)