FAcilitates Chromatin Transactions Research Articles

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome byengaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana

BackgroundH2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction.ResultsH2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA.ConclusionsOur genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

FACT interacts with Set3 HDAC and fine-tunes GAL1 transcription in response to environmental stimulation.

The histone chaperone facilitates chromatin transactions (FACT) functions in various DNA transactions. How FACT performs these multiple functions remains largely unknown. Here, we found, for the first time, that the N-terminal domain of its Spt16 subunit interacts with the Set3 histone deacetylase complex (Set3C) and that FACT and Set3C function in the same pathway to regulate gene expression in some settings. We observed that Spt16-G132D mutant proteins show defects in binding to Set3C but not other reported FACT interactors. At the permissive temperature, induction of the GAL1 and GAL10 genes is reduced in both spt16-G132D and set3Δ cells, whereas transient upregulation of GAL10 noncoding RNA (ncRNA), which is transcribed from the 3′ end of the GAL10 gene, is elevated. Mutations that inhibit GAL10 ncRNA transcription reverse the GAL1 and GAL10 induction defects in spt16-G132D and set3Δ mutant cells. Mechanistically, set3Δ and FACT (spt16-G132D) mutants show reduced histone acetylation and increased nucleosome occupancy at the GAL1 promoter under inducing conditions and inhibition of GAL10 ncRNA transcription also partially reverses these chromatin changes. These results indicate that FACT interacts with Set3C, which in turn prevents uncontrolled GAL10 ncRNA expression and fine-tunes the expression of GAL genes upon a change in carbon source.

Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes.

The FACT (FAcilitates Chromatin Transactions) complex influences transcription initiation and enables passage of RNA polymerase (Pol) II through gene body nucleosomes during elongation. In the budding yeast, ∼280 noncoding RNA genes highly transcribed in vivo by Pol III are found in the nucleosome-free regions bordered by positioned nucleosomes. The downstream nucleosome dynamics was found to regulate transcription via controlling the gene terminator accessibility and hence, terminator-dependent Pol III recycling. As opposed to the enrichment at the 5′-ends of Pol II-transcribed genes, our genome-wide mapping found transcription-dependent enrichment of the FACT subunit Spt16 near the 3′-end of all Pol III-transcribed genes. Spt16 physically associates with the Pol III transcription complex and shows gene-specific occupancy levels on the individual genes. On the non-tRNA Pol III-transcribed genes, Spt16 facilitates transcription by reducing the nucleosome occupany on the gene body. On the tRNA genes, it maintains the position of the nucleosome at the 3′ gene end and affects transcription in a gene-specific manner. Under nutritional stress, Spt16 enrichment is abolished in the gene downstream region of all Pol III-transcribed genes and reciprocally changed on the induced or repressed Pol II-transcribed ESR genes. Under the heat and replicative stress, its occupancy on the Pol III-transcribed genes increases significantly. Our results show that Spt16 elicits a differential, gene-specific and stress-responsive dynamics, which provides a novel stress-sensor mechanism of regulating transcription against external stress. By primarily influencing the nucleosomal organization, FACT links the downstream nucleosome dynamics to transcription and environmental stress on the Pol III-transcribed genes.

Evidence that dissociation of Spt16 from transcribed genes is partially dependent on RNA Polymerase II termination

ABSTRACTFACT (FAcilitates Chromatin Transactions) is a highly conserved histone chaperone complex in eukaryotic cells that can interact and manipulate nucleosomes in order to promote a variety of DNA-based processes and to maintain the integrity of chromatin throughout the genome. Whereas key features of the physical interactions that occur between FACT and nucleosomes in vitro have been elucidated in recent years, less is known regarding FACT functional dynamics in vivo. Using the Saccharomyces cerevisiae system, we now provide evidence that at least at some genes dissociation of the FACT subunit Spt16 from their 3′ ends is partially dependent on RNA Polymerase II (Pol II) termination. Combined with other studies, our results are consistent with a two-phase mechanism for FACT dissociation from genes, one that occurs upstream from Pol II dissociation and is Pol II termination-independent and the other that occurs further downstream and is dependent on Pol II termination.

The structural basis of human Spt16 N-terminal domain interaction with histone (H3-H4)2 tetramer

FACT (Facilitates Chromatin Transactions) is a heterodimeric protein complex involved in RNA polymerase II transcription elongation, playing essential roles in chromatin remodeling during transcription, replication, and DNA damage repair. The FACT subunit hSpt16 is essential for nucleosome reorganization. The N-terminal domain of hSpt16 (hSpt16-NTD) was recently described as a histone (H3-H4)2-binding domain; however, its mode of interaction remains unknown. In this study, we solved the structure of hSpt16-NTD437 at 2.19 Å and found that a long-disordered region (hSpt16-LDR), after the main body of hSpt16-NTD, is a novel histone-binding motif. Furthermore, hSpt16-LDR interaction with (H3-H4)2 is H3 N-terminal tail-independent. Therefore, Spt16-NTD is a histone H3-H4-specific binding domain with a distinct mechanism of interaction between histones and histone chaperones.

FACT Inhibition Blocks Induction But Not Maintenance of Pluripotency.

The histone chaperone facilitates chromatin transactions (FACT) is associated with nuclear processes, including DNA transcription, replication, and repair. We previously showed that FACT is transiently recruited to pluripotency-associated target genes by newly bound Oct4. In this study, we tested the effects of FACT depletion by knockout or chemical inhibition on the induction and maintenance of pluripotency. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated deletion of the FACT subunit Spt16 did not affect the viability or proliferation of fibroblasts but blocked their ability to form induced pluripotent stem cells. Similarly, a small molecule inhibitor of FACT blocked the induction of pluripotency at an early step in reprogramming, without affecting the viability, proliferation, undifferentiated state, or the expression of core pluripotency genes. Notably, trypsinization and passage of pluripotent cells transiently reintroduced a requirement for FACT. Although FACT has been considered to be an essential transcription elongation factor, these results contribute to the emerging view that it instead promotes transitions between stable chromatin states, including during reprogramming to pluripotency.

Transcription Promotes the Interaction of the FAcilitates Chromatin Transactions (FACT) Complex with Nucleosomes in Saccharomyces cerevisiae.

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 (RNAP)-disrupted nucleosomes. In this study, we used high-resolution analysis of FACT occupancy in Saccharomyces cerevisiae to test this hypothesis. We demonstrate that FACT interacts with nucleosomes in vivo and that its interaction with chromatin is dependent on transcription by any of the three RNAPs. Deep sequencing of micrococcal nuclease-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 dinucleosomes," 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 RNAP-disrupted nucleosomes.

FACT complex is required for DNA demethylation at heterochromatin during reproduction in Arabidopsis

The DEMETER (DME) DNA glycosylase catalyzes genome-wide DNA demethylation and is required for endosperm genomic imprinting and embryo viability. Targets of DME-mediated DNA demethylation reside in small, euchromatic, AT-rich transposons and at the boundaries of large transposons, but how DME interacts with these diverse chromatin states is unknown. The STRUCTURE SPECIFIC RECOGNITION PROTEIN 1 (SSRP1) subunit of the chromatin remodeler FACT (facilitates chromatin transactions), was previously shown to be involved in the DME-dependent regulation of genomic imprinting in Arabidopsis endosperm. Therefore, to investigate the interaction between DME and chromatin, we focused on the activity of the two FACT subunits, SSRP1 and SUPPRESSOR of TY16 (SPT16), during reproduction in Arabidopsis We found that FACT colocalizes with nuclear DME in vivo, and that DME has two classes of target sites, the first being euchromatic and accessible to DME, but the second, representing over half of DME targets, requiring the action of FACT for DME-mediated DNA demethylation genome-wide. Our results show that the FACT-dependent DME targets are GC-rich heterochromatin domains with high nucleosome occupancy enriched with H3K9me2 and H3K27me1. Further, we demonstrate that heterochromatin-associated linker histone H1 specifically mediates the requirement for FACT at a subset of DME-target loci. Overall, our results demonstrate that FACT is required for DME targeting by facilitating its access to heterochromatin.

The Histone Chaperone FACT Contributes to DNA Replication-Coupled Nucleosome Assembly.

DNA replication-coupled (RC) nucleosome assembly is mediated by histone chaperones and is fundamental for epigenetic inheritance and maintenance of genomic integrity. The mechanisms that promote this process are only partially understood. Here, we show that the histone chaperone FACT (facilitates chromatin transactions), consisting of Spt16 and Pob3, promotes newly synthesized histone H3-H4 deposition. We describe an allele of Spt16 (spt16-m) that has a defect in binding to H3-H4 and impairs their deposition onto DNA. Consistent with a direct role for FACT in RC nucleosome assembly, spt16-m displays synthetic defects with other histone chaperones associated with this process, CAF-1 and Rtt106. Importantly, we show that FACT physically associates with Rtt106 and that the acetylation of H3K56, a mark on newly synthesized H3, modulates this interaction. Therefore, FACT collaborates with CAF-1 and Rtt106 in RC nucleosome assembly.

Pluripotency Transcription Factor Oct4 Mediates Stepwise Nucleosome Demethylation and Depletion

The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes the accumulation of local H3K9me2 and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. Chromatin immunoprecipitation (ChIP) time course experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT (facilitates chromatin transactions) complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion.

The FACT Histone Chaperone Guides Histone H4 Into Its Nucleosomal Conformation in Saccharomyces cerevisiae

The pob3-Q308K mutation alters the small subunit of the Saccharomyces cerevisiae histone/nucleosome chaperone Facilitates Chromatin Transactions (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 to promote reorganization of nucleosomes into a more open form, but neither the pob3-Q308K mutation nor the compensating histone mutations affect this activity. Instead, purified mutant FACT complexes fail to release from nucleosomes efficiently, and the histone mutations correct this flaw. We confirm that pob3-T252E also suppresses pob3-Q308K and show that combining two suppressor mutations can be detrimental, further demonstrating the importance of balance between association and dissociation for efficient FACT:nucleosome interactions. To explain our results, we propose that histone H4 can adopt multiple conformations, most of which are incompatible with nucleosome assembly. FACT guides H4 to adopt appropriate conformations, and this activity can be enhanced or diminished by mutations in Pob3 or histones. FACT can therefore destabilize nucleosomes by favoring the reorganized state, but it can also promote assembly by tethering histones and DNA together and maintaining them in conformations that promote canonical nucleosome formation.

A Nucleosomal Region Important for Ensuring Proper Interactions Between the Transcription Elongation Factor Spt16 and Transcribed Genes inSaccharomyces cerevisiae

The highly conserved FACT (FAcilitates Chromatin Transactions) histone chaperone assists in the transcription elongation process first by facilitating the removal of histones in front of transcribing RNA polymerase II (Pol II) and then by contributing to nucleosome reassembly in the wake of Pol II passage. Whereas it is well established that FACT localizes across actively transcribed genes, the mechanisms that regulate FACT recruitment to and disengagement from chromatin during transcription still remain to be elucidated. Using the Saccharomyces cerevisiae model system, we previously showed that a histone H3 mutant—H3-L61W—greatly perturbs interactions between the yeast FACT (yFACT) complex and chromatin during transcription, resulting in a pronounced shift in yFACT occupancy toward the 3′ ends of transcribed genes. In the present study we report that two histone H4 mutants—H4-R36A and H4-K31E—alter the association pattern of the yFACT subunit Spt16 across transcribed genes in a fashion similar to that seen for H3-L61W. Interestingly, H4-R36, H4-K31, and H3-L61 are in close proximity to each other on the side of the nucleosome. We also provide evidence that the H4-R36A and H3-L61W mutants impair proper Spt16−chromatin interactions by perturbing a common process. Collectively, our results suggest that a nucleosomal region encompassing the H4-R36, H4-K31, and H3-L61 residues plays an important role in ensuring proper association of yFACT across transcribed genes.

Identification of Mutant Versions of the Spt16 Histone Chaperone That Are Defective for Transcription-Coupled Nucleosome Occupancy in Saccharomyces cerevisiae.

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.

The C‐terminal domain of the Spt16 subunit of FACT affects transcription elongation

In the budding yeast S. cerevisiae, the abundant nuclear complex FACT (Facilitates Chromatin Transactions) is a heterodimer of two essential proteins: Spt16 and Pob3. FACT is highly conserved among eukaryotes, and has been implicated in transcription, DNA replication and DNA repair. To learn more about the roles FACT plays in these processes, genome-wide synthetic-lethal analysis of several mutant alleles of SPT16 was carried out. These mutant alleles were all identified by a dominant Spt¯ phenotype, yet they yielded radically different patterns of genetic interactions. Those alleles that display interactions with genes involved in transcription elongation all contain mutations affecting the C-terminal portion of Spt16; this clustering of mutations may identify a region of Spt16 responsible for the proposed histone-chaperone activity of FACT. Further studies with one mutant allele that affects the C-terminal domain, spt16-E857K, demonstrate that this mutant protein interacts more weakly with histone H2B than does wild-type Spt16 or a mutant Spt16 without C-terminal-domain alterations. We therefore propose a model in which weakened interactions between FACT and histones negatively affects transcription elongation. This work has been supported by the Natural Sciences and Engineering Research Council (NSERC) and the Canadian Institutes for Health Reseach (CIHR).