Spt16 is the essential large subunit of FACT (FAcilitates Chromatin Transactions), the conserved ATP-independent histone chaperone that it forms with Pob3 (pob3 is not essential in fission yeast). FACT reorganizes nucleosomes ahead of RNA polymerase II during transcription elongation, helping the polymerase through chromatin together with the Fun30 remodeller Fft3, and restores nucleosomes behind it, which keeps H3 chromatin intact on transcribed genes and suppresses cryptic and antisense transcription. FACT can make nucleosomal DNA accessible without obligatory eviction of an H2A-H2B dimer. The Spt16 N-terminal domain has an aminopeptidase P fold that has lost catalytic activity and instead binds histone H3-H4 cores and tails; the middle domain binds Pob3; and the acidic C-terminal region binds H2A-H2B. In the nucleus Spt16 is most abundant on euchromatin, but FACT also maintains centromeric heterochromatin independently of RNAi, limits histone turnover in heterochromatin so that H3K9me2 can be converted to H3K9me3 and heterochromatin can spread, prevents CENP-A(Cnp1) from being deposited outside centromeres, keeps nucleosomes in place at subtelomeres, and helps transfer parental H3-H4 to both daughter strands during DNA replication.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006337 nucleosome disassembly | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the eukaryote-wide FACT node PTN000360139 (all Spt16 orthologs, PANTHER PTHR13980:SF15, which includes O94267). Consistent with S. pombe data showing that FACT and the Fun30 remodeller Fft3 together account for much of RNAPII-mediated nucleosome disassembly in transcribed regions. FACT also reassembles nucleosomes behind the polymerase, so disassembly is one half of its nucleosome reorganization role rather than its sole outcome. Reason: Phylogenetic placement is sound (O94267 is a bona fide member of the Spt16 subfamily), and pombe-specific genetic evidence supports FACT participation in nucleosome disassembly during transcription. Supporting Evidence: PMID:28218250 Fun30Fft3 cooperates with FACT to induce nucleosome disassembly at transcribing regions, which accounts for a large fraction of RNAPII-mediated nucleosome disassembly. |
| GO:0032784 regulation of DNA-templated transcription elongation | IBA GO_REF:0000033 | ACCEPT | Summary: Correct. FACT is a well-established regulator of transcription elongation, facilitating Pol II passage through chromatin. Supported by multiple S. pombe studies showing Spt16 cooperates with Fft3 and is modulated by H2Bub during elongation. Supporting Evidence: PMID:28218250 Fun30Fft3 cooperates with FACT to induce nucleosome disassembly at transcribing regions, which accounts for a large fraction of RNAPII-mediated nucleosome disassembly. PMID:31837996 ...FACT and H2Bub globally repress antisense transcripts near the 5' end of genes and inside gene bodies, respectively... |
| GO:0035101 FACT complex | IBA GO_REF:0000033 | ACCEPT | Summary: Redundant with IDA annotation from PMID:17614284 below. Correct annotation. |
| GO:0031491 nucleosome binding | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the eukaryote-wide Spt16 node PTN000360139 (PAINT IBD seeded by Drosophila dre4), which contains O94267 (PTHR13980:SF15). FACT engages nucleosomes through multiple histone-binding modules; in S. pombe the Spt16 N-terminal domain binds H3-H4 cores and tails, and the C-terminal acidic region binds H2A-H2B. Pombe-specific evidence is for histone (not intact-nucleosome) binding, so this rests on the phylogenetic inference. Reason: The PAINT node placement is sound and nucleosome binding is a conserved property of FACT needed for its chaperone mechanism; no evidence of divergence in fission yeast. Supporting Evidence: PMID:18579787 ...the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails... |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: Correct but redundant with IDA evidence from PMID:17614284. Nuclear localization is well established. |
| GO:0005694 chromosome | IEA GO_REF:0000044 | ACCEPT | Summary: Reasonable inference from UniProt subcellular location. FACT associates with chromatin at transcribing regions, centromeres, and subtelomeres. |
| GO:0010468 regulation of gene expression | IEA GO_REF:0000117 | MODIFY | Summary: ARBA-derived term. True of FACT, but far less specific than what the evidence supports for how Spt16 affects gene expression. Reason: Correct but general. FACT affects gene expression by facilitating RNA polymerase II elongation through chromatin and by restoring chromatin behind the polymerase (suppressing cryptic and antisense transcription). Replace with regulation of DNA-templated transcription elongation (GO:0032784), an is_a descendant of GO:0010468 that the gene already carries via IBA. Proposed replacements: regulation of DNA-templated transcription elongation Supporting Evidence: PMID:28218250 Fun30Fft3 associates with RNAPII and collaborates with the histone chaperone, FACT, which facilitates RNAPII elongation through chromatin, to induce nucleosome disassembly at transcribing regions during RNAPII transcription. |
| GO:0034728 nucleosome organization | IEA GO_REF:0000117 | ACCEPT | Summary: Correct. FACT reorganizes nucleosomes during transcription and maintains nucleosome occupancy genome-wide. Redundant with NAS annotation from PMID:17614284. |
| GO:0035101 FACT complex | IEA GO_REF:0000120 | ACCEPT | Summary: Redundant with IDA annotation from PMID:17614284. Correct. |
| GO:0005515 protein binding | IPI PMID:18579787 The FACT Spt16 "peptidase" domain is a histone H3-H4 binding... | MODIFY | Summary: Experimental interaction between the Spt16 N-terminal (aminopeptidase P-like) domain and a core histone H3 or H4 (S. pombe or Drosophila histone used in the binding assays). The interaction is real, but bare protein binding carries no functional information. Reason: Per the protein-binding policy, replace bare GO:0005515 with the informative term the paper supports. PMID:18579787 shows that Spt16-N is a dedicated histone H3-H4 binding module (core domains and N-terminal tails), so histone binding (GO:0042393) is the appropriate molecular function. The interaction partner is a histone in every one of these rows (pombe H4 P09322 hhf1/2/3, pombe H3 P09988 hht1/2, pombe H3.3 P10651 hht3, Drosophila H3 P02299, Drosophila H4 P84040). Proposed replacements: histone binding Supporting Evidence: PMID:18579787 the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails |
| GO:0006261 DNA-templated DNA replication | NAS PMID:17614284 The chromatin-remodeling factor FACT contributes to centrome... | KEEP AS NON CORE | Summary: Supported by NAS from PMID:17614284 which notes sensitivity of pob3 deletion to HU (hydroxyurea), implicating FACT in DNA replication. Additionally, PMID:38479839 directly demonstrates FACT role in parental histone transfer during replication. However, the direct replication role is better captured by the more specific term DNA replication-dependent chromatin assembly (GO:0006335). Reason: GO:0006335 is not a descendant of GO:0006261 (it sits under chromatin organization), so the two terms describe different aspects. FACT travels with the replisome and handles parental histones as forks pass, which is a genuine contribution to replication of a chromatin template, but Spt16 does not catalyze DNA synthesis; its replication-coupled role is chromatin handling. The pombe evidence for the replication term itself is indirect (HU sensitivity of pob3 deletion, NAS), so the term is retained as non-core rather than as a core function. Supporting Evidence: PMID:17614284 Cells lacking Pob3 are sensitive to HU, CPT, UV and (mildly) to 6-AU, suggesting DNA replication, DNA repair and transcription phenotypes PMID:38479839 ...the FACT histone chaperone regulates parental histone transfer to both strands and collaborates with Mcm2 and Dpb3/4 to maintain parental histone H3-H4 density and faithful heterochromatin inheritance... |
| GO:0006335 DNA replication-dependent chromatin assembly | NAS PMID:38479839 Coordination of histone chaperones for parental histone segr... | ACCEPT | Summary: Well supported. PMID:38479839 directly demonstrates that FACT regulates parental histone H3-H4 transfer to both daughter strands during DNA replication and collaborates with Mcm2 and Dpb3/4 for epigenetic inheritance. Supporting Evidence: PMID:38479839 ...the FACT histone chaperone regulates parental histone transfer to both strands and collaborates with Mcm2 and Dpb3/4 to maintain parental histone H3-H4 density and faithful heterochromatin inheritance... |
| GO:0006338 chromatin remodeling | IMP PMID:31837996 The Chaperone FACT and Histone H2B Ubiquitination Maintain S... | ACCEPT | Summary: Supported. PMID:31837996 demonstrates via MNase-seq and H3 ChIP-seq that FACT mutants show defects in nucleosome positioning and phasing, especially at +1 and TSS-proximal nucleosomes, and loss of nucleosomes at subtelomeres. This constitutes chromatin remodeling activity. Supporting Evidence: PMID:31837996 ...FACT maintains nucleosomes in subtelomeric regions, which is crucial for their compaction... |
| GO:0034728 nucleosome organization | NAS PMID:17614284 The chromatin-remodeling factor FACT contributes to centrome... | ACCEPT | Summary: Supported. PMID:17614284 establishes FACT as a chromatin remodeling factor in S. pombe, and nucleosome organization is a core function of FACT. Supporting Evidence: PMID:17614284 ...our genetic and biochemical data implicate the chromatin remodeling complex FACT in forming functional centromeres... |
| GO:0000511 H2A-H2B histone complex chaperone activity | IC GO_REF:0000111 | ACCEPT | Summary: Inferred from FACT complex membership. Consistent with direct experimental evidence in PMID:31837996 and PMID:34731638. Supporting Evidence: PMID:31837996 we performed nucleosome chaperoning assays with recombinant FACT and recombinant histone octamers |
| GO:0140673 transcription elongation-coupled chromatin remodeling | IC GO_REF:0000111 | ACCEPT | Summary: Correct. Inferred from FACT complex membership. FACT's primary role is to reorganize nucleosomes during Pol II elongation, which is precisely this process. Supporting Evidence: PMID:28218250 Fun30Fft3 associates with RNAPII and collaborates with the histone chaperone, FACT, which facilitates RNAPII elongation through chromatin, to induce nucleosome disassembly at transcribing regions during RNAPII transcription. |
| GO:0006335 DNA replication-dependent chromatin assembly | IC GO_REF:0000111 | ACCEPT | Summary: Inferred from FACT complex membership. Consistent with experimental evidence from PMID:38479839. |
| GO:0000511 H2A-H2B histone complex chaperone activity | EXP PMID:31837996 The Chaperone FACT and Histone H2B Ubiquitination Maintain S... | ACCEPT | Summary: The activity is correct for Spt16 as a FACT subunit (contributes_to is appropriate for a heterodimer-level activity), and PAINT places GO:0000511 at the eukaryote-wide Spt16 node PTN000360139. However, the in vitro EMSA and nucleosome chaperoning assays in this paper used recombinant human FACT (SPT16/SSRP1), not S. pombe Spt16-Pob3. The pombe-specific data are in vivo (H2Bub alters FACT association with histones), which support histone binding rather than a direct chaperone assay. Reason: The function is clearly correct for the gene (conserved FACT activity, IC and IBA-node support, and the H2A-H2B dimers are bound by the Spt16 and Pob3 C-terminal acidic regions). The evidence code is questionable because the biochemical assay was done with the human orthologous complex; recorded as a suspected source defect for PomBase to check. Supporting Evidence: PMID:31837996 we performed nucleosome chaperoning assays with recombinant FACT and recombinant histone octamers PMID:31837996 In particular, H2A-H2B dimers are bound by the unstructured C-terminal domains of Spt16 and Pob3 which compete with DNA binding to histones |
| GO:0000511 H2A-H2B histone complex chaperone activity | EXP PMID:34731638 The histone chaperone FACT facilitates heterochromatin sprea... | ACCEPT | Summary: The term is correct for Spt16 (conserved FACT H2A-H2B chaperone activity; see the IC row and PMID:31837996). PMID:34731638 itself shows that FACT limits heterochromatic histone turnover (measured with an H3 turnover reporter), which is in vivo evidence for histone chaperone function in general rather than H2A-H2B-specific handling. Reason: Defer to the PomBase curator, who read the full paper; the activity is well established for the Spt16 subfamily. The paper's data more directly support the parent term histone chaperone activity (GO:0140713), which the gene also carries. Supporting Evidence: PMID:34731638 ...FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading... |
| GO:0140719 constitutive heterochromatin formation | IMP PMID:34731638 The histone chaperone FACT facilitates heterochromatin sprea... | ACCEPT | Summary: Well supported. PMID:34731638 demonstrates that FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes near heterochromatin boundaries. FACT promotes heterochromatin spreading by suppressing histone turnover. Supporting Evidence: PMID:34731638 ...FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries... |
| GO:0000791 euchromatin | EXP PMID:34731638 The histone chaperone FACT facilitates heterochromatin sprea... | ACCEPT | Summary: Supported. Genome-wide ChIP of Spt16 and Pob3 shows the highest FACT occupancy in euchromatin, lower at subtelomeres and lowest at pericentromeres, consistent with its transcription-coupled role. Supporting Evidence: PMID:34731638 we examined the genome-wide distribution of Pob3 and Spt16 (Figure S1E). This analysis showed the highest levels of FACT at euchromatin, lower levels at subtelomeres, and the lowest levels at pericentromeres. |
| GO:0140713 histone chaperone activity | IMP PMID:23028377 Factors that promote H3 chromatin integrity during transcrip... | ACCEPT | Summary: Well supported. PMID:23028377 demonstrates that FACT mutants impair maintenance of H3 chromatin on transcribed regions and promote widespread ectopic CENP-A(Cnp1) incorporation, directly showing histone chaperone function for maintaining H3 nucleosome integrity during transcription. Supporting Evidence: PMID:23028377 ...Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites... |
| GO:0006325 chromatin organization | EXP PMID:23028377 Factors that promote H3 chromatin integrity during transcrip... | MODIFY | Summary: Broadly correct. PMID:23028377 shows FACT maintains chromatin organization by preserving H3 chromatin integrity during transcription. This is a parent term of more specific annotations like nucleosome organization and chromatin remodeling that are also annotated. Reason: Correct but general. The experimental finding is loss of H3 chromatin integrity and cryptic intragenic transcription on transcribed regions in FACT mutants, i.e. a failure of transcription-coupled nucleosome reorganization. Replace with transcription elongation-coupled chromatin remodeling (GO:0140673) and nucleosome organization (GO:0034728), both is_a descendants of GO:0006325 that the gene already carries. Proposed replacements: transcription elongation-coupled chromatin remodeling nucleosome organization Supporting Evidence: PMID:23028377 ...Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites... |
| GO:0005634 nucleus | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: High-throughput localization study confirming nuclear localization. Consistent with IDA from PMID:17614284. Supporting Evidence: PMID:16823372 ...we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome... |
| GO:0005515 protein binding | IPI PMID:17614284 The chromatin-remodeling factor FACT contributes to centrome... | REMOVE | Summary: Spt16 interacts with Pob3 (SPBC609.05), its FACT heterodimer partner; the interaction requires the Spt16 middle domain. The interaction is real but is an intra-complex subunit contact. Reason: Per the protein-binding policy, bare GO:0005515 is uninformative and is removed; removal does not mean the interaction is false. The Spt16-Pob3 association is fully captured by the FACT complex (GO:0035101) IDA annotation from the same paper, and no more specific molecular-function term describes a structural subunit contact within a heterodimer. Supporting Evidence: PMID:17614284 ...Spt16 and Pob3 associate in vitro and...the interaction requires the Spt16-M domain... |
| GO:0005634 nucleus | IDA PMID:17614284 The chromatin-remodeling factor FACT contributes to centrome... | ACCEPT | Summary: Directly demonstrated by GFP-tagged Spt16 imaging showing nuclear localization. Supporting Evidence: PMID:17614284 ...Pob3-GFP and Spt16-GFP are nuclear factors... |
| GO:0035101 FACT complex | IDA PMID:17614284 The chromatin-remodeling factor FACT contributes to centrome... | ACCEPT | Summary: Co-purification and interaction experiments identify Spt16 and Pob3 as the fission-yeast FACT complex. Supporting Evidence: PMID:17614284 ...Mass spectrometry analysis identifies the two bands as Pob3 and Spt16...Spt16 and Pob3 associate in vitro and...the interaction requires the Spt16-M domain... |
| GO:0005515 protein binding | IPI PMID:18579787 The FACT Spt16 "peptidase" domain is a histone H3-H4 binding... | MODIFY | Summary: Experimental interaction between the Spt16 N-terminal (aminopeptidase P-like) domain and a core histone H3 or H4 (S. pombe or Drosophila histone used in the binding assays). The interaction is real, but bare protein binding carries no functional information. Reason: Per the protein-binding policy, replace bare GO:0005515 with the informative term the paper supports. PMID:18579787 shows that Spt16-N is a dedicated histone H3-H4 binding module (core domains and N-terminal tails), so histone binding (GO:0042393) is the appropriate molecular function. The interaction partner is a histone in every one of these rows (pombe H4 P09322 hhf1/2/3, pombe H3 P09988 hht1/2, pombe H3.3 P10651 hht3, Drosophila H3 P02299, Drosophila H4 P84040). Proposed replacements: histone binding Supporting Evidence: PMID:18579787 the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails |
| GO:0005515 protein binding | IPI PMID:18579787 The FACT Spt16 "peptidase" domain is a histone H3-H4 binding... | MODIFY | Summary: Experimental interaction between the Spt16 N-terminal (aminopeptidase P-like) domain and a core histone H3 or H4 (S. pombe or Drosophila histone used in the binding assays). The interaction is real, but bare protein binding carries no functional information. Reason: Per the protein-binding policy, replace bare GO:0005515 with the informative term the paper supports. PMID:18579787 shows that Spt16-N is a dedicated histone H3-H4 binding module (core domains and N-terminal tails), so histone binding (GO:0042393) is the appropriate molecular function. The interaction partner is a histone in every one of these rows (pombe H4 P09322 hhf1/2/3, pombe H3 P09988 hht1/2, pombe H3.3 P10651 hht3, Drosophila H3 P02299, Drosophila H4 P84040). Proposed replacements: histone binding Supporting Evidence: PMID:18579787 the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails |
| GO:0005515 protein binding | IPI PMID:18579787 The FACT Spt16 "peptidase" domain is a histone H3-H4 binding... | MODIFY | Summary: Experimental interaction between the Spt16 N-terminal (aminopeptidase P-like) domain and a core histone H3 or H4 (S. pombe or Drosophila histone used in the binding assays). The interaction is real, but bare protein binding carries no functional information. Reason: Per the protein-binding policy, replace bare GO:0005515 with the informative term the paper supports. PMID:18579787 shows that Spt16-N is a dedicated histone H3-H4 binding module (core domains and N-terminal tails), so histone binding (GO:0042393) is the appropriate molecular function. The interaction partner is a histone in every one of these rows (pombe H4 P09322 hhf1/2/3, pombe H3 P09988 hht1/2, pombe H3.3 P10651 hht3, Drosophila H3 P02299, Drosophila H4 P84040). Proposed replacements: histone binding Supporting Evidence: PMID:18579787 the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails |
| GO:0005515 protein binding | IPI PMID:18579787 The FACT Spt16 "peptidase" domain is a histone H3-H4 binding... | MODIFY | Summary: Experimental interaction between the Spt16 N-terminal (aminopeptidase P-like) domain and a core histone H3 or H4 (S. pombe or Drosophila histone used in the binding assays). The interaction is real, but bare protein binding carries no functional information. Reason: Per the protein-binding policy, replace bare GO:0005515 with the informative term the paper supports. PMID:18579787 shows that Spt16-N is a dedicated histone H3-H4 binding module (core domains and N-terminal tails), so histone binding (GO:0042393) is the appropriate molecular function. The interaction partner is a histone in every one of these rows (pombe H4 P09322 hhf1/2/3, pombe H3 P09988 hht1/2, pombe H3.3 P10651 hht3, Drosophila H3 P02299, Drosophila H4 P84040). Proposed replacements: histone binding Supporting Evidence: PMID:18579787 the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails |
| GO:0005515 protein binding | IPI PMID:18579787 The FACT Spt16 "peptidase" domain is a histone H3-H4 binding... | MODIFY | Summary: Experimental interaction between the Spt16 N-terminal (aminopeptidase P-like) domain and a core histone H3 or H4 (S. pombe or Drosophila histone used in the binding assays). The interaction is real, but bare protein binding carries no functional information. Reason: Per the protein-binding policy, replace bare GO:0005515 with the informative term the paper supports. PMID:18579787 shows that Spt16-N is a dedicated histone H3-H4 binding module (core domains and N-terminal tails), so histone binding (GO:0042393) is the appropriate molecular function. The interaction partner is a histone in every one of these rows (pombe H4 P09322 hhf1/2/3, pombe H3 P09988 hht1/2, pombe H3.3 P10651 hht3, Drosophila H3 P02299, Drosophila H4 P84040). Proposed replacements: histone binding Supporting Evidence: PMID:18579787 the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails |
| GO:0005515 protein binding | IPI PMID:18579787 The FACT Spt16 "peptidase" domain is a histone H3-H4 binding... | MODIFY | Summary: Experimental interaction between the Spt16 N-terminal (aminopeptidase P-like) domain and a core histone H3 or H4 (S. pombe or Drosophila histone used in the binding assays). The interaction is real, but bare protein binding carries no functional information. Reason: Per the protein-binding policy, replace bare GO:0005515 with the informative term the paper supports. PMID:18579787 shows that Spt16-N is a dedicated histone H3-H4 binding module (core domains and N-terminal tails), so histone binding (GO:0042393) is the appropriate molecular function. The interaction partner is a histone in every one of these rows (pombe H4 P09322 hhf1/2/3, pombe H3 P09988 hht1/2, pombe H3.3 P10651 hht3, Drosophila H3 P02299, Drosophila H4 P84040). Proposed replacements: histone binding Supporting Evidence: PMID:18579787 the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails |
| GO:0005515 protein binding | IPI PMID:18579787 The FACT Spt16 "peptidase" domain is a histone H3-H4 binding... | MODIFY | Summary: Experimental interaction between the Spt16 N-terminal (aminopeptidase P-like) domain and a core histone H3 or H4 (S. pombe or Drosophila histone used in the binding assays). The interaction is real, but bare protein binding carries no functional information. Reason: Per the protein-binding policy, replace bare GO:0005515 with the informative term the paper supports. PMID:18579787 shows that Spt16-N is a dedicated histone H3-H4 binding module (core domains and N-terminal tails), so histone binding (GO:0042393) is the appropriate molecular function. The interaction partner is a histone in every one of these rows (pombe H4 P09322 hhf1/2/3, pombe H3 P09988 hht1/2, pombe H3.3 P10651 hht3, Drosophila H3 P02299, Drosophila H4 P84040). Proposed replacements: histone binding Supporting Evidence: PMID:18579787 the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails |
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Download this section (compressed HTML)Q: Does Spt16 have functions independent of Pob3 in S. pombe beyond controlling 3' gene ends, given that spt16 is essential but pob3 is not?
Suggested experts: Ladurner AG, Braun S
Q: What is the precise mechanism by which FACT suppresses histone turnover at heterochromatic regions -- does it involve direct competition with Epe1 for nucleosome access?
Suggested experts: Murawska M, Al-Sady B
Q: How does FACT coordinate with the Mcm2 and Dpb3/4 histone chaperones during DNA replication fork passage to achieve balanced parental histone distribution?
Suggested experts: Jia S, Zhang Z
Experiment: ChIP-seq of Spt16 in spt16 temperature-sensitive mutants at permissive and restrictive temperatures to map genome-wide FACT occupancy changes, particularly at heterochromatin boundaries and subtelomeres.
Hypothesis: FACT occupancy at heterochromatin boundaries correlates with H3K9me3 spreading capacity.
Experiment: eSPAN (enrichment and sequencing of protein-associated nascent DNA) analysis in spt16 conditional mutants to directly measure parental histone distribution asymmetry at replication forks.
Hypothesis: FACT impairment causes asymmetric parental histone distribution favoring the leading strand.
Experiment: Proximity ligation assay or cross-linking mass spectrometry to identify direct Spt16 interaction partners at heterochromatin versus euchromatin, testing whether Spt16 engages distinct co-factors in different chromatin contexts.
Hypothesis: Spt16 interacts with Clr4/Swi6 at heterochromatin but with elongation factors (Spt4/5/6) at euchromatin.
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