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FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner [ChIP-exo]

ABSTRACT: The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models for how FACT finds its way to transcriptionally active chromatin include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails and binding partially disassembled nucleosomes. Here, we systematically tested these and other scenarios in Saccharomyces cerevisiae and found that FACT binds transcribed chromatin, not RNAPII. Through a combination of experimental and mathematical modeling evidence, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE155138 | GEO | 2021/07/26

REPOSITORIES: GEO

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FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner [ChIP-chip]

Project description:The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models for how FACT finds its way to transcriptionally active chromatin include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails and binding partially disassembled nucleosomes. Here, we systematically tested these and other scenarios in Saccharomyces cerevisiae and found that FACT binds transcribed chromatin, not RNAPII. Through a combination of experimental and mathematical modeling evidence, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.

2021-07-26 | GSE155110 | GEO

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner [MNase-ChIP-Seq]

2021-07-26 | GSE155143 | GEO

Project description:Specialization of RSC to mobilize partially unwrapped nucleosomes

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ChIP-seq for Spt16 before and after transcription inhibition

Project description:The FACT (FAcilitates Chromatin Transactions) complex is a conserved complex that maintains chromatin structure on transcriptionally active genes. Consistent with this, FACT is enriched on highly expressed genes, but how it is targeted to these regions is unknown. In vitro, FACT binds destabilized nucleosomes, supporting the hypothesis that FACT is targeted to transcribed chromatin through recognition of RNA polymerase-disrupted nucleosomes. In this study, we used high resolution analysis of FACT occupancy in S. cerevisiae to test this hypothesis. We demonstrate that FACT interacts with nucleosomes in vivo and its interaction with chromatin is dependent on transcription by any of the three RNA polymerases. Deep sequencing of micrococcal nuclease (MNase)-resistant fragments shows that FACT-bound nucleosomes exhibit differing nuclease sensitivity compared to bulk chromatin, consistent with a modified nucleosome structure being the preferred ligand for this complex. Interestingly, a subset of FACT-bound nucleosomes may be “overlapping di-nucleosomes”, in which one histone octamer invades the ~147 bp territory normally occupied by the adjacent nucleosome. While the differing nuclease sensitivity of FACT-bound nucleosomes could also be explained by the demonstrated ability of FACT to alter nucleosome structure, transcription inhibition restores nuclease resistance suggesting that it is not due to FACT interaction alone. Collectively these results are consistent with a model in which FACT is targeted to transcribed genes through preferential interaction with RNA polymerase-disrupted nucleosomes.

2018-09-26 | GSE111426 | GEO

ChIP-seq for RNAPII before and after transcription inhibition

2018-09-26 | GSE110286 | GEO

Histone acetylation targeting to transcribed chromatin through RNAPII

Project description:Histone acetylation targeting to transcribed chromatin through RNAPII

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Effect of FACT depletion on the cell composition of bone marrow and intestine in mice

Project description:Preservation of nucleosomes during replication has been extensively studied, while the maintenance of nucleosomes during transcription has gotten less attention. The histone chaperone FACT is involved in transcription elongation, although whether it disassembles or assembles nucleosomes during the passage of RNA polymerase is still unclear. We deleted the FACT subunit in adult mice to clarify the function of FACT in mammals. FACT loss was lethal due to bone marrow and intestinal failure, where the earliest progenitors completely vanished, while several other cell types were increased or decreased. Using cells isolated from several tissues, we showed that FACT loss was lethal only for stem cells but not cells differentiated in vitro. FACT depletion led to increased chromatin accessibility in a transcription-dependent manner, suggesting that nucleosomes are lost from transcribed regions in the absence of FACT. The most prominent response to the loss of chromatin integrity was the activation of interferon signaling and the accumulation of immunocytes in sensitive organs. FACT maintained chromatin integrity during transcription in mammalian adult stem cells, suggesting that chromatin transcription in these cells is different from more differentiated cells.

2021-12-02 | GSE189866 | GEO

Effect of Ssrp1 KO on transcriptome in mouse adipose stem cells [RNA-seq]

2021-11-30 | GSE189662 | GEO

Effect of Ssrp1 KO on chromatin accessibility in mouse adipose stem cells [ATAC-seq]

2021-11-30 | GSE189659 | GEO

Epigenome characterization at single base-pair resolution

Project description:We have combined standard micrococcal (MNase) digestion of nuclei with a modified protocol for construction paired-end DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including eviction of nucleosomes. Our protocol and mapping method provide a general strategy for characterizing full epigenomes. We used micrococcal nuclease mapping, chromatin immunoprecipitation and paired-end sequencing to determine the structure of yeast centromeres at single base-pair resolution.

2011-10-26 | E-GEOD-30551 | biostudies-arrayexpress

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