The histone chaperone FACT facilitates heterochromatin spreading through regulation of histone turnover and H3K9 methylation states
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ABSTRACT:
PROVIDER: PRJNA730872 | ENA |
REPOSITORIES: ENA
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Project description:Heterochromatin formation requires several steps involving: nucleation followed by spreading of large, silent domains and its faithful re-establishment and maintenance after each replication cycle. The essential histone chaperone FACT has been implicated in heterochromatin silencing, however, little is known about the specificity of FACT in this process. Here, by applying single cell assays, ChIP and genetics, we show that FACT mutants have defects in the spreading of heterochromatin at subtelomeres and mating type locus in fission yeast. Further, we demonstrate that the transition of H3K9me2 to me3 is impaired in the FACT mutant and we link the spreading function of FACT to its role in histone turnover suppression. We also identify Epe1 as a specific factor that counteracts heterochromatin spreading defects in the FACT mutants. Together, our studies identify FACT as a first histone chaperone that promotes heterochromatin spreading and add to the current models on histone turnover in propagation of epigenetic marks.
2021-10-12 | GSE174641 | GEO
Project description: Not available
| S-EPMC8608617 | biostudies-literature
Project description:The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we find that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identify Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone FACT, as CENP-W binding partners through a proteomic screen. We find that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres and site directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.
2016-06-14 | PXD004099 | Pride
Project description:Parental histone recycling is essential for the restoration of chromatin-based epigenetic information during chromatin replication; however, the specific mechanisms underlying the local recycling of parental histones remain poorly understood. Here, we reveal an unexpected role of the Spt16-N domain in histone chaperone FACT during parental histone recycling and transfer in budding yeast. We found that depletion of Spt16 or mutations in the Spt16 middle domain leads to defects in both parental histone recycling and new histone deposition, affecting both the leading and lagging strands of DNA replication forks, highlighting the essential role of the FACT complex in both parental histone recycling and new histone deposition. Surprisingly, Spt16-N deletion results in an apparent defect in parental histone recycling, with a more pronounced defect on the lagging strand than the corresponding leading strand. Mechanistically, the Spt16-N domain acts as a protective barrier, shielding FACT-bound histone H3-H4 and facilitating its interaction with Mcm2, which ensures efficient local parental histone recycling. Collectively, the Spt16-N domain provides a protein–protein interaction module allowing FACT to act as a shuttle chaperone, cooperate with multiple replisome components, which act as co-chaperones, to form a complex involving the shuttle chaperone, histones, and co-chaperones, during parental histone recycling and transfer.
2024-01-26 | PXD043392 | Pride
Project description:Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A-H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A-H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A-H2B deposition by FACT. 6 samples, 2 inputs and 4 ChIP samples for histone H2B (2 for wild-type and 2 for an H2B â30-37 mutant)
2016-08-10 | E-GEOD-80831 | biostudies-arrayexpress
Project description:The histone chaperone FACT and histone H2B ubiquitination (H2Bub) facilitate RNA Polymerase II passage through chromatin. It remains unknown to what extent they cooperate in vivo. Here, we used genome-wide, genetic, biochemical and microscopic approaches to dissect their interplay in the fission yeast Schizosaccharomyces pombe. We show that FACT and H2Bub repress antisense transcripts near the 5’ end and inside gene bodies, respectively. The accumulation of these transcripts accompanies with changes at genic nucleosomes in FACT and H2Bub mutants. Moreover, in the H2Bub mutant, FACT binding to histones is stabilized. Interestingly, analysis of FACT-H2Bub double mutants revealed that FACT depletion suppresses genic nucleosome loss in the H2Bub mutant. In addition, FACT, unlike H2Bub, maintains nucleosomes in intergenic regions. This is especially important for subtelomeric gene repression and in compacting chromatin into ‘knobs’. Collectively, our study demonstrates that FACT and H2Bub functionally interact with each other in a genome-context dependent manner. Further, our data indicate an involvement of FACT in higher order chromatin formation outside of euchromatin.
2019-12-11 | GSE135766 | GEO
Project description:The histone chaperone FACT and histone H2B ubiquitination (H2Bub) facilitate RNA Polymerase II passage through chromatin. It remains unknown to what extent they cooperate in vivo. Here, we used genome-wide, genetic, biochemical and microscopic approaches to dissect their interplay in the fission yeast Schizosaccharomyces pombe. We show that FACT and H2Bub repress antisense transcripts near the 5’ end and inside gene bodies, respectively. The accumulation of these transcripts accompanies with changes at genic nucleosomes in FACT and H2Bub mutants. Moreover, in the H2Bub mutant, FACT binding to histones is stabilized. Interestingly, analysis of FACT-H2Bub double mutants revealed that FACT depletion suppresses genic nucleosome loss in the H2Bub mutant. In addition, FACT, unlike H2Bub, maintains nucleosomes in intergenic regions. This is especially important for subtelomeric gene repression and in compacting chromatin into ‘knobs’. Collectively, our study demonstrates that FACT and H2Bub functionally interact with each other in a genome-context dependent manner. Further, our data indicate an involvement of FACT in higher order chromatin formation outside of euchromatin.
2019-12-11 | GSE124091 | GEO
Project description:The histone chaperone FACT and histone H2B ubiquitination (H2Bub) facilitate RNA Polymerase II passage through chromatin. It remains unknown to what extent they cooperate in vivo. Here, we used genome-wide, genetic, biochemical and microscopic approaches to dissect their interplay in the fission yeast Schizosaccharomyces pombe. We show that FACT and H2Bub repress antisense transcripts near the 5’ end and inside gene bodies, respectively. The accumulation of these transcripts accompanies with changes at genic nucleosomes in FACT and H2Bub mutants. Moreover, in the H2Bub mutant, FACT binding to histones is stabilized. Interestingly, analysis of FACT-H2Bub double mutants revealed that FACT depletion suppresses genic nucleosome loss in the H2Bub mutant. In addition, FACT, unlike H2Bub, maintains nucleosomes in intergenic regions. This is especially important for subtelomeric gene repression and in compacting chromatin into ‘knobs’. Collectively, our study demonstrates that FACT and H2Bub functionally interact with each other in a genome-context dependent manner. Further, our data indicate an involvement of FACT in higher order chromatin formation outside of euchromatin.
2019-12-11 | GSE124090 | GEO
Project description:The heterodimeric histone chaperone FACT consisting of SSRP1 and SPT16 contributes to dynamic nucleosome rearrangements during various DNA-dependent processes including transcription. In search of post-translational modifications that may regulate the activity of FACT, SSRP1 and SPT16 were isolated from Arabidopsis cells and analysed by mass spectrometry. Four acetylated lysine residues could be mapped within the basic C-terminal region of SSRP1, while three phosphorylated serine/threonine residues were identified in the acidic C-terminal region of SPT16. Mutational analysis of the SSRP1 acetylation sites revealed only mild effects. However, phosphorylation of SPT16 that is catalysed by protein kinase CK2, modulates histone interactions. A non-phosphorylatable version of SPT16 displayed reduced histone binding and (unlike the phosphorylatable wild-type and phosphomimic versions) proved inactive in complementing the growth and developmental phenotypes of spt16 mutant plants. In plants expressing the non-phosphorylatable SPT16 version we detected at a subset of genes enrichment of histone H3 directly upstream of RNA polymerase II transcriptional start sites (TSSs) in a region that usually is nucleosome-depleted. This is associated with altered transcript levels, suggesting that some genes require phosphorylation of the SPT16 acidic region for establishing the correct nucleosome occupancy at the TSS as a prerequisite for proper transcription.
2022-06-09 | PXD028534 | Pride
Project description:Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A-H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A-H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A-H2B deposition by FACT.
2016-08-10 | GSE80831 | GEO