Nucleosome Reassembly Research Articles

The FACT Histone Chaperone: Tuning Gene Transcription in the Chromatin Context to Modulate Plant Growth and Development.

FACT is a heterodimeric histone chaperone consisting of the SSRP1 and SPT16 proteins and is conserved among eukaryotes. It interacts with the histones H2A-H2B and H3-H4 as well as with DNA. Based on in vitro and in vivo studies mainly in yeast and mammalian cells, FACT can mediate nucleosome disassembly and reassembly and thus facilitates in the chromatin context DNA-dependent processes including transcription, replication and repair. In plants, primarily the role of FACT related to RNA polymerase II transcription has been examined. FACT was found to associate with elongating Arabidopsis RNA polymerase II (RNAPII) as part of the transcript elongation complex and it was identified as repressor of aberrant intragenic transcriptional initiation. Arabidopsis mutants depleted in FACT subunits exhibit various defects in vegetative and reproductive development. Strikingly, FACT modulates important developmental transitions by promoting expression of key repressors of these processes. Thus, FACT facilitates expression of DOG1 and FLC adjusting the switch from seed dormancy to germination and from vegetative to reproductive development, respectively. In the central cell of the female gametophyte, FACT can facilitate DNA demethylation especially within heterochromatin, and thereby contributes to gene imprinting during Arabidopsis reproduction. This review discusses results particularly from the plant perspective about the contribution of FACT to processes that involve reorganisation of nucleosomes with a main focus on RNAPII transcription and its implications for diverse areas of plant biology.

FACT caught in the act of manipulating the nucleosome.

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone ‘FAcilitates Chromatin Transcription’ (FACT, consisting of subunits SPT16 and SSRP11) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication, and repair2. The mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-EM structures of human FACT in complex with partially assembled ‘sub-nucleosomes’, with supporting biochemical and hydrogen-deuterium exchange (HDX) data. FACT is engaged in extensive interactions with nucleosomal DNA and all histones. The large DNA-binding surface on FACT appears to be protected by the C-terminal domains of both subunits, and this inhibition is released by interaction with H2A-H2B, allowing FACT-H2A-H2B to dock onto a (H3-H4)2-DNA complex3. SPT16 binds nucleosomal DNA and tethers H2A-H2B through its C-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A-H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating H2A-H2B dimer removal, stabilizing intermediate ‘subnucleosomal’ states, and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.

Distinct transcriptional roles for Histone H3-K56 acetylation during the cell cycle in Yeast

Dynamic disruption and reassembly of promoter-proximal nucleosomes is a conserved hallmark of transcriptionally active chromatin. Histone H3-K56 acetylation (H3K56Ac) enhances these turnover events and promotes nucleosome assembly during S phase. Here we sequence nascent transcripts to investigate the impact of H3K56Ac on transcription throughout the yeast cell cycle. We find that H3K56Ac is a genome-wide activator of transcription. While H3K56Ac has a major impact on transcription initiation, it also appears to promote elongation and/or termination. In contrast, H3K56Ac represses promiscuous transcription that occurs immediately following replication fork passage, in this case by promoting efficient nucleosome assembly. We also detect a stepwise increase in transcription as cells transit S phase and enter G2, but this response to increased gene dosage does not require H3K56Ac. Thus, a single histone mark can exert both positive and negative impacts on transcription that are coupled to different cell cycle events.

The Arabidopsis Histone Chaperone FACT: Role of the HMG-Box Domain of SSRP1

Histone chaperones play critical roles in regulated structural transitions of chromatin in eukaryotic cells that involve nucleosome disassembly and reassembly. The histone chaperone FACT is a heterodimeric complex consisting in plants and metazoa of SSRP1/SPT16 and is involved in dynamic nucleosome reorganization during various DNA-dependent processes including transcription, replication and repair. The C-terminal HMG-box domain of the SSRP1 subunit mediates interactions with DNA and nucleosomes in vitro, but its relevance in vivo is unclear. Here, we demonstrate that Arabidopsis ssrp1–2 mutant plants express a C-terminally truncated SSRP1 protein. Although the structure of the truncated HMG-box domain is distinctly disturbed, it still exhibits residual DNA-binding activity, but has lost DNA-bending activity. Since ssrp1–2 plants are phenotypically affected but viable, the HMG-box domain may be functionally non-essential. To examine this possibility, SSRP1∆HMG completely lacking the HMG-box domain was studied. SSRP1∆HMG in vitro did not bind to DNA and its interactions with nucleosomes were severely reduced. Nevertheless, the protein showed a nuclear mobility and protein interactions similar to SSRP1. Interestingly, expression of SSRP1∆HMG is almost as efficient as that of full-length SSRP1 in supporting normal growth and development of the otherwise non-viable Arabidopsis ssrp1–1 mutant. SSRP1∆HMG is structurally similar to the fungal ortholog termed Pob3 that shares clear similarity with SSRP1, but it lacks the C-terminal HMG-box. Therefore, our findings indicate that the HMG-box domain conserved among SSRP1 proteins is not critical in Arabidopsis, and thus, the functionality of SSRP1/SPT16 in plants/metazoa and Pob3/Spt16 in fungi is perhaps more similar than anticipated.

Spt6 Association with RNA Polymerase II Directs mRNA Turnover During Transcription

Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 also associates with RNA polymerase II (RNAPII) via a tandem Src2 homology domain. However, the significance of Spt6-RNAPII interaction is not well understood. Here, we show that Spt6 recruitment togenes and the nucleosome reassembly functions of Spt6 can still occur in the absence of its association with RNAPII. Surprisingly, we found that Spt6-RNAPII association is required for efficient recruitment of the Ccr4-Not de-adenylation complex to transcribed genes for essential degradation of a range of mRNAs, including mRNAs required for cell-cycle progression. These findings reveal an unexpected control mechanism for mRNA turnover during transcription facilitated by a histone chaperone.

HIF-dependent and reversible nucleosome disassembly in hypoxia-inducible gene promoters

Hypoxia causes dramatic changes in gene expression profiles, and the mechanism of hypoxia-inducible transcription has been analyzed for use as a model system of stress-inducible gene regulation. In this study, changes in chromatin organization in promoters of hypoxia-inducible genes were investigated during hypoxia-reoxygenation conditions. Most of the hypoxia-inducible gene promoters were hypersensitive to DNase I under both normal and hypoxic conditions, and our data indicate an immediate recruitment of transcription factors under hypoxic conditions. In some of the hypoxia-inducible promoters, nucleosome-free DNA regions (NFRs) were established in parallel with hypoxia-induced transcription. We also show that the hypoxia-inducible formation of NFRs requires that hypoxia-inducible transcription factors (HIFs) bind to the promoters together with the transcriptional coactivator CBP. Within 1 h after the hypoxia exposure was ended (reoxygenation), HIF complexes were dissociated from the promoter regions. Within 24 h of reoxygenation, the hypoxia-induced transcription returned to basal levels and the nucleosome structure was reassembled in the hypoxia-inducible NFRs. Nucleosome reassembly required the function of the transcriptional coregulator SIN3A. Thus, reversible changes in nucleosome organization mediated by transcription factors are notable features of stress-inducible gene regulation.

Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A

Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro. Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.

RRM3 regulates epigenetic conversions in Saccharomyces cerevisiae in conjunction with Chromatin Assembly Factor I

ABSTRACTChromatin structures are transmitted to daughter cells through a complex system of nucleosome disassembly and re-assembly at the advancing replication forks. However, the role of replication pausing in the transmission and perturbation of chromatin structures has not been addressed. RRM3 encodes a DNA helicase, which facilitates replication at sites covered with non-histone protein complexes (tRNA genes, active gene promoters, telomeres) in Saccharomyces cerevisiae. In this report we show that the deletion of RRM3 reduces the frequency of epigenetic conversions in the subtelomeric regions of the chromosomes. This phenotype is strongly dependent on 2 histone chaperones, CAF-I and ASF1, which are involved in the reassembly of nucleosomes behind replication forks, but not on the histone chaperone HIR1. We also show that the deletion of RRM3 increases the spontaneous mutation rates in conjunction with CAF-I and ASF1, but not HIR1. Finally, we demonstrate that Rrm3p and CAF-I compete for the binding to the DNA replication clamp PCNA (Proliferating Cell Nuclear Antigen). We propose that the stalling of DNA replication predisposes to epigenetic conversions and that RRM3 and CAF-I play key roles in this process.

Histone chaperones: Variety and functions

Histone chaperones are required for the formation of the nucleosome—the basic unit of chromatin that consists of the DNA and histones. In this review, participation of histone chaperones CAF-1, ASF1, NAP1, and FACT in key cellular processes is discussed. Being multifunctional factors, histone chaperones take part in DNA replication, transcription, and repair. During replication, histone chaperones are required to form chromatin structure on both the mother and daughter DNA. They are involved at different stages of genome packaging: from histone transport into the nucleus to nucleosome formation. During transcription, histone chaperones reduce a nucleosome barrier for RNA polymerases accelerating the rate of RNA synthesis and promote nucleosome reassembly. During DNA repair, histone chaperones provide access to the damaged genome region for the repair enzymes, and participate in the chromatin assembly after DNA repair. Mutations in histone chaperones typically result in multiple defects in the cell, underlying the functional importance of these proteins.

Regulation of chaperone binding and nucleosome dynamics by key residues within the globular domain of histone H3.

BackgroundNucleosomes have an important role in modulating access of DNA by regulatory factors. The role specific histone residues have in this process has been shown to be an important mechanism of transcription regulation. Previously, we identified eight amino acids in histones H3 and H4 that are required for nucleosome occupancy over highly transcribed regions of the genome.ResultsWe investigate the mechanism through which three of these previously identified histone H3 amino acids regulate nucleosome architecture. We find that histone H3 K122, Q120, and R49 are required for Spt2, Spt6, and Spt16 occupancies at genomic locations where transcription rates are high, but not over regions of low transcription rates. Furthermore, substitution at one residue, K122, located on the dyad axis of the nucleosome, results in improper reassembly and disassembly of nucleosomes, likely accounting for the transcription rate-dependent regulation by these mutant histones.ConclusionsThese data show that when specific amino acids of histone proteins are substituted, Spt2, Spt6, and Spt16 occupancies are reduced and nucleosome dynamics are altered. Therefore, these data support a mechanism for histone chaperone binding where these factors interact with histone proteins to promote their activities during transcription.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0066-4) contains supplementary material, which is available to authorized users.

Roles of the Histone Chaperone FACT in Drosophila Gene Regulation and Chromatin Architecture

The highly conserved histone chaperone FACT (Facilitates Chromatin Transcription) is thought to contribute to the disassembly and reassembly of nucleosomes in the wake of RNA polymerase II passage through chromatin. In yeast, mutation of FACT subunits leads to decreased nucleosome occupancy at the promoters and transcribed regions of genes. In addition, genetic experiments suggest that yeast FACT plays a critical role in restricting initiation of transcription to normal promoters, ensuring that only appropriate mRNAs are synthesized. However, FACT's roles in chromatin biology and transcriptional regulation in higher eukaryotes are not well understood. Using Drosophila S2 cells as a model, we observe that depletion of the FACT complex results in aberrant transcriptional regulation. Surprisingly, we observe in genome‐wide experiments that FACT depletion has little effect on total nucleosome occupancy or position, but dramatically alters the distribution of various histone modifications, including histone 3 lysine 4 trimethylation (H3K4me3), histone 2A variant (H2Av) and histone 3 lysine 36 trimethylation (H3K36me3), in the promoters and transcribed regions of genes. Our results thus far provide valuable insight into roles of FACT in transcriptional regulation and chromatin dynamics in higher eukaryotes.Support or Funding InformationStowers Institute for Medical Research

Structural basis for histone H2B deubiquitination by the SAGA DUB module

Monoubiquitinated histone H2B plays multiple roles in transcription activation. H2B is deubiquitinated by the Spt‐Ada‐Gcn5 acetyltransferase (SAGA) coactivator, which contains a four‐protein subcomplex known as the deubiquitinating (DUB) module. The crystal structure of the Ubp8/Sgf11/Sus1/Sgf73 DUB module bound to a ubiquitinated nucleosome reveals that the DUB module primarily contacts H2A/H2B, with an arginine cluster on the Sgf11 zinc finger domain docking on the conserved H2A/H2B acidic patch. The Ubp8 catalytic domain mediates additional contacts with H2B as well as with the conjugated ubiquitin. We find that the DUB module deubiquitinates H2B both in the context of the nucleosome and in H2A/H2B dimers complexed with the histone chaperone, FACT, suggesting that SAGA could target H2B at multiple stages of nucleosome disassembly and reassembly during transcription.Support or Funding InformationSupported by grant GM‐095822 from the National Institute of General Medical Sciences (C.W.) and by a Ruth L. Kirschstein National Research Service Award (M.M.). A. Brik is a Neubauer Professor. GM/CA @ APS has been funded by the National Cancer Institute (Y1‐CO‐1020) and the National Institute of General Medical Sciences (Y1‐GM‐1104).

Transcription Pausing Regulates Hematopoietic Stem Cell Emergence through Fine-Tuning of Signaling Pathways

Regulation of RNA polymerase II (Pol II)-mediated gene transcription is a fundamental mechanism to control cell fate during developmental organogenesis, including the emergence of hematopoietic stem cells (HSCs) in vertebrate embryos. Increasing evidences in recent years have demonstrated that transcriptionally engaged Pol II often pauses shortly after initiation by pausing factors such as DSIF and NELF, and requires positive elongation factors such as P-TEFb for pause release. How this pause-to-elongation transition contributes to HSC development is largely unknown. In a zebrafish spt5 mutant, we detected a dramatic loss of HSCs in early embryos. Spt5 is a key subunit of DSIF, which plays a dual role in both Pol II pausing and elongation. The mutation of the zebrafish spt5 mutant has been reported to specifically disrupt the pausing function of DSIF. Similarly, reduction of HSCs was also detected in embryos lacking the other pausing factor NELF. Consistent with the loss-of-pausing in Spt5- and NELF-deficient embryos, we found that the HSC defect could be rescued by inhibiting Pol II pause-to-elongation transition with either P-TEFb inhibitors or knockdown of the P-TEFb subunit cdk9. Intriguingly, we identified that two essential signaling pathways involved in HSC development, the TGFβ signaling pathway and the JAK-STAT mediated proinflammation pathway, were oppositely regulated by Pol II pausing. In spt5 mutant embryos, the TGFβ signaling pathway is upregulated with an enhanced transcription elongation at several TGFβ signaling regulators, as revealed by Pol II ChIP assays; whereas the transcription of multiple genes involved in proinflammation signaling pathways, especially the genes regulating the interferon signaling, are downregulated. Pol II ChIP and nucleosome mapping studies revealed an increase of nucleosome occupancy at the promoters of proinflammation genes, leading to attenuated transcription initiation. These results suggest that paused Pol II can compete with nucleosomes for occupancy on some promoters to maintain accessible chromatin thus the basal transcription level of these genes. Consistent with the change of signaling activity, the HSC defect in mutant embryos could be effectively rescued by TGFβ inhibitors or by activation of JAK-STAT, the downstream effector of IFNγ signaling. Thus Pol II pausing affects HSC emergence through differentially regulating the transcription of distinct signaling regulators: while they negatively regulate the HSC inhibitory signaling (such as TGFβ) by preventing premature transcription elongation, they are also required to maintain the basal transcription of HSC-promoting signaling (such as the inflammation signaling) by preventing promoter nucleosome reassembly. In summary, our study suggests that Pol II pausing/elongation regulators and developmental signals converge at the paused Pol II to regulate HSC emergence in vertebrate embryos. DisclosuresNo relevant conflicts of interest to declare.

The Histone Chaperones FACT and Spt6 Restrict H2A.Z from Intragenic Locations

H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here we provide genomic and biochemical evidence that the RNA polymerase II (RNA Pol II) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of efficient nucleosome reassembly coupled to pervasive incorporation of H2A.Z by mislocalized SWR-C alters chromatin composition and contributes to cryptic initiation. Therefore, chaperone-mediated H2A.Z confinement is crucial for restricting the chromatin signature of gene promoters that otherwise may license or promote cryptic transcription.

ASF1 and the SWI/SNF complex interact functionally during nucleosome displacement, while FACT is required for nucleosome reassembly at yeast heat shock gene promoters during sustained stress.

Histone chaperones are an integral part of the transcription regulatory machinery. We investigated the involvement of histone chaperones and their functional interactions with ATP-dependent chromatin remodeling complexes in the regulation of yeast heat shock genes. Strong functional interaction between the histone chaperone ASF1 and the ATP-dependent chromatin remodeling complex SWI/SNF is exhibited in synergistic diminishment of nucleosome displacement during heat shock in the ΔASF1/ΔSNF2 strain in comparison to individual ASF1 or SNF2 inactivation. A similar but less pronounced effect was observed for ISW1/ASF1 inactivation but not for ASF1/STH1 (RSC complex) combinatorial inactivation. The depletion of Spt16, which is a major subunit of the FACT histone chaperone complex, leads to a severe growth defect phenotype associated with unusual thermotolerance. The acquired thermotolerance in the Spt16-depleted strain is associated with a defect in the reassembly of nucleosomes at the promoters of heat shock genes during sustained heat stress, leading to increased recruitment of the transcriptional activator HSF and RNA polymerase II. The defect in nucleosome assembly associated with Spt16 depletion also leads to an increased tolerance to stress due to an increased concentration of NaCl.

H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly.

The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.

Mutant versions of histone H3 are defective for nucleosome reassembly and histone chaperone recruitment during transcription (944.3)

Expression of the Saccharomyces cerevisiae SER3 gene is regulated by transcription interference. In the presence of serine, transcription of SRG1 ncDNA is initiated upstream of the adjacent SER3 gene and extends across the SER3 promoter. Our previous studies provided evidence that during SRG1 transcription, Spt6 and Spt16 histone chaperones reassemble nucleosomes over the SER3 promoter after the passage of RNA Pol II, which then interfere with transcription factor binding resulting in SER3 repression. In response to serine starvation, SRG1 transcription is reduced causing nucleosome depletion over the SER3 promoter, which in turn allows transcription factors to bind the SER3 promoter and activate SER3 transcription. We recently reported the identification of mutations altering eight histone residues that strongly increase SER3 expression without reducing transcription of intergenic SRG1 ncDNA. Nucleosome occupancy across SRG1 in these mutants was reduced to degrees that correlated well with the level of SER3 derepression. Additional analyses of these mutants indicated a broader defect in transcription‐coupled nucleosome occupancy at highly transcribed genes, but not lowly transcribed genes. Taken together, these data defined a new class of histone mutants that are defective for transcription‐dependent nucleosome occupancy. Further analysis focused on a subset of these histone mutants revealed a reduced rate of nucleosome reassembly during transcription in vivo. Moreover, we provide evidence that Spt6 and Spt16 histone chaperones fail to be recruited to genes being transcribed by RNA Pol II in these mutants. These results suggest that amino acids in histone H3 and H4 promote transcription‐coupled nucleosome assembly by facilitating the recruitment or retention of Spt6 and/or Spt16 histone chaperones.Grant Funding Source: Supported by NIGMS

FACT and the H2B N Tail

The FACT histone chaperone/nucleosome reorganization factor plays key roles in nucleosome dynamics during transcription. A new study has linked a specific domain in the H2B N terminus to the activity of FACT in regulating nucleosome disassembly at promoters during transcription activation and nucleosome reassembly at coding regions during transcription elongation.

Reassembly of Nucleosomes at the MLH1 Promoter Initiates Resilencing Following Decitabine Exposure

Hypomethylating agents reactivate tumor suppressor genes that are epigenetically silenced in cancer. Inevitably these genes are resilenced, leading to drug resistance. Using the MLH1 tumor suppressor gene as a model, we showed that decitabine-induced re-expression was dependent upon demethylation and eviction of promoter nucleosomes. Following decitabine withdrawal, MLH1 was rapidly resilenced despite persistent promoter demethylation. Single molecule analysis at multiple time points showed that gene resilencing was initiated by nucleosome reassembly on demethylated DNA and only then was followed by remethylation and stable silencing. Taken together, these data establish the importance of nucleosome positioning in mediating resilencing of drug-induced gene reactivation and suggest a role for therapeutic targeting of nucleosome assembly as a mechanism to overcome drug resistance.

UpSETing chromatin during non-coding RNA production

The packaging of eukaryotic DNA into nucleosomal arrays permits cells to tightly regulate and fine-tune gene expression. The ordered disassembly and reassembly of these nucleosomes allows RNA polymerase II (RNAPII) conditional access to the underlying DNA sequences. Disruption of nucleosome reassembly following RNAPII passage results in spurious transcription initiation events, leading to the production of non-coding RNA (ncRNA). We review the molecular mechanisms involved in the suppression of these cryptic initiation events and discuss the role played by ncRNAs in regulating gene expression.