clr4

UniProt ID: O60016
Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Review Status: DRAFT
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Gene Description

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.

Existing Annotations Review

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.
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.
Supporting Evidence:
PMID:10949293
encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro

Core Functions

Sole histone H3 lysine 9 methyltransferase of fission yeast; catalyzes H3K9 mono-, di- and trimethylation via its SET catalytic domain to establish the defining heterochromatic histone mark.

Molecular Function:
histone H3K9 methyltransferase activity
Directly Involved In:
heterochromatin formation
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.
  • PMID:34524082
    Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe

Reader-writer of H3K9 methylation: the N-terminal chromodomain binds pre-existing H3K9me2/3 and tethers the SET domain to methylate neighboring nucleosomes, driving sequence-independent spreading and epigenetic maintenance of heterochromatin.

Molecular Function:
histone H3K9me2/3 reader activity
Directly Involved In:
heterochromatin formation
Supporting Evidence:
  • PMID:18345014
    the ability of Clr4 to both 'write' and 'read' H3K9me facilitates heterochromatin maintenance through successive cell divisions.

Catalytic subunit of the CLRC complex, a CRL4-type cullin-RING E3 ubiquitin ligase (with Rik1, Cul4/Pcu4, Raf1/Dos1, Raf2/Dos2 and Pip1) that ubiquitylates histone H3K14; H3K14ub is read by the Clr4 catalytic-domain UBR and strongly stimulates H3K9 methylation.

Molecular Function:
ubiquitin-modified histone reader activity
In Complex:
CLRC complex
Supporting Evidence:
  • PMID:16024659
    subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1 and Clr4
  • PMID:34524082
    the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold.

Effector of RNAi-directed and RNA-elimination-directed heterochromatin formation, mediating transcriptional gene silencing at centromeres, telomeres/subtelomeres, the silent mating-type region and facultative heterochromatin islands at meiotic genes.

Directly Involved In:
siRNA-mediated pericentric heterochromatin formation
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
  • PMID:22144463
    RNA elimination machinery is enriched at meiotic loci and interacts with Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly.

References

GO_REF:0000002
Gene Ontology annotation through association of InterPro records with GO terms
GO_REF:0000044
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
GO_REF:0000116
Automatic Gene Ontology annotation based on Rhea mapping
GO_REF:0000117
Electronic Gene Ontology annotations created by ARBA machine learning models
GO_REF:0000120
Combined Automated Annotation using Multiple IEA Methods
PMID:10949293
Regulation of chromatin structure by site-specific histone H3 methyltransferases.
  • SUV39H1/Suv39h1, mammalian homologs of S. pombe clr4, are histone H3-specific methyltransferases that selectively methylate H3K9 via the SET domain plus adjacent cysteine-rich regions.
    "encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro. We mapped the catalytic motif to the evolutionarily conserved SET domain, which requires adjacent cysteine-rich regions to confer histone methyltransferase activity."
PMID:11283354
Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly.
  • Clr4 preferentially methylates H3K9 at heterochromatic regions in vivo, and both the chromo- and SET domains are required; H3K9me is needed for Swi6 localization.
    "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:15615848
RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production.
  • A self-enforcing RNAi loop couples siRNA production to heterochromatin assembly; RITS/Rdp1 localization at centromeric repeats depends on Clr4 and Swi6.
    "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"
PMID:16024659
A Rik1-associated, cullin-dependent E3 ubiquitin ligase is essential for heterochromatin formation.
  • A Rik1-/Clr4-associated cullin-dependent E3 ubiquitin ligase (with Raf1, Raf2, Pcu4, Pip1) is essential for H3K9 methylation and heterochromatin formation.
    "subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1 and Clr4, and Rik1-TAP preparations exhibit robust E3 ubiquitin ligase activity."
PMID:16127433
Ubiquitin ligase component Cul4 associates with Clr4 histone methyltransferase to assemble heterochromatin.
  • Clr4 directly associates with the cullin Cul4/Pcu4; Cul4 is required for Clr4 localization, H3K9 methylation, Swi6 recruitment and silencing.
    "We show that Clr4 associates with Cul4, a cullin family protein that serves as a scaffold for assembling ubiquitin ligases. Mutations in Cul4 result in defective localization of Clr4 and loss of silencing at heterochromatic loci."
PMID:16823372
ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe.
  • Genome-wide GFP localization study; Clr4 localizes to the nucleus.
    "ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe."
PMID:17512405
RNAi-dependent and -independent RNA turnover mechanisms contribute to heterochromatic gene silencing.
  • RNAi and RNA turnover (Cid14/TRAMP-like) cooperate in heterochromatic gene silencing at centromeric repeats.
    "the RNAi pathway is required for heterochromatin-dependent silencing of transgene insertions at centromeric repeats"
PMID:18345014
Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin.
  • ClrC nucleates and spreads heterochromatin; the Clr4 chromodomain binds H3K9me to enable spreading, and Clr4's write/read of H3K9me underlies maintenance.
    "the chromodomain of Clr4 binds specifically to H3K9me that is essential for the spreading of heterochromatin... the ability of Clr4 to both 'write' and 'read' H3K9me facilitates heterochromatin maintenance through successive cell divisions."
PMID:19136623
Phosphorylation of Swi6/HP1 regulates transcriptional gene silencing at heterochromatin.
  • CK2-dependent Swi6 phosphorylation controls transcriptional gene silencing downstream of Clr4-mediated heterochromatin.
    "CK2-dependent Swi6 phosphorylation specifically controls TGS in heterochromatin."
PMID:20211136
Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity.
  • Stc1 bridges the RNAi effector Ago1 to the CLRC complex, recruiting Clr4 to promote H3K9 methylation.
    "This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation."
PMID:21436456
Clr4/Suv39 and RNA quality control factors cooperate to trigger RNAi and suppress antisense RNA.
  • Clr4 interacts with Mlo3 (and methylates it); loss of Clr4 impairs RITS-Mlo3 interaction required for centromeric siRNA production and antisense suppression.
    "Clr4 and the RNAi effector RITS (RNA-induced transcriptional silencing) interact with Mlo3, a protein related to mRNA quality control and export factors. Loss of Clr4 impairs RITS interaction with Mlo3, which is required for centromeric siRNA production and antisense suppression."
PMID:22144463
RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation.
  • Facultative heterochromatin islands form at meiotic genes via RNA elimination factors (Mmi1/Red1) that interact with Clr4/SUV39h.
    "RNA elimination machinery is enriched at meiotic loci and interacts with Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly."
PMID:22727667
Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for heterochromatic gene silencing.
  • This study characterizes intrinsic nucleic-acid binding by fission-yeast chromodomains. While the title emphasizes Chp1, the paper also assays the Clr4 chromodomain and reports that Clr4 (the SUV39H homolog) binds nucleic acid via its chromodomain, supporting the PomBase dsDNA/ssDNA/ssRNA-binding IDA annotations to clr4 (a non-core ancillary property of the chromodomain).
    "Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for heterochromatic gene silencing."
PMID:24240238
Elimination of shelterin components bypasses RNAi for pericentric heterochromatin assembly.
  • Deletion of telomere shelterin components restores pericentric heterochromatin in RNAi mutants by releasing Swi6 for RNAi-independent assembly; informs subtelomeric heterochromatin formation by Clr4.
    "deletion of telomere shelterin components restores pericentric heterochromatin and its functions in RNAi mutants"
PMID:24449894
CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed surface of Dos1 for heterochromatin silencing.
  • Clr4 is the only H3K9 methyltransferase in S. pombe and a subunit of the CRL4-like CLRC complex (Cul4, Pip1, Rik1, Dos1); H3K9me deposition depends on the RNAi pathway.
    "Cryptic loci regulator 4 (Clr4), the only known H3K9 methyltransferase in this organism, is a subunit of the Clr4 methyltransferase complex (CLRC), whose composition is reminiscent of a CRL4 type cullin-RING ubiquitin ligase"
PMID:28143796
Clr4 specificity and catalytic activity beyond H3K9 methylation.
  • Clr4 introduces H3K9 di-/tri-methylation and also methylates non-histone substrates including Mlo3 and additional target sites.
    "The enzyme introduces histone 3 lysine 9 (H3K9) di- and tri-methylation, a central heterochromatic histone modification, and later it was also found to methylate the Mlo3 protein, which has a role in heterochromatin formation as well."
PMID:30051891
Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability.
  • An internal autoinhibitory loop blocks the H3K9 substrate pocket; automethylation of K455 (and K472) triggers a conformational switch that activates Clr4 and prevents aberrant heterochromatin spreading.
    "an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4."
PMID:31165882
Disordered region of H3K9 methyltransferase Clr4 binds the nucleosome and contributes to its activity.
  • The Clr4 chromodomain binds the H3K9me3 tail and, together with the disordered linker, binds the nucleosome core to contribute to H3K9 methylation in vitro and in vivo.
    "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."
PMID:31468675
H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly.
  • CLRC (Clr4 + Cul4) ubiquitylates H3K14, and H3K14ub promotes Clr4 H3K9 methylation for heterochromatin assembly.
    "H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly."
PMID:32269268
Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin.
  • The bromodomain AAA-ATPase Abo1 is required for the H3K9me2-to-H3K9me3 transition deposited by Clr4 in heterochromatin.
    "Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin."
PMID:34010645
The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin.
  • H3K9M and H3K14ub together selectively sequester Clr4 at pericentric heterochromatin, demonstrating Clr4 reading of ubiquitin-modified histone.
    "The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin."
PMID:34524082
SUV39 SET domains mediate crosstalk of heterochromatic histone marks.
  • The Clr4 catalytic domain contains a ubiquitin-binding region that binds H3K14ub and stimulates the enzyme >250-fold; disrupting it abolishes heterochromatin silencing like clr4 deletion. Clr4 is the sole H3K9me2/3 methyltransferase of S. pombe.
    "the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion."
PMID:40446033
Mechanistic insights into the stimulation of the histone H3K9 methyltransferase Clr4 by proximal H3K14 ubiquitination.
  • Crystal structures of the Clr4 KMT (pre-SET/SET/post-SET) catalytic domain bound to H3 peptide and SAM cofactor reveal the SAM-dependent H3K9 methyltransferase active site and the multivalent ubiquitin interface by which proximal H3K14 ubiquitination stimulates H3K9me2/3 deposition.
    "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:40923761
Intrinsically disordered region of Clr4/Suv39 regulates its enzymatic activity and ensures heterochromatin spreading.
  • The intrinsically disordered region of Clr4 regulates its H3K9 methyltransferase activity and is required for heterochromatin spreading.
    "Intrinsically disordered region of Clr4/Suv39 regulates its enzymatic activity and ensures heterochromatin spreading."
PMID:8001791
Three additional linkage groups that repress transcription and meiotic recombination in the mating-type region of Schizosaccharomyces pombe.
  • clr4 (with clr2, clr3) is a trans-acting locus required for transcriptional and recombinational silencing at the mating-type region.
    "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."

Suggested Questions for Experts

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?

Suggested Experiments

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.

📚 Additional Documentation

Notes

(clr4-notes.md)

clr4 (Cryptic loci regulator 4 / Kmt1) — S. pombe — Curation Notes

UniProt: O60016 (CLR4_SCHPO). PomBase SPBC428.08c. 490 aa. Synonym kmt1.
EC 2.1.1.355 (H3K9 trimethyl), 2.1.1.366 (H3K9me2), 2.1.1.367 (H3K9 monomethyl), plus protein-lysine activities.

Core identity

Clr4 is the SOLE histone H3 lysine 9 (H3K9) methyltransferase of fission yeast and the catalytic
subunit of the CLRC (Clr4 methyltransferase complex). It is the homolog of metazoan SUV39H1/2 and
Drosophila Su(var)3-9. Domain architecture: N-terminal chromodomain (8–69), disordered linker,
pre-SET (258–325), SET (328–452), post-SET (473–489).

Catalytic activity / methylation states

Catalyzes H3K9 mono-, di-, and trimethylation. Also methylates non-histone substrates (Mlo3 and
others) — "protein-lysine N-methyltransferase activity".
- PMID:28143796
- PMID:28143796

Reader/writer (chromodomain)

The chromodomain reads H3K9me; together with the SET writer it gives a read-write feedback that
spreads/maintains heterochromatin.
- PMID:18345014
- PMID:31165882

CLRC complex (E3 ligase / methyltransferase)

Clr4 + Rik1 + Cul4(Pcu4) + Raf1/Dos1 + Raf2/Dos2 + Rbx1/Pip1; resembles a CRL4 cullin-RING ligase.
Preferred ub substrate is H3K14 (H3K14ub), which stimulates Clr4 H3K9 methylation.
- PMID:16127433
- PMID:16024659
- PMID:31468675
- PMID:34524082
- PMID:34010645

Autoregulation (automethylation)

An internal autoregulatory loop autoinhibits the SET pocket; automethylation of Lys455 switches it on.
- PMID:30051891
- IDR also regulates activity: PMID:40923761

Biological processes (silencing / heterochromatin)

H3K9me recruits Swi6/HP1 -> silencing at centromeres, telomeres, mating-type locus, rDNA.
- Mating-type silencing: PMID:8001791
- Pericentric/silencing via RNAi self-enforcing loop: PMID:15615848
- Co-transcriptional gene silencing / RNA turnover: PMID:17512405 (Clr4 IMP for GO:0033562)
- Facultative heterochromatin islands at meiotic genes: PMID:22144463
- Subtelomeric: [PMID:24240238 deletion of shelterin restores pericentric heterochromatin in RNAi mutants — Clr4 IMP for subtelomeric formation] ; PMID:32269268
- siRNA processing/antisense suppression via Mlo3: PMID:21436456

Localization

Nucleus; active in pericentric, mating-type, subtelomeric heterochromatin. SPB localization from a
single high-throughput GFP study (16823372) — weak/likely non-core; nucleus is the functional site.
- [PMID:16823372 global GFP localization study] ; [PMID:18345014 ClrC components distributed throughout heterochromatic domains]

DNA/RNA/nucleosome binding

PMID:22727667 is actually about the Chp1 chromodomain, not Clr4 — the dsDNA/ssDNA/ssRNA binding IDA
annotations attributed to clr4 from this paper look mis-attributed/over-annotated (paper title and
abstract concern Chp1). Treat with caution. Nucleosome binding (31165882) is genuine and core-relevant.
ssRNA binding ARBA IEA is generic.

Stc1 interaction (GO:0005515 IPI, PMID:20211136)

Stc1 links RNAi (Ago1) to CLRC; bridges RITS to Clr4 complex. Adapter/recruitment interaction.
- PMID:20211136

Summary of action plan

  • Core MF: histone H3K9 methyltransferase (mono/di/tri) — ACCEPT specific terms; generalize generic
    "methyltransferase activity" and "histone methyltransferase activity".
  • Core MF: H3K9me reader / chromodomain (GO:0062072) — ACCEPT.
  • Core BP: heterochromatin formation at pericentric, subtelomeric, mating-type; RNAi-related — ACCEPT/KEEP.
  • protein binding (GO:0005515) bare terms — replace/mark; capture as complex membership / adapter.
  • DNA binding IEA, ssRNA IEA, zinc IEA — keep_as_non_core / accept low-value structural.
  • SPB localization — KEEP_AS_NON_CORE (single HTP study).
  • 22727667 DNA/RNA binding IDA — likely mis-attributed (paper is about Chp1) -> MARK_AS_OVER_ANNOTATED / UNDECIDED.

📄 View Raw YAML

 
id: O60016
gene_symbol: clr4
product_type: PROTEIN
status: DRAFT
taxon:
  id: NCBITaxon:284812
  label: Schizosaccharomyces pombe (strain 972 / ATCC 24843)
description: >-
  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.
existing_annotations:
- term:
    id: GO:0003677
    label: DNA binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.)
    supported_by:
    - reference_id: PMID:22727667
      supporting_text: >-
        Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required
        for heterochromatic gene silencing.
      reference_section_type: TITLE
- term:
    id: GO:0003727
    label: single-stranded RNA binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.)
    supported_by:
    - reference_id: PMID:22727667
      supporting_text: >-
        Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required
        for heterochromatic gene silencing.
      reference_section_type: TITLE
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: located_in
  review:
    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.
    action: ACCEPT
    reason: Nuclear localization is well established experimentally and by SubCell mapping.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: ClrC components are distributed throughout heterochromatic domains.
      reference_section_type: ABSTRACT
- term:
    id: GO:0005694
    label: chromosome
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    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.
    action: KEEP_AS_NON_CORE
    reason: Correct but general; more specific heterochromatin location terms are also annotated.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: ClrC components are distributed throughout heterochromatic domains.
      reference_section_type: ABSTRACT
- term:
    id: GO:0005721
    label: pericentric heterochromatin
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: located_in
  review:
    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.
    action: ACCEPT
    reason: Correct localization, also supported by experimental IDA annotations.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: ClrC components are distributed throughout heterochromatic domains.
      reference_section_type: ABSTRACT
- term:
    id: GO:0005816
    label: spindle pole body
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    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.
    action: MARK_AS_OVER_ANNOTATED
    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.
    supported_by:
    - reference_id: PMID:16823372
      supporting_text: >-
        ORFeome cloning and global analysis of protein localization in the
        fission yeast Schizosaccharomyces pombe.
      reference_section_type: TITLE
- term:
    id: GO:0008270
    label: zinc ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    reason: >-
      Structurally verified zinc binding required for SET-domain catalysis;
      supporting rather than core molecular function.
    supported_by:
    - reference_id: PMID:10949293
      supporting_text: >-
        We mapped the catalytic motif to the evolutionarily conserved SET
        domain, which requires adjacent cysteine-rich regions to confer histone
        methyltransferase activity.
      reference_section_type: ABSTRACT
    - reference_id: PMID:40446033
      supporting_text: >-
        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).
      reference_section_type: RESULTS
- term:
    id: GO:0016279
    label: protein-lysine N-methyltransferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000116
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct general activity, supported experimentally; the H3K9-specific
      methyltransferase term is the core function.
    supported_by:
    - reference_id: PMID:28143796
      supporting_text: >-
        later it was also found to methylate the Mlo3 protein, which has a role
        in heterochromatin formation as well
      reference_section_type: ABSTRACT
- term:
    id: GO:0030466
    label: silent mating-type cassette heterochromatin formation
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    reason: Core silencing function corroborated by experimental IMP evidence (PMID:8001791, PMID:11283354).
    supported_by:
    - reference_id: PMID:8001791
      supporting_text: >-
        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.
      reference_section_type: ABSTRACT
- term:
    id: GO:0031509
    label: subtelomeric heterochromatin formation
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: involved_in
  review:
    summary: >-
      Subtelomeric heterochromatin formation is a core role of Clr4 and is also
      supported by experimental IMP evidence (PMID:24240238).
    action: ACCEPT
    reason: Core heterochromatin role; corroborated by experimental annotation.
    supported_by:
    - reference_id: PMID:24240238
      supporting_text: >-
        deletion of telomere shelterin components restores pericentric
        heterochromatin and its functions in RNAi mutants
      reference_section_type: ABSTRACT
- term:
    id: GO:0031934
    label: mating-type region heterochromatin
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: located_in
  review:
    summary: >-
      Localization to mating-type region heterochromatin is correct and is also
      supported experimentally (is_active_in IDA, PMID:18345014).
    action: ACCEPT
    reason: Correct site of action, also supported by experimental IDA.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: ClrC components are distributed throughout heterochromatic domains.
      reference_section_type: ABSTRACT
- term:
    id: GO:0031981
    label: nuclear lumen
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: located_in
  review:
    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.
    action: KEEP_AS_NON_CORE
    reason: Correct but over-general compartment; subsumed by more specific nuclear/heterochromatin annotations.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: ClrC components are distributed throughout heterochromatic domains.
      reference_section_type: ABSTRACT
- term:
    id: GO:0042054
    label: histone methyltransferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    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.
    action: MARK_AS_OVER_ANNOTATED
    reason: Over-general parent of the well-supported H3K9-specific methyltransferase activity.
    proposed_replacement_terms:
    - id: GO:0046974
      label: histone H3K9 methyltransferase activity
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the
        Clr4 protein at heterochromatin-associated regions in fission yeast.
      reference_section_type: ABSTRACT
- term:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Core molecular function, supported by numerous direct assays.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        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.
      reference_section_type: ABSTRACT
    - reference_id: PMID:40446033
      supporting_text: >-
        Clr4 is the sole known methyltransferase catalyzing H3K9 methylation
        in Schizosaccharomyces pombe.
      reference_section_type: ABSTRACT
    - reference_id: PMID:40446033
      supporting_text: >-
        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.
      reference_section_type: RESULTS
- term:
    id: GO:0062072
    label: histone H3K9me2/3 reader activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: enables
  review:
    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).
    action: ACCEPT
    reason: Genuine reader function of the chromodomain, also experimentally supported.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: >-
        the chromodomain of Clr4 binds specifically to H3K9me that is essential
        for the spreading of heterochromatin.
      reference_section_type: ABSTRACT
- term:
    id: GO:0140720
    label: subtelomeric heterochromatin
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: located_in
  review:
    summary: >-
      Localization to subtelomeric heterochromatin is correct and is also
      supported by experimental IDA (PMID:18345014, PMID:32269268).
    action: ACCEPT
    reason: Correct site of action, also supported experimentally.
    supported_by:
    - reference_id: PMID:32269268
      supporting_text: >-
        Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin.
      reference_section_type: TITLE
- term:
    id: GO:0140727
    label: siRNA-mediated pericentric heterochromatin formation
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: involved_in
  review:
    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).
    action: ACCEPT
    reason: Core RNAi-directed heterochromatin function, supported experimentally.
    supported_by:
    - reference_id: PMID:15615848
      supporting_text: >-
        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
      reference_section_type: ABSTRACT
- term:
    id: GO:0140947
    label: histone H3K9me2 methyltransferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      Clr4 catalyzes H3K9 dimethylation; this Rhea/EC-derived specific activity
      is correct and supported by experimental EXP evidence (PMID:30051891).
    action: ACCEPT
    reason: Specific catalytic activity within the core H3K9 methyltransferase function; experimentally supported.
    supported_by:
    - reference_id: PMID:30051891
      supporting_text: >-
        automethylation of specific lysines in this loop promotes a
        conformational switch that enhances the H3K9me activity of Clr4.
      reference_section_type: ABSTRACT
- term:
    id: GO:0140948
    label: histone H3K9 monomethyltransferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      Clr4 catalyzes H3K9 monomethylation; this specific activity is correct and
      supported by experimental EXP evidence (PMID:10949293, PMID:11283354).
    action: ACCEPT
    reason: Specific catalytic activity within the core function; experimentally supported.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the
        Clr4 protein
      reference_section_type: ABSTRACT
- term:
    id: GO:0140949
    label: histone H3K9 trimethyltransferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      Clr4 catalyzes H3K9 trimethylation (the heterochromatic H3K9me3 mark); this
      specific activity is correct and supported by experimental EXP evidence
      (PMID:11283354).
    action: ACCEPT
    reason: Specific catalytic activity within the core function; experimentally supported.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        Role of histone H3 lysine 9 methylation in epigenetic control of
        heterochromatin assembly.
      reference_section_type: TITLE
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:16024659
  qualifier: enables
  review:
    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.
    action: REMOVE
    reason: >-
      Uninformative bare protein-binding term; the underlying Clr4-Rik1
      interaction is better represented by the CLRC complex membership annotation.
    supported_by:
    - reference_id: PMID:16024659
      supporting_text: >-
        subunits of a cullin-dependent E3 ubiquitin ligase are associated with
        Rik1 and Clr4
      reference_section_type: ABSTRACT
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:16127433
  qualifier: enables
  review:
    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.
    action: REMOVE
    reason: >-
      Uninformative bare protein-binding term; the Clr4-Cul4/Pcu4 interaction is
      better represented by CLRC complex membership.
    supported_by:
    - reference_id: PMID:16127433
      supporting_text: >-
        Clr4 associates with Cul4, a cullin family protein that serves as a
        scaffold for assembling ubiquitin ligases.
      reference_section_type: ABSTRACT
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:20211136
  qualifier: enables
  review:
    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.
    action: REMOVE
    reason: >-
      Uninformative bare protein-binding term; the Clr4/CLRC-Stc1 interaction is
      better represented within the RNAi-directed heterochromatin process terms.
    supported_by:
    - reference_id: PMID:20211136
      supporting_text: >-
        This leads to recruitment of the CLRC complex, including the histone
        methyltransferase Clr4, promoting H3K9 methylation and heterochromatin
        formation.
      reference_section_type: ABSTRACT
- term:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:40923761
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Direct assay of the core catalytic function.
    supported_by:
    - reference_id: PMID:40923761
      supporting_text: >-
        Intrinsically disordered region of Clr4/Suv39 regulates its enzymatic
        activity and ensures heterochromatin spreading.
      reference_section_type: TITLE
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: EXP
  original_reference_id: PMID:18345014
  qualifier: located_in
  review:
    summary: >-
      Experimentally supported nuclear localization, consistent with Clr4's
      chromatin function.
    action: ACCEPT
    reason: Experimentally supported correct compartment.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: ClrC components are distributed throughout heterochromatic domains.
      reference_section_type: ABSTRACT
- term:
    id: GO:0005694
    label: chromosome
  evidence_type: EXP
  original_reference_id: PMID:18345014
  qualifier: located_in
  review:
    summary: >-
      Experimentally supported chromosome localization (ChIP across
      heterochromatic domains). Correct but general relative to the specific
      heterochromatin terms.
    action: KEEP_AS_NON_CORE
    reason: Correct but general; more specific heterochromatin location terms preferred.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: ClrC components are distributed throughout heterochromatic domains.
      reference_section_type: ABSTRACT
- term:
    id: GO:0016279
    label: protein-lysine N-methyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:28143796
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    reason: Real non-histone methyltransferase activity; supporting/secondary to the H3K9 core function.
    supported_by:
    - reference_id: PMID:28143796
      supporting_text: >-
        Peptide methylation was observed on Mlo3 and 7 novel target sites
      reference_section_type: ABSTRACT
- term:
    id: GO:0140947
    label: histone H3K9me2 methyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:30051891
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported specific catalytic activity within the core function.
    supported_by:
    - reference_id: PMID:30051891
      supporting_text: >-
        automethylation of specific lysines in this loop promotes a
        conformational switch that enhances the H3K9me activity of Clr4.
      reference_section_type: ABSTRACT
    - reference_id: PMID:40446033
      supporting_text: >-
        The side chain of histone H3 K9Nle inserts into a hydrophobic tunnel
        and attacks the S-methyl group of SAM
      reference_section_type: RESULTS
    - reference_id: PMID:40446033
      supporting_text: >-
        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.
      reference_section_type: ABSTRACT
- term:
    id: GO:0140948
    label: histone H3K9 monomethyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:10949293
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported specific catalytic activity within the core function.
    supported_by:
    - reference_id: PMID:10949293
      supporting_text: >-
        encode histone H3-specific methyltransferases that selectively methylate
        lysine 9 of the amino terminus of histone H3 in vitro
      reference_section_type: ABSTRACT
    - reference_id: PMID:40446033
      supporting_text: >-
        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.
      reference_section_type: RESULTS
    - reference_id: PMID:40446033
      supporting_text: >-
        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
      reference_section_type: RESULTS
- term:
    id: GO:0140948
    label: histone H3K9 monomethyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:11283354
  qualifier: enables
  review:
    summary: >-
      Experimentally supported H3K9 monomethyltransferase activity, with
      catalytic mutagenesis (R320H, G378S, G486D) abolishing activity.
    action: ACCEPT
    reason: Experimentally supported specific catalytic activity within the core function.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the
        Clr4 protein
      reference_section_type: ABSTRACT
- term:
    id: GO:0140949
    label: histone H3K9 trimethyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:11283354
  qualifier: enables
  review:
    summary: >-
      Experimentally supported H3K9 trimethyltransferase activity producing the
      hallmark H3K9me3 heterochromatic mark.
    action: ACCEPT
    reason: Experimentally supported specific catalytic activity within the core function.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        Role of histone H3 lysine 9 methylation in epigenetic control of
        heterochromatin assembly.
      reference_section_type: TITLE
- term:
    id: GO:0031491
    label: nucleosome binding
  evidence_type: EXP
  original_reference_id: PMID:31165882
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Directly demonstrated nucleosome binding contributing to catalysis and de novo H3K9me deposition.
    supported_by:
    - reference_id: PMID:31165882
      supporting_text: >-
        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.
      reference_section_type: ABSTRACT
- term:
    id: GO:0031491
    label: nucleosome binding
  evidence_type: IPI
  original_reference_id: PMID:31165882
  qualifier: enables
  review:
    summary: >-
      IPI annotation of nucleosome (histone) binding from the same study,
      supported by direct binding assays with reconstituted nucleosomes.
    action: ACCEPT
    reason: Direct nucleosome-binding interaction evidence; same well-supported function.
    supported_by:
    - reference_id: PMID:31165882
      supporting_text: >-
        both, the chromodomain and the disordered region connecting the
        chromodomain and the SET domain, bind the nucleosome core.
      reference_section_type: ABSTRACT
- term:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  evidence_type: IMP
  original_reference_id: PMID:31165882
  qualifier: enables
  review:
    summary: >-
      IMP support for H3K9 methyltransferase activity: disordered-region mutants
      reduce nucleosome binding and H3K9 methylation in vivo. Confirms the core
      function.
    action: ACCEPT
    reason: Mutational support for the core catalytic function.
    supported_by:
    - reference_id: PMID:31165882
      supporting_text: >-
        interaction of the disordered region with the nucleosome core is
        independent of H3K9me and contributes to H3K9me in vitro and in vivo.
      reference_section_type: ABSTRACT
- term:
    id: GO:0005721
    label: pericentric heterochromatin
  evidence_type: NAS
  original_reference_id: PMID:24449894
  qualifier: located_in
  review:
    summary: >-
      Pericentric heterochromatin localization (NAS, ComplexPortal). Correct and
      also supported by experimental IDA; pericentric heterochromatin is a
      principal site of CLRC/Clr4 action.
    action: ACCEPT
    reason: Correct localization, corroborated by experimental IDA evidence.
    supported_by:
    - reference_id: PMID:24449894
      supporting_text: >-
        Repressive histone H3 lysine 9 methylation (H3K9me) and its recognition
        by HP1 proteins are necessary for pericentromeric heterochromatin
        formation.
      reference_section_type: ABSTRACT
- term:
    id: GO:0043494
    label: CLRC complex
  evidence_type: NAS
  original_reference_id: PMID:16024659
  qualifier: part_of
  review:
    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.
    action: ACCEPT
    reason: Core complex membership; the central organizing fact of Clr4 biology.
    supported_by:
    - reference_id: PMID:16024659
      supporting_text: >-
        subunits of a cullin-dependent E3 ubiquitin ligase are associated with
        Rik1 and Clr4
      reference_section_type: ABSTRACT
- term:
    id: GO:0140727
    label: siRNA-mediated pericentric heterochromatin formation
  evidence_type: NAS
  original_reference_id: PMID:24449894
  qualifier: involved_in
  review:
    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).
    action: ACCEPT
    reason: Core RNAi-directed heterochromatin process, corroborated experimentally.
    supported_by:
    - reference_id: PMID:24449894
      supporting_text: H3K9me deposition depends on the RNAi pathway.
      reference_section_type: ABSTRACT
- term:
    id: GO:0005721
    label: pericentric heterochromatin
  evidence_type: IDA
  original_reference_id: PMID:18345014
  qualifier: is_active_in
  review:
    summary: >-
      Direct evidence that Clr4/ClrC is active in pericentric heterochromatin,
      where it nucleates and spreads H3K9me. Core site of action.
    action: ACCEPT
    reason: Direct experimental evidence of Clr4 activity at pericentric heterochromatin.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: >-
        The Clr4 methyltransferase complex (ClrC) is responsible for nucleation
        and spreading of heterochromatin
      reference_section_type: ABSTRACT
- term:
    id: GO:0031934
    label: mating-type region heterochromatin
  evidence_type: IDA
  original_reference_id: PMID:18345014
  qualifier: is_active_in
  review:
    summary: >-
      Direct evidence that Clr4 is active in mating-type region heterochromatin,
      a principal silencing domain. Core site of action.
    action: ACCEPT
    reason: Direct experimental evidence of Clr4 activity at the mating-type heterochromatin.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: >-
        The Clr4 methyltransferase complex (ClrC) is responsible for nucleation
        and spreading of heterochromatin
      reference_section_type: ABSTRACT
- term:
    id: GO:0062072
    label: histone H3K9me2/3 reader activity
  evidence_type: EXP
  original_reference_id: PMID:18345014
  qualifier: enables
  review:
    summary: >-
      Experimentally supported reader activity: the Clr4 chromodomain binds
      H3K9me2/3, enabling the read-write feedback that spreads and maintains
      heterochromatin. Core function.
    action: ACCEPT
    reason: Experimentally supported chromodomain reader function central to spreading/maintenance.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: >-
        the ability of Clr4 to both 'write' and 'read' H3K9me facilitates
        heterochromatin maintenance through successive cell divisions.
      reference_section_type: ABSTRACT
- term:
    id: GO:0140720
    label: subtelomeric heterochromatin
  evidence_type: IDA
  original_reference_id: PMID:18345014
  qualifier: is_active_in
  review:
    summary: >-
      Direct evidence that Clr4 is active in subtelomeric heterochromatin, one of
      the major H3K9me domains. Core site of action.
    action: ACCEPT
    reason: Direct experimental evidence of Clr4 activity at subtelomeric heterochromatin.
    supported_by:
    - reference_id: PMID:18345014
      supporting_text: >-
        The Clr4 methyltransferase complex (ClrC) is responsible for nucleation
        and spreading of heterochromatin
      reference_section_type: ABSTRACT
- term:
    id: GO:0031508
    label: pericentric heterochromatin formation
  evidence_type: IMP
  original_reference_id: PMID:19136623
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    reason: Core heterochromatin process, well established across the literature.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic
        regions is dependent on H3 Lys9 methylation.
      reference_section_type: ABSTRACT
- term:
    id: GO:0031509
    label: subtelomeric heterochromatin formation
  evidence_type: IMP
  original_reference_id: PMID:24240238
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    reason: Core subtelomeric heterochromatin function, experimentally supported.
    supported_by:
    - reference_id: PMID:24240238
      supporting_text: >-
        deletion of telomere shelterin components restores pericentric
        heterochromatin and its functions in RNAi mutants
      reference_section_type: ABSTRACT
- term:
    id: GO:1902794
    label: siRNA-independent facultative heterochromatin formation
  evidence_type: IMP
  original_reference_id: PMID:22144463
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported facultative heterochromatin role via RNA elimination machinery.
    supported_by:
    - reference_id: PMID:22144463
      supporting_text: >-
        RNA elimination machinery is enriched at meiotic loci and interacts with
        Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly.
      reference_section_type: ABSTRACT
- term:
    id: GO:0140720
    label: subtelomeric heterochromatin
  evidence_type: IDA
  original_reference_id: PMID:32269268
  qualifier: is_active_in
  review:
    summary: >-
      Direct evidence of Clr4 activity at subtelomeric heterochromatin in the
      context of the H3K9me2-to-me3 transition. Core site of action.
    action: ACCEPT
    reason: Direct experimental localization/activity at subtelomeric heterochromatin.
    supported_by:
    - reference_id: PMID:32269268
      supporting_text: >-
        Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin.
      reference_section_type: TITLE
- term:
    id: GO:0033562
    label: co-transcriptional gene silencing by RNA interference machinery
  evidence_type: IMP
  original_reference_id: PMID:17512405
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported role in RNAi-coupled co-transcriptional gene silencing.
    supported_by:
    - reference_id: PMID:17512405
      supporting_text: >-
        the RNAi pathway is required for heterochromatin-dependent silencing of
        transgene insertions at centromeric repeats
      reference_section_type: ABSTRACT
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:21436456
  qualifier: enables
  review:
    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.
    action: REMOVE
    reason: >-
      Uninformative bare protein-binding term; the Clr4-Mlo3 interaction is better
      represented by the protein-lysine methyltransferase activity and process
      annotations.
    supported_by:
    - reference_id: PMID:21436456
      supporting_text: >-
        Clr4 and the RNAi effector RITS (RNA-induced transcriptional silencing)
        interact with Mlo3, a protein related to mRNA quality control and export
        factors.
      reference_section_type: ABSTRACT
- term:
    id: GO:0016279
    label: protein-lysine N-methyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:21436456
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    reason: Real non-histone methyltransferase activity; supporting/secondary to the H3K9 core function.
    supported_by:
    - reference_id: PMID:21436456
      supporting_text: >-
        Clr4 and the RNAi effector RITS (RNA-induced transcriptional silencing)
        interact with Mlo3, a protein related to mRNA quality control and export
        factors.
      reference_section_type: ABSTRACT
- term:
    id: GO:0140727
    label: siRNA-mediated pericentric heterochromatin formation
  evidence_type: EXP
  original_reference_id: PMID:15615848
  qualifier: involved_in
  review:
    summary: >-
      Experimentally supported role in the RNAi self-enforcing loop coupling
      siRNA production to pericentric heterochromatin assembly. Core RNAi-directed
      heterochromatin function.
    action: ACCEPT
    reason: Experimentally supported core RNAi-directed pericentric heterochromatin role.
    supported_by:
    - reference_id: PMID:15615848
      supporting_text: >-
        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
      reference_section_type: ABSTRACT
- term:
    id: GO:0043130
    label: ubiquitin binding
  evidence_type: EXP
  original_reference_id: PMID:34524082
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Experimentally demonstrated ubiquitin binding via the catalytic-domain UBR.
    supported_by:
    - reference_id: PMID:34524082
      supporting_text: >-
        the H3K14ub substrate binds specifically and tightly to the catalytic
        domain of Clr4, and thereby stimulates the enzyme by over 250-fold.
      reference_section_type: ABSTRACT
- term:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:34524082
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported core catalytic function.
    supported_by:
    - reference_id: PMID:34524082
      supporting_text: Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe
      reference_section_type: ABSTRACT
- term:
    id: GO:0140006
    label: histone H3 reader activity
  evidence_type: EXP
  original_reference_id: PMID:34524082
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported histone H3 reading (H3K14ub sensing) coupled to catalysis.
    supported_by:
    - reference_id: PMID:34524082
      supporting_text: >-
        the Clr4 SET domain harbors a conserved sensor for H3K14ub, which
        mediates licensing of heterochromatin formation.
      reference_section_type: ABSTRACT
- term:
    id: GO:0061649
    label: ubiquitin-modified histone reader activity
  evidence_type: IPI
  original_reference_id: PMID:34010645
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported reading of H3K14-ubiquitylated histone via the catalytic-domain UBR.
    supported_by:
    - reference_id: PMID:34010645
      supporting_text: >-
        The histone H3K9M mutation synergizes with H3K14 ubiquitylation to
        selectively sequester histone H3K9 methyltransferase Clr4 at
        heterochromatin.
      reference_section_type: TITLE
- term:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:31468675
  qualifier: enables
  review:
    summary: >-
      Direct demonstration of Clr4 H3K9 methyltransferase activity stimulated by
      CLRC-mediated H3K14 ubiquitylation. Confirms the core catalytic function
      and its regulation.
    action: ACCEPT
    reason: Direct assay of the core catalytic function in the context of H3K14ub regulation.
    supported_by:
    - reference_id: PMID:31468675
      supporting_text: H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly.
      reference_section_type: TITLE
- term:
    id: GO:0030466
    label: silent mating-type cassette heterochromatin formation
  evidence_type: IMP
  original_reference_id: PMID:11283354
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported core mating-type heterochromatin function.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        Both the conserved chromo- and SET domains of Clr4 are required for H3
        Lys9 methylation in vivo.
      reference_section_type: ABSTRACT
- term:
    id: GO:0031508
    label: pericentric heterochromatin formation
  evidence_type: IMP
  original_reference_id: PMID:11283354
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    reason: Experimentally supported core pericentric heterochromatin function.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic
        regions is dependent on H3 Lys9 methylation.
      reference_section_type: ABSTRACT
- term:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:11283354
  qualifier: enables
  review:
    summary: >-
      Direct in vivo demonstration that Clr4 methylates H3K9 at heterochromatic
      regions; chromo- and SET-domain dependent. Core catalytic function.
    action: ACCEPT
    reason: Direct experimental evidence for the core catalytic function.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the
        Clr4 protein at heterochromatin-associated regions in fission yeast.
      reference_section_type: ABSTRACT
- term:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  evidence_type: IMP
  original_reference_id: PMID:11283354
  qualifier: enables
  review:
    summary: >-
      IMP support for H3K9 methyltransferase activity, from catalytic mutants
      (R320H, G378S, G486D) that abolish methylation and silencing. Core function.
    action: ACCEPT
    reason: Mutational evidence supporting the core catalytic function.
    supported_by:
    - reference_id: PMID:11283354
      supporting_text: >-
        Both the conserved chromo- and SET domains of Clr4 are required for H3
        Lys9 methylation in vivo.
      reference_section_type: ABSTRACT
- term:
    id: GO:0003690
    label: double-stranded DNA binding
  evidence_type: IDA
  original_reference_id: PMID:22727667
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.)
    supported_by:
    - reference_id: PMID:22727667
      supporting_text: >-
        Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required
        for heterochromatic gene silencing.
      reference_section_type: TITLE
- term:
    id: GO:0003697
    label: single-stranded DNA binding
  evidence_type: IDA
  original_reference_id: PMID:22727667
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.)
    supported_by:
    - reference_id: PMID:22727667
      supporting_text: >-
        Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required
        for heterochromatic gene silencing.
      reference_section_type: TITLE
- term:
    id: GO:0003727
    label: single-stranded RNA binding
  evidence_type: IDA
  original_reference_id: PMID:22727667
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.)
    supported_by:
    - reference_id: PMID:22727667
      supporting_text: >-
        Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required
        for heterochromatic gene silencing.
      reference_section_type: TITLE
- term:
    id: GO:0030466
    label: silent mating-type cassette heterochromatin formation
  evidence_type: IMP
  original_reference_id: PMID:8001791
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    reason: Foundational genetic evidence for the core mating-type silencing role.
    supported_by:
    - reference_id: PMID:8001791
      supporting_text: >-
        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.
      reference_section_type: ABSTRACT
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: HDA
  original_reference_id: PMID:16823372
  qualifier: is_active_in
  review:
    summary: >-
      High-throughput GFP localization placing Clr4 in the nucleus, consistent
      with its chromatin function. Correct compartment.
    action: ACCEPT
    reason: Correct nuclear localization, consistent with all functional data.
    supported_by:
    - reference_id: PMID:16823372
      supporting_text: >-
        ORFeome cloning and global analysis of protein localization in the
        fission yeast Schizosaccharomyces pombe.
      reference_section_type: TITLE
- term:
    id: GO:0043494
    label: CLRC complex
  evidence_type: IDA
  original_reference_id: PMID:16127433
  qualifier: part_of
  review:
    summary: >-
      Direct evidence (co-purification, direct Clr4-Cul4/Pcu4 interaction) for
      Clr4 as a subunit of the CLRC complex. Core complex membership.
    action: ACCEPT
    reason: Direct experimental evidence for the core CLRC complex membership.
    supported_by:
    - reference_id: PMID:16127433
      supporting_text: >-
        Clr4 associates with Cul4, a cullin family protein that serves as a
        scaffold for assembling ubiquitin ligases.
      reference_section_type: ABSTRACT
- term:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:10949293
  qualifier: enables
  review:
    summary: >-
      Direct in vitro demonstration that the SUV39/Clr4 family selectively
      methylates H3K9. Founding evidence for the core catalytic function.
    action: ACCEPT
    reason: Direct experimental evidence for the core catalytic function.
    supported_by:
    - reference_id: PMID:10949293
      supporting_text: >-
        encode histone H3-specific methyltransferases that selectively methylate
        lysine 9 of the amino terminus of histone H3 in vitro
      reference_section_type: ABSTRACT
- term:
    id: GO:0008168
    label: methyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:10949293
  qualifier: enables
  review:
    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.
    action: MARK_AS_OVER_ANNOTATED
    reason: Over-general term; the specific H3K9 methyltransferase activity is the well-supported core function.
    proposed_replacement_terms:
    - id: GO:0046974
      label: histone H3K9 methyltransferase activity
    supported_by:
    - reference_id: PMID:10949293
      supporting_text: >-
        encode histone H3-specific methyltransferases that selectively methylate
        lysine 9 of the amino terminus of histone H3 in vitro
      reference_section_type: ABSTRACT
core_functions:
- description: >-
    Sole histone H3 lysine 9 methyltransferase of fission yeast; catalyzes H3K9
    mono-, di- and trimethylation via its SET catalytic domain to establish the
    defining heterochromatic histone mark.
  supported_by:
  - reference_id: PMID:11283354
    supporting_text: >-
      lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4
      protein at heterochromatin-associated regions in fission yeast.
    reference_section_type: ABSTRACT
  - reference_id: PMID:34524082
    supporting_text: Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe
    reference_section_type: ABSTRACT
  molecular_function:
    id: GO:0046974
    label: histone H3K9 methyltransferase activity
  directly_involved_in:
  - id: GO:0031507
    label: heterochromatin formation
- description: >-
    Reader-writer of H3K9 methylation: the N-terminal chromodomain binds
    pre-existing H3K9me2/3 and tethers the SET domain to methylate neighboring
    nucleosomes, driving sequence-independent spreading and epigenetic
    maintenance of heterochromatin.
  supported_by:
  - reference_id: PMID:18345014
    supporting_text: >-
      the ability of Clr4 to both 'write' and 'read' H3K9me facilitates
      heterochromatin maintenance through successive cell divisions.
    reference_section_type: ABSTRACT
  molecular_function:
    id: GO:0062072
    label: histone H3K9me2/3 reader activity
  directly_involved_in:
  - id: GO:0031507
    label: heterochromatin formation
- description: >-
    Catalytic subunit of the CLRC complex, a CRL4-type cullin-RING E3 ubiquitin
    ligase (with Rik1, Cul4/Pcu4, Raf1/Dos1, Raf2/Dos2 and Pip1) that
    ubiquitylates histone H3K14; H3K14ub is read by the Clr4 catalytic-domain UBR
    and strongly stimulates H3K9 methylation.
  supported_by:
  - reference_id: PMID:16024659
    supporting_text: >-
      subunits of a cullin-dependent E3 ubiquitin ligase are associated with
      Rik1 and Clr4
    reference_section_type: ABSTRACT
  - reference_id: PMID:34524082
    supporting_text: >-
      the H3K14ub substrate binds specifically and tightly to the catalytic
      domain of Clr4, and thereby stimulates the enzyme by over 250-fold.
    reference_section_type: ABSTRACT
  molecular_function:
    id: GO:0061649
    label: ubiquitin-modified histone reader activity
  in_complex:
    id: GO:0043494
    label: CLRC complex
- description: >-
    Effector of RNAi-directed and RNA-elimination-directed heterochromatin
    formation, mediating transcriptional gene silencing at centromeres,
    telomeres/subtelomeres, the silent mating-type region and facultative
    heterochromatin islands at meiotic genes.
  supported_by:
  - reference_id: PMID:15615848
    supporting_text: >-
      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
    reference_section_type: ABSTRACT
  - reference_id: PMID:22144463
    supporting_text: >-
      RNA elimination machinery is enriched at meiotic loci and interacts with
      Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly.
    reference_section_type: ABSTRACT
  directly_involved_in:
  - id: GO:0140727
    label: siRNA-mediated pericentric heterochromatin formation
proposed_new_terms: []
suggested_questions:
- question: >-
    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?
- question: >-
    What is the full in vivo repertoire of non-histone Clr4 substrates (beyond
    Mlo3 and automethylation), and what are their biological consequences?
- question: >-
    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?
suggested_experiments:
- description: >-
    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.
- description: >-
    Reconstitution of CLRC on nucleosomal arrays to measure how H3K14
    ubiquitylation, Clr4 automethylation and chromodomain engagement
    cooperatively control processive H3K9 methylation and spreading kinetics.
- description: >-
    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.
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO
    terms
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
    vocabulary mapping, accompanied by conservative changes to GO terms applied by
    UniProt
  findings: []
- id: GO_REF:0000116
  title: Automatic Gene Ontology annotation based on Rhea mapping
  findings: []
- id: GO_REF:0000117
  title: Electronic Gene Ontology annotations created by ARBA machine learning models
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:10949293
  title: Regulation of chromatin structure by site-specific histone H3 methyltransferases.
  findings:
  - statement: >-
      SUV39H1/Suv39h1, mammalian homologs of S. pombe clr4, are histone
      H3-specific methyltransferases that selectively methylate H3K9 via the SET
      domain plus adjacent cysteine-rich regions.
    supporting_text: >-
      encode histone H3-specific methyltransferases that selectively methylate
      lysine 9 of the amino terminus of histone H3 in vitro. We mapped the
      catalytic motif to the evolutionarily conserved SET domain, which requires
      adjacent cysteine-rich regions to confer histone methyltransferase activity.
    reference_section_type: ABSTRACT
- id: PMID:11283354
  title: Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin
    assembly.
  findings:
  - statement: >-
      Clr4 preferentially methylates H3K9 at heterochromatic regions in vivo, and
      both the chromo- and SET domains are required; H3K9me is needed for Swi6
      localization.
    supporting_text: >-
      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.
    reference_section_type: ABSTRACT
- id: PMID:15615848
  title: RNA-dependent RNA polymerase is an essential component of a self-enforcing
    loop coupling heterochromatin assembly to siRNA production.
  findings:
  - statement: >-
      A self-enforcing RNAi loop couples siRNA production to heterochromatin
      assembly; RITS/Rdp1 localization at centromeric repeats depends on Clr4 and
      Swi6.
    supporting_text: >-
      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
    reference_section_type: ABSTRACT
- id: PMID:16024659
  title: A Rik1-associated, cullin-dependent E3 ubiquitin ligase is essential for
    heterochromatin formation.
  findings:
  - statement: >-
      A Rik1-/Clr4-associated cullin-dependent E3 ubiquitin ligase (with Raf1,
      Raf2, Pcu4, Pip1) is essential for H3K9 methylation and heterochromatin
      formation.
    supporting_text: >-
      subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1
      and Clr4, and Rik1-TAP preparations exhibit robust E3 ubiquitin ligase
      activity.
    reference_section_type: ABSTRACT
- id: PMID:16127433
  title: Ubiquitin ligase component Cul4 associates with Clr4 histone methyltransferase
    to assemble heterochromatin.
  findings:
  - statement: >-
      Clr4 directly associates with the cullin Cul4/Pcu4; Cul4 is required for
      Clr4 localization, H3K9 methylation, Swi6 recruitment and silencing.
    supporting_text: >-
      We show that Clr4 associates with Cul4, a cullin family protein that serves
      as a scaffold for assembling ubiquitin ligases. Mutations in Cul4 result in
      defective localization of Clr4 and loss of silencing at heterochromatic loci.
    reference_section_type: ABSTRACT
- id: PMID:16823372
  title: ORFeome cloning and global analysis of protein localization in the fission
    yeast Schizosaccharomyces pombe.
  findings:
  - statement: Genome-wide GFP localization study; Clr4 localizes to the nucleus.
    supporting_text: >-
      ORFeome cloning and global analysis of protein localization in the fission
      yeast Schizosaccharomyces pombe.
    reference_section_type: TITLE
- id: PMID:17512405
  title: RNAi-dependent and -independent RNA turnover mechanisms contribute to heterochromatic
    gene silencing.
  findings:
  - statement: >-
      RNAi and RNA turnover (Cid14/TRAMP-like) cooperate in heterochromatic gene
      silencing at centromeric repeats.
    supporting_text: >-
      the RNAi pathway is required for heterochromatin-dependent silencing of
      transgene insertions at centromeric repeats
    reference_section_type: ABSTRACT
- id: PMID:18345014
  title: Roles of the Clr4 methyltransferase complex in nucleation, spreading and
    maintenance of heterochromatin.
  findings:
  - statement: >-
      ClrC nucleates and spreads heterochromatin; the Clr4 chromodomain binds
      H3K9me to enable spreading, and Clr4's write/read of H3K9me underlies
      maintenance.
    supporting_text: >-
      the chromodomain of Clr4 binds specifically to H3K9me that is essential for
      the spreading of heterochromatin... the ability of Clr4 to both 'write' and
      'read' H3K9me facilitates heterochromatin maintenance through successive
      cell divisions.
    reference_section_type: ABSTRACT
- id: PMID:19136623
  title: Phosphorylation of Swi6/HP1 regulates transcriptional gene silencing at heterochromatin.
  findings:
  - statement: >-
      CK2-dependent Swi6 phosphorylation controls transcriptional gene silencing
      downstream of Clr4-mediated heterochromatin.
    supporting_text: >-
      CK2-dependent Swi6 phosphorylation specifically controls TGS in
      heterochromatin.
    reference_section_type: ABSTRACT
- id: PMID:20211136
  title: 'Stc1: a critical link between RNAi and chromatin modification required for
    heterochromatin integrity.'
  findings:
  - statement: >-
      Stc1 bridges the RNAi effector Ago1 to the CLRC complex, recruiting Clr4 to
      promote H3K9 methylation.
    supporting_text: >-
      This leads to recruitment of the CLRC complex, including the histone
      methyltransferase Clr4, promoting H3K9 methylation and heterochromatin
      formation.
    reference_section_type: ABSTRACT
- id: PMID:21436456
  title: Clr4/Suv39 and RNA quality control factors cooperate to trigger RNAi and
    suppress antisense RNA.
  findings:
  - statement: >-
      Clr4 interacts with Mlo3 (and methylates it); loss of Clr4 impairs RITS-Mlo3
      interaction required for centromeric siRNA production and antisense
      suppression.
    supporting_text: >-
      Clr4 and the RNAi effector RITS (RNA-induced transcriptional silencing)
      interact with Mlo3, a protein related to mRNA quality control and export
      factors. Loss of Clr4 impairs RITS interaction with Mlo3, which is required
      for centromeric siRNA production and antisense suppression.
    reference_section_type: ABSTRACT
- id: PMID:22144463
  title: RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin
    formation.
  findings:
  - statement: >-
      Facultative heterochromatin islands form at meiotic genes via RNA
      elimination factors (Mmi1/Red1) that interact with Clr4/SUV39h.
    supporting_text: >-
      RNA elimination machinery is enriched at meiotic loci and interacts with
      Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly.
    reference_section_type: ABSTRACT
- id: PMID:22727667
  title: Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required
    for heterochromatic gene silencing.
  findings:
  - statement: >-
      This study characterizes intrinsic nucleic-acid binding by fission-yeast
      chromodomains. While the title emphasizes Chp1, the paper also assays the Clr4
      chromodomain and reports that Clr4 (the SUV39H homolog) binds nucleic acid via its
      chromodomain, supporting the PomBase dsDNA/ssDNA/ssRNA-binding IDA annotations to
      clr4 (a non-core ancillary property of the chromodomain).
    supporting_text: >-
      Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for
      heterochromatic gene silencing.
    reference_section_type: TITLE
- id: PMID:24240238
  title: Elimination of shelterin components bypasses RNAi for pericentric heterochromatin
    assembly.
  findings:
  - statement: >-
      Deletion of telomere shelterin components restores pericentric
      heterochromatin in RNAi mutants by releasing Swi6 for RNAi-independent
      assembly; informs subtelomeric heterochromatin formation by Clr4.
    supporting_text: >-
      deletion of telomere shelterin components restores pericentric
      heterochromatin and its functions in RNAi mutants
    reference_section_type: ABSTRACT
- id: PMID:24449894
  title: CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed
    surface of Dos1 for heterochromatin silencing.
  findings:
  - statement: >-
      Clr4 is the only H3K9 methyltransferase in S. pombe and a subunit of the
      CRL4-like CLRC complex (Cul4, Pip1, Rik1, Dos1); H3K9me deposition depends on
      the RNAi pathway.
    supporting_text: >-
      Cryptic loci regulator 4 (Clr4), the only known H3K9 methyltransferase in
      this organism, is a subunit of the Clr4 methyltransferase complex (CLRC),
      whose composition is reminiscent of a CRL4 type cullin-RING ubiquitin ligase
    reference_section_type: ABSTRACT
- id: PMID:28143796
  title: Clr4 specificity and catalytic activity beyond H3K9 methylation.
  findings:
  - statement: >-
      Clr4 introduces H3K9 di-/tri-methylation and also methylates non-histone
      substrates including Mlo3 and additional target sites.
    supporting_text: >-
      The enzyme introduces histone 3 lysine 9 (H3K9) di- and tri-methylation, a
      central heterochromatic histone modification, and later it was also found to
      methylate the Mlo3 protein, which has a role in heterochromatin formation as
      well.
    reference_section_type: ABSTRACT
- id: PMID:30051891
  title: Automethylation-induced conformational switch in Clr4 (Suv39h) maintains
    epigenetic stability.
  findings:
  - statement: >-
      An internal autoinhibitory loop blocks the H3K9 substrate pocket;
      automethylation of K455 (and K472) triggers a conformational switch that
      activates Clr4 and prevents aberrant heterochromatin spreading.
    supporting_text: >-
      an internal loop in Clr4 inhibits the catalytic activity of this enzyme by
      blocking the histone H3K9 substrate-binding pocket, and that automethylation
      of specific lysines in this loop promotes a conformational switch that
      enhances the H3K9me activity of Clr4.
    reference_section_type: ABSTRACT
- id: PMID:31165882
  title: Disordered region of H3K9 methyltransferase Clr4 binds the nucleosome and
    contributes to its activity.
  findings:
  - statement: >-
      The Clr4 chromodomain binds the H3K9me3 tail and, together with the
      disordered linker, binds the nucleosome core to contribute to H3K9
      methylation in vitro and in vivo.
    supporting_text: >-
      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.
    reference_section_type: ABSTRACT
- id: PMID:31468675
  title: H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly.
  findings:
  - statement: >-
      CLRC (Clr4 + Cul4) ubiquitylates H3K14, and H3K14ub promotes Clr4 H3K9
      methylation for heterochromatin assembly.
    supporting_text: H3K14 ubiquitylation promotes H3K9 methylation for heterochromatin assembly.
    reference_section_type: TITLE
- id: PMID:32269268
  title: Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin.
  findings:
  - statement: >-
      The bromodomain AAA-ATPase Abo1 is required for the H3K9me2-to-H3K9me3
      transition deposited by Clr4 in heterochromatin.
    supporting_text: Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin.
    reference_section_type: TITLE
- id: PMID:34010645
  title: The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively
    sequester histone H3K9 methyltransferase Clr4 at heterochromatin.
  findings:
  - statement: >-
      H3K9M and H3K14ub together selectively sequester Clr4 at pericentric
      heterochromatin, demonstrating Clr4 reading of ubiquitin-modified histone.
    supporting_text: >-
      The histone H3K9M mutation synergizes with H3K14 ubiquitylation to
      selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin.
    reference_section_type: TITLE
- id: PMID:34524082
  title: SUV39 SET domains mediate crosstalk of heterochromatic histone marks.
  findings:
  - statement: >-
      The Clr4 catalytic domain contains a ubiquitin-binding region that binds
      H3K14ub and stimulates the enzyme >250-fold; disrupting it abolishes
      heterochromatin silencing like clr4 deletion. Clr4 is the sole H3K9me2/3
      methyltransferase of S. pombe.
    supporting_text: >-
      the H3K14ub substrate binds specifically and tightly to the catalytic domain
      of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that
      disrupt this mechanism lead to a loss of H3K9me2/3 and abolish
      heterochromatin silencing similar to clr4 deletion.
    reference_section_type: ABSTRACT
- id: PMID:40446033
  title: Mechanistic insights into the stimulation of the histone H3K9 methyltransferase
    Clr4 by proximal H3K14 ubiquitination.
  findings:
  - statement: >-
      Crystal structures of the Clr4 KMT (pre-SET/SET/post-SET) catalytic domain
      bound to H3 peptide and SAM cofactor reveal the SAM-dependent H3K9
      methyltransferase active site and the multivalent ubiquitin interface by
      which proximal H3K14 ubiquitination stimulates H3K9me2/3 deposition.
    supporting_text: >-
      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.
    reference_section_type: RESULTS
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      PubMed-verified crystal structure of the Clr4 KMT catalytic domain bound
      to H3 peptide and SAM, supporting H3K9 methyltransferase activity, the
      pre-SET zinc-binding catalytic module, and H3K14ub-stimulated H3K9
      methylation.
- id: PMID:40923761
  title: Intrinsically disordered region of Clr4/Suv39 regulates its enzymatic activity
    and ensures heterochromatin spreading.
  findings:
  - statement: >-
      The intrinsically disordered region of Clr4 regulates its H3K9
      methyltransferase activity and is required for heterochromatin spreading.
    supporting_text: >-
      Intrinsically disordered region of Clr4/Suv39 regulates its enzymatic
      activity and ensures heterochromatin spreading.
    reference_section_type: TITLE
- id: PMID:8001791
  title: Three additional linkage groups that repress transcription and meiotic recombination
    in the mating-type region of Schizosaccharomyces pombe.
  findings:
  - statement: >-
      clr4 (with clr2, clr3) is a trans-acting locus required for transcriptional
      and recombinational silencing at the mating-type region.
    supporting_text: >-
      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.
    reference_section_type: ABSTRACT