Insight Into the Mechanism of Nucleosome Reorganization From Histone Mutants That Suppress Defects in the FACT Histone Chaperone

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112, USA.
Genetics (Impact Factor: 5.96). 05/2011; 188(4):835-46. DOI: 10.1534/genetics.111.128769
Source: PubMed


FACT (FAcilitates Chromatin Transcription/Transactions) plays a central role in transcription and replication in eukaryotes by both establishing and overcoming the repressive properties of chromatin. FACT promotes these opposing goals by interconverting nucleosomes between the canonical form and a more open reorganized form. In the forward direction, reorganization destabilizes nucleosomes, while the reverse reaction promotes nucleosome assembly. Nucleosome destabilization involves disrupting contacts among histone H2A-H2B dimers, (H3-H4)(2) tetramers, and DNA. Here we show that mutations that weaken the dimer:tetramer interface in nucleosomes suppress defects caused by FACT deficiency in vivo in the yeast Saccharomyces cerevisiae. Mutating the gene that encodes the Spt16 subunit of FACT causes phenotypes associated with defects in transcription and replication, and we identify histone mutants that selectively suppress those associated with replication. Analysis of purified components suggests that the defective version of FACT is unable to maintain the reorganized nucleosome state efficiently, whereas nucleosomes with mutant histones are reorganized more easily than normal. The genetic suppression observed when the FACT defect is combined with the histone defect therefore reveals the importance of the dynamic reorganization of contacts within nucleosomes to the function of FACT in vivo, especially to FACT's apparent role in promoting progression of DNA replication complexes. We also show that an H2B mutation causes different phenotypes, depending on which of the two similar genes that encode this protein are altered, revealing unexpected functional differences between these duplicated genes and calling into question the practice of examining the effects of histone mutants by expressing them from a single plasmid-borne allele.

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Available from: Laura Mccullough, Oct 13, 2014
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    • "Given the function of FACT as a chromatin reorganizer, our study raises the possibility that R loops promote specific chromatin modifications that may demand the obligatory action of FACT for replication to reorganize chromatin. It is worth noting that the thermosensitivity and HU sensitivity of spt16-11 mutant are suppressed by histone mutations that weaken the interface between histones within nucleosomes (McCullough et al. 2011), consistent with the idea that the spt16-11 FACT mutant indeed may have difficulties in reorganizing nucleosomes and thereby may hamper the progression of RF through R-loop-enriched chromatin. Interestingly, R loops have recently been detected at centromeres (Nakama et al. 2012), in which we clearly observed that FACT is required to prevent RF stalling (Fig. 5D). "
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    ABSTRACT: FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP-chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription-replication conflicts, likely associated with a specific chromatin organization.
    Genes & development 03/2014; 28(7). DOI:10.1101/gad.234070.113 · 10.80 Impact Factor
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    • "To address the specificity of the Spt16 ChIP signals, the spt16-11 negative control ChIP (Fig. 4D, open bars) was performed because the specific enrichment of wild type FACT at any particular gene relative to control is usually mild, probably due to FACT binding activity throughout the genome. Inactive mutant FACT protein levels are probably quite reduced at this semi-permissive temperature in mutant cells, based on the reduced levels seen in Western blots at the non-permissive temperature of 37°C [62], and loss of FACT function under these conditions has been observed in other assays [63]. The low enrichment values for FACT binding across PDR5 in the spt16-11 strain grown at 34°C thus define ChIP background signals. "
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    ABSTRACT: Xenobiotic drugs induce Pleiotropic Drug Resistance (PDR) genes via the orthologous Pdr1/Pdr3 transcription activators. We previously identified the Mediator transcription co-activator complex as a key target of Pdr1 orthologs and demonstrated that Pdr1 interacts directly with the Gal11/Med15 subunit of the Mediator complex. Based on an interaction between Pdr1 and the FACT complex, we show that strains with spt16 or pob3 mutations are sensitive to xenobiotic drugs and display diminished PDR gene induction. Although FACT acts during the activation of some genes by assisting in the nucleosomes eviction at promoters, PDR promoters already contain nucleosome-depleted regions (NDRs) before induction. To determine the function of FACT at PDR genes, we examined the kinetics of RNA accumulation and changes in nucleosome occupancy following exposure to a xenobiotic drug in wild type and FACT mutant yeast strains. In the presence of normal FACT, PDR genes are transcribed within 5 minutes of xenobiotic stimulation and transcription returns to basal levels by 30-40 min. Nucleosomes are constitutively depleted in the promoter regions, are lost from the open reading frames during transcription, and the ORFs are wholly repopulated with nucleosomes as transcription ceases. While FACT mutations cause minor delays in activation of PDR genes, much more pronounced and significant defects in nucleosome repopulation in the ORFs are observed in FACT mutants upon transcription termination. FACT therefore has a major role in nucleosome redeposition following cessation of transcription at the PDR genes, the opposite of its better-known function in nucleosome disassembly.
    PLoS ONE 01/2014; 9(1):e84092. DOI:10.1371/journal.pone.0084092 · 3.23 Impact Factor
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    • "Although the precise molecular functions of FACT in transcription elongation remain under investigation, several studies have strongly implicated FACT in facilitating the nucleosome dynamics that occur during transcription elongation. These studies suggest that FACT associates with a nucleosome in front of RNA Pol II resulting in the reorganization of histones that eventually lead to the displacement of H2A-H2B dimers and the passage of RNA Pol II (Belotserkovskaya et al. 2003; McCullough et al. 2011; Orphanides et al. 1998). Once RNA Pol II has passed, FACT is also required to assist in the reassembly of nucleosomes to protect recently transcribed DNA from spurious transcription from cryptic intragenic promoters (Belotserkovskaya et al. 2003; Formosa et al. 2002; Jamai et al. 2009; Orphanides et al. 1999; Schwabish and Struhl 2004; Stuwe et al. 2008; VanDemark et al. 2008). "
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    ABSTRACT: The highly conserved FACT (Facilitates Chromatin Transactions) complex performs essential functions in eukaryotic cells through the reorganization of nucleosomes. During transcription, FACT reorganizes nucleosomes to allow passage of RNA Polymerase II and then assists in restoring these nucleosomes after RNA Polymerase II has passed. We have previously shown, consistent with this function, that Spt16 facilitates repression of the Saccharomyces cerevisiae SER3 gene by maintaining nucleosome occupancy over the promoter of this gene as a consequence of intergenic transcription of SRG1 noncoding DNA. In this study, we report the results of a genetic screen to identify mutations in SPT16 that derepress SER3. Twenty-five spt16 mutant alleles were found to derepress SER3 without causing significant reductions in either SRG1 RNA levels or Spt16 protein levels. Additional phenotypic assays indicate that these mutants have general transcription defects related to altered chromatin structure. Our analyses of a subset of these spt16 mutants reveal defects in SRG1 transcription-coupled nucleosome occupancy over the SER3 promoter. We provide evidence that these mutants broadly impair transcription-coupled nucleosome occupancy at highly transcribed genes but not at lowly transcribed genes. Finally, we show that one consequence shared by these mutations is the reduced binding of mutant Spt16 proteins across SRG1 and other highly transcribed genes. Taken together, our results highlight an important role for Spt16 in orchestrating transcription-coupled nucleosome assembly at highly transcribed regions of the genome, possibly by facilitating the association of Spt16 during this process.
    G3-Genes Genomes Genetics 05/2012; 2(5):555-67. DOI:10.1534/g3.112.002451 · 3.20 Impact Factor
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