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Effect of mutations on total and soluble histone H3 levels. Histone levels were measured by Western blotting (Materials and Methods) using whole cell extracts (total) or after removing chromatin by centrifugation to measure the free pool of histones (soluble). Signals were normalized in each case to the level observed with strains carrying wild-type POB3 and H4; the absolute level of soluble histone was 0.6% of the total level. Error bars indicate the standard deviation from three independent measurements.
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The pob3-Q308K mutation alters the small subunit of the Saccharomyces cerevisiae histone/nucleosome chaperone FACT, causing defects in both transcription and DNA replication. We describe histone mutations that suppress some of these defects, providing new insight into the mechanism of FACT activity in vivo. FACT is primarily known for its ability t...
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Context 1
... adapted a cell lysis and centrifugation assay to separate histones associated with chromatin from soluble histones likely to be associated with chaperones but not with chromatin (Feser et al. 2010) and then used quantitative Western blots to determine the amount of H3 in each fraction (see Materials and Methods). The results showed a slight decrease in the amount of soluble H3 in a pob3-Q308K strain, but no other significant changes that would explain the suppression (Fig- ure 4). We conclude that suppression of pob3-Q308K is not related to changes in the total amounts or the soluble pools of histones. ...
Context 2
... FACT:nucleosome complexes are rapidly reversible, converting 5% of the complexes to a persistently blocked form could also have a disproportionate effect on the yield of nucleosomes (and therefore histone H3 molecules) that copurify with FACT in an immunoprecipitation, as observed ( Zunder et al. 2012). We did not detect an increase in the total or soluble pools of H3 in a pob3-Q308K strain (Figure 4), and we did not see an increase in affinity of Pob3-Q308K protein for nucleosomes or H3-H4, so we propose that the excess H3 that copurifies with Pob3-Q308K protein is due to persistently associated FACT:nucleosome complexes. ...
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Citations
... It was reported that the Pob3-Q308K mutation increased the interaction of FACT with histone H3-H4 and the mutant FACT complex showed an inefficient release from the nucleosomes. 51,52 So, we introduced the equivalent Q265K mutation of SSRP1 in mESC by CRISPR-Cas9-based genomic editing to test whether the dynamic binding of FACT to chromatin is critical for cell fate transition in early development ( Figure S1A). The SSRP1-Q265 residue is located in the middle domain (MD) of SSRP1 and is highly conserved among eukaryotes ( Figures 1A-1C). ...
... Like yeast Pob3-Q308K mutation, we found that FACT SSRP1-Q265K shows an increased binding ability with histone H3-H4. A previous study showed that purified yeast FACT pob3-Q308K fails to release from nucleosomes efficiently, 51 indicating a reduced FACT turnover on chromatin. Consistent with this observation, our single-molecular magnetic tweezer experiments revealed that FACT SSRP1-Q265K requires a more potent force to disassemble nucleosomes, probably due to its increased interaction with histones. ...
The differentiation of embryonic stem cells (ESCs) begins with the transition from the naive to the primed state. The formative state was recently established as a critical intermediate between the two states. Here, we demonstrate the role of the histone chaperone FACT in regulating the naive-to-formative transition. We found that the Q265K mutation in the FACT subunit SSRP1 increased the binding of FACT to histone H3-H4, impaired nucleosome disassembly in vitro, and reduced the turnover of FACT on chromatin in vivo. Strikingly, mouse ESCs harboring this mutation showed elevated naive-to-formative transition. Mechanistically, the SSRP1-Q265K mutation enriched FACT at the enhancers of formative-specific genes to increase targeted gene expression. Together, these findings suggest that the turnover of FACT on chromatin is crucial for regulating the enhancers of formative-specific genes, thereby mediating the naive-to-formative transition. This study highlights the significance of FACT in fine-tuning cell fate transition during early development.
... The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11192931/s1, Figure S1: Electrophoresis of Hmo1 complexes with nucleosomes, which were constructed with a 181 bp DNA fragment labeled with Cy5 (green) based on the nucleosome positioning sequence from sea urchin 5S rDNA and yeast histones labeled with Oregon Green on H2A-Q114C; Figure S2: Competitor DNA itself does not affect nucleosome structure; Figure S3: FACT itself does not interact with a nucleosome; Figure S4: spFRET analysis of FACT interactions with N35/11 nucleosomes (~1 nM) in the presence of Hmo1 at different Hmo1:FACT ratios; Figure S5: Deletion of HMO1 causes synthetic effects when combined with defective alleles of the FACT subunits. References [63][64][65] ...
Yeast Hmo1 is a high mobility group B (HMGB) protein that participates in the transcription of ribosomal protein genes and rDNA, and also stimulates the activities of some ATP-dependent remodelers. Hmo1 binds both DNA and nucleosomes and has been proposed to be a functional yeast analog of mammalian linker histones. We used EMSA and single particle Förster resonance energy transfer (spFRET) microscopy to characterize the effects of Hmo1 on nucleosomes alone and with the histone chaperone FACT. Hmo1 induced a significant increase in the distance between the DNA gyres across the nucleosomal core, and also caused the separation of linker segments. This was opposite to the effect of the linker histone H1, which enhanced the proximity of linkers. Similar to Nhp6, another HMGB factor, Hmo1, was able to support large-scale, ATP-independent, reversible unfolding of nucleosomes by FACT in the spFRET assay and partially support FACT function in vivo. However, unlike Hmo1, Nhp6 alone does not affect nucleosome structure. These results suggest physiological roles for Hmo1 that are distinct from Nhp6 and possibly from other HMGB factors and linker histones, such as H1.
... Specific mutations in FACT subunits cause different activity defects in vitro, and different phenotypes in vivo, indicating distinct activities and functional roles for individual domains (119,120). For example, FACT containing the mutant Pob3-Q308K protein binds to and reorganizes nucleosomes normally, but it fails to dissociate efficiently, leading to abnormal persistence of complexes (126). Notably, the homologous residue in human SSRP1 is in a loop that contacts DNA (45), and while both pob3-Q308K and pob3-Q308R were isolated multiple times in a screen for pob3 mutations that cause sensitivity to the DNA replication toxin hydroxyurea, pob3-Q308A had no phenotype (127), suggesting that introduction of a positive charge near the DNA causes the reduced dissociation efficiency. ...
... In contrast, FACT with the Spt16-11 mutant subunit binds to and releases from nucleosomes normally but reorganizes them inefficiently (51). The phenotypes caused by these mutations are suppressed and enhanced by different profiles of histone mutations that affect different domains of nucleosomes (51,126). These and other genetic and biochemical studies suggest that promoting and resolving changes in nucleosome structure have separable functions in distinct processes in vivo. ...
FACT (FAcilitates Chromatin Transcription) has long been considered to be a transcription elongation factor whose ability to destabilize nucleosomes promotes RNAPII progression on chromatin templates. However, this is just one function of this histone chaperone, as FACT also functions in DNA replication. While broadly conserved among eukaryotes and essential for viability in many organisms, dependence on FACT varies widely, with some differentiated cells proliferating normally in its absence. It is therefore unclear what the core functions of FACT are, whether they differ in different circumstances, and what makes FACT essential in some situations but not others. Here, we review recent advances and propose a unifying model for FACT activity. By analogy to DNA repair, we propose that the ability of FACT to both destabilize and assemble nucleosomes allows it to monitor and restore nucleosome integrity as part of a system of chromatin repair, in which disruptions in the packaging of DNA are sensed and returned to their normal state. The requirement for FACT then depends on the level of chromatin disruption occurring in the cell, and the cell's ability to tolerate packaging defects. The role of FACT in transcription would then be just one facet of a broader system for maintaining chromatin integrity.
... SP-3N did not bind nucleosomes when competitor was added first (not shown), so this reveals slow or inefficient dissociation of SP-3N:nucleosome complexes. A similar phenomenon was observed with Spt16-Pob3-Q308K complexes previously (42), and was interpreted as failed resolution of the reorganized state back to canonical nucleosomes. A similar interpretation here would suggest that fusing three Nhp6 modules to Pob3 promotes formation of complexes, but also inhibits the successful reassembly of the nucleosome, and that this resolution is necessary for release of FACT. ...
... SP-3N failed to release efficiently from nucleosomes in vitro, and it was toxic in vivo (Figs 2,5). The pob3-Q308K allele also causes multiple defects in vivo (47) and inefficient release from nucleosomes in vitro (42), which was interpreted as failure of a proposed nucleosome assembly checkpoint in which release of FACT from complexes is dependent on successful assembly of a canonical nucleosome. Extending this interpretation, SP-3N may be unable to complete nucleosome assembly effectively because its enhanced DNA binding capacity makes it too prone to initiate another cycle of reorganization or unable to satisfy some test of the quality of the assembly process. ...
... The DNA in form 2 is tightly coiled, producing the same spFRET and nuclease sensitivity profiles as canonical nucleosomes (11), but this form is not fully resolved and can promote active processes like transcription or remodeling. Complete resolution requires coordinated assessment of the integrity of the nucleosome, with FACT variants like SP-3N ( Fig 5) and Spt16-Pob3-Q308K failing to release efficiently due to inefficient completion of this checkpoint (42). Factors are drawn approximately to scale using the color scheme in Fig 1 and ...
The essential histone chaperone FAcilitates Chromatin Transcription (FACT) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization in vitro, indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic in vivo. Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.
... We further explored the interaction between SSRP1 and CENP-T/-W. SSRP1 contains five well-characterized domains (Supplemental Fig. S4a; McCullough et al. 2013). Using expressed and purified recombinant GST-tagged truncation mutants of SSRP1, we found that only those that contained the acidic intrinsic disordered domain (IDD) of SSRP1 (amino acids 432-514) bound to the CENP-T HFD /-W complex (Supplemental Fig. S4c). ...
... To understand how Spt16 interacts with CENP-T-/W, we next tested which domains of Spt16 could bind to CENP-T HFD /-W. Spt16 contains several well-characterized functional domains ( Fig. 3A; McCullough et al. 2013). We expressed GST-tagged truncated forms of Spt16 that spanned the middle and C-terminal acidic domains (Fig. 3A). ...
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 found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found 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.
... DNA replication requires construction of about 75,000 new nucleosomes during S phase, with about 150,000 molecules of each histone being synthesized, shuttled to replication forks, and assembled. These processes are carefully regulated, as excess histones are detrimental (MEEKS-WAGNER AND HARTWELL 1986) and the pool of soluble histone proteins that is not associated with chromatin is small (we previously estimated the free pool of H3 to be about 0.6% of the total H3 in an asynchronous culture; MCCULLOUGH et al. 2013). Given the potential importance of concentration in understanding the effects of chaperone and histone mutations, we next investigated the abundance of these factors. ...
Saccharomyces cerevisiae Spt6 protein is a conserved chromatin factor with several distinct functional domains including a natively unstructured 30 residue N-terminal region that binds competitively with Spn1 or nucleosomes. To uncover physiological roles of these interactions, we isolated histone mutations that suppress defects caused by weakening Spt6:Spn1 binding with the spt6-F249K mutation. The strongest suppressor was H2A-N39K, which perturbs the point of contact between the two H2A-H2B dimers in an assembled nucleosome. Substantial suppression was also observed when the H2A-H2B interface with H3-H4 was altered, and many members of this class of mutations also suppressed a defect in another essential histone chaperone, FACT. Spt6 is best known as an H3-H4 chaperone, but we found that it binds with similar affinity to H2A-H2B or H3-H4. Like FACT, Spt6 is therefore capable of binding each of the individual components of a nucleosome, but unlike FACT Spt6 did not produce endonuclease-sensitive reorganized nucleosomes and did not displace H2A-H2B dimers from nucleosomes. Spt6 and FACT therefore have distinct activities, but defects can be suppressed by overlapping histone mutations. We also found that Spt6 and FACT together are nearly as abundant as nucleosomes, with ~24,000 Spt6 molecules, ~42,000 FACT molecules, and ~75,000 nucleosomes per cell. Histone mutations that destabilize interfaces within nucleosomes therefore reveal multiple spatial regions that have both common and distinct roles in the functions of these two essential, abundant histone chaperones. We discuss these observations in terms of different potential roles for chaperones in both promoting the assembly of nucleosomes and monitoring their quality.
... These chaperones incorporate canonical H3.1/H4 into DNA during replication, and ASF1 also collaborates with HIRA to exchange H3.1 for H3.3 at transcriptionally active sites. Processes of H3/H4 histone loading require a specific conformation that is facilitated by histone chaperones FACT facilitates chromatin transcription 1 or NAP1 nucleosome assembly protein 1 (Bowman et al., 2011;McCullough et al., 2013). These factors are primarily involved in deposition of the other core histone components (H2A/H2B dimers) (Ishimi et al., 1985;Ito et al., 1996;Hondele et al., 2013). ...
Telomeres and genes coding for 45S ribosomal RNA (rDNA) are frequent neighbours on eukaryotic chromosomes. Although their primary roles are different, they reveal an almost striking blend in their features and additional functions. Both genome domains show remarkable dynamics in their chromatin structures. They both are hypersensitive to dysfunctional histone chaperones, responding at the genomic and epigenomic levels. Both generate non-coding transcripts which, besides their epigenetic roles, may induce gross chromosomal rearrangements. Both can give rise to chromosomal fragile sites since their replication is intrinsically problematic. However, at the same time, both are essential for maintenance of genomic stability and integrity. Here we discuss data demonstrating structural and functional interconnectivity of telomeres and rDNA with a focus on recent results obtained in plants. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Using a combination of in vitro biochemistry, genetics, and genomics, McCullough and Pham et al. explore how the histone:DNA contact at the entry/exit site of nucleosomes affects the functions of the histone chaperone FACT in the yeast...
FACT (FAcilitates Chromatin Transcription/Transactions) is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone–DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome reorganization activity in vitro, and chromatin integrity and transcript output in vivo. Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT activity as expected, but the same mutations had surprisingly similar effects on global transcript levels. The results are consistent with emerging models that emphasize FACT’s importance in establishing global chromatin architecture prior to transcription, promoting transitions among different states as transcription profiles change, and restoring chromatin integrity after it is disturbed. Optimal FACT activity required the availability of both modified and unmodified states of H3-K56. Perturbing this balance was especially detrimental for maintaining repression of genes with high nucleosome occupancy over their promoters and for blocking antisense transcription at the +1 nucleosome. The results reveal a complex collaboration between H3-K56 modification status and multiple FACT functions, and support roles for nucleosome reorganization by FACT before, during, and after transcription.
Using affinity purification mass spectrometry (AP-MS) approaches, we have identified a novel role for CKII in the modification
of the Polymerase Associated Factor Complex (PAF-C). Our data indicates that the FAcilitates Chromatin Transcription complex
(FACT) interacts with CKII and may facilitate PAF Complex phosphorylation. Post-translational modification analysis of affinity
isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C although CKII subunits were not detected
as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate
PAF-C in vitro whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with SILAC
quantitation for PAF-C phosphorylation from wild−type and CKII temperature sensitive strains (cka1Δ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation - a modification dependent on FACT and PAF-C. Taken
together, our results define a coordinated role of CKII and FACT in the regulation of RNAPII transcription through chromatin
via phosphorylation of PAF-C.
