Histone Chaperone Research Articles

TOR inactivation triggers heterochromatin formation in rDNA during glucose starvation

In response to environmental cues, such as nutrient starvation, living organisms modulate gene expression through mechanisms involving histone modifications. Specifically, nutrient depletion inactivates the TOR (target of rapamycin) pathway, leading to reduced expression of ribosomal genes. While these regulatory mechanisms are well elucidated in budding yeast Saccharomyces cerevisiae, their conservation across diverse organisms remains unclear. In this study, we demonstrate that fission yeast Schizosaccharomyces pombe cells repress ribosomal gene transcription through a different mechanism. TORC1, which accumulates in the rDNA region, dissociates upon starvation, resulting in enhanced methylation of H3K9 and heterochromatin formation, facilitated by dissociation of the stress-responsive transcription factor Atf1 and accumulation of the histone chaperone FACT. We propose that this mechanism might be adapted in mammals that possess Suv39H1 and HP1, which are absent in budding yeast.

Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-modal active/silent state at the sub-telomeres of S. cerevisiae

BackgroundClassical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. In this study, we used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the different roles of PCNA and histone chaperones in the stability and the propagation of repressed states at the sub-telomeres of S. cerevisiae.ResultsWe show position dependent transient repression or bi-modal expression of reporter genes at the VIIL sub-telomere. We also show that mutations in the replicative clamp POL30 (PCNA) or the deletion of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the deletion of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. We analyze the physical interaction of CAF1 and RRM3 with PCNA and discuss the implications of these findings for our understanding of the stability and transmission of the epigenetic state of the genes.ConclusionsThere are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. We characterise the roles of CAF1, RRM3 and ASF1 in these modes of gene repression. We suggest that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing.

The N-terminus of Spt16 anchors FACT to MCM2-7 for parental histone recycling.

Parental histone recycling is vital for maintaining chromatin-based epigenetic information during replication, yet its underlying mechanisms remain unclear. Here, we uncover an unexpected role of histone chaperone FACT and its N-terminus of the Spt16 subunit during parental histone recycling and transfer in budding yeast. Depletion of Spt16 and mutations at its middle domain that impair histone binding compromise parental histone recycling on both the leading and lagging strands of DNA replication forks. Intriguingly, deletion of the Spt16-N domain impairs parental histone recycling, with a more pronounced defect observed on the lagging strand. Mechanistically, the Spt16-N domain interacts with the replicative helicase MCM2-7 and facilitates the formation of a ternary complex involving FACT, histone H3/H4and Mcm2 histone binding domain, critical for the recycling and transfer of parental histones to lagging strands. Lack of the Spt16-N domain weakens the FACT-MCM interaction and reduces parental histone recycling. We propose that the Spt16-N domain acts as a protein-protein interaction module, enabling FACT to function as a shuttle chaperone in collaboration with Mcm2 and potentially other replisome components for efficient local parental histone recycling and inheritance.

Different elongation factors distinctly modulate RNA polymerase II transcription in Arabidopsis.

Various transcript elongation factors (TEFs) including modulators of RNA polymerase II (RNAPII) activity and histone chaperones tune the efficiency of transcription in the chromatin context. TEFs are involved in establishing gene expression patterns during growth and development in Arabidopsis, while little is known about the genomic distribution of the TEFs and the way they facilitate transcription. We have mapped the genome-wide occupancy of the elongation factors SPT4-SPT5, PAF1C and FACT, relative to that of elongating RNAPII phosphorylated at residues S2/S5 within the carboxyterminal domain. The distribution of SPT4-SPT5 along transcribed regions closely resembles that of RNAPII-S2P, while the occupancy of FACT and PAF1C is rather related to that of RNAPII-S5P. Under transcriptionally challenging heat stress conditions, mutant plants lacking the corresponding TEFs are differentially impaired in transcript synthesis. Strikingly, in plants deficient in PAF1C, defects in transcription across intron/exon borders are observed that are cumulative along transcribed regions. Upstream of transcriptional start sites, the presence of FACT correlates with nucleosomal occupancy. Under stress conditions FACT is particularly required for transcriptional upregulation and to promote RNAPII transcription through+1 nucleosomes. Thus, Arabidopsis TEFs are differently distributed along transcribed regions, and are distinctly required during transcript elongation especially upon transcriptional reprogramming.

The histone chaperone ANP32B regulates chromatin incorporation of the atypical human histone variant macroH2A

All vertebrate genomes encode for three large histone H2A variants that have an additional metabolite-binding globular macrodomain module, macroH2A. MacroH2A variants impact heterochromatin organization and transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how macroH2A is incorporated into chromatin and the identity of any chaperones required for histone deposition remain elusive. Here, we develop a split-GFP-based assay for chromatin incorporation and use it to conduct a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We show that the histone chaperone ANP32B is a regulator of macroH2A deposition. ANP32B associates with macroH2A in cells and invitro binds to histones with low nanomolar affinity. Invitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetrasomes. In cells, depletion of ANP32B strongly affects global macroH2A chromatin incorporation, revealing ANP32B as a macroH2A histone chaperone.

Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication

ATAD2 is a non-canonical ATP-dependent histone chaperone and a major cancer target. Despite widespread efforts to design drugs targeting the ATAD2 bromodomain, little is known about the overall structural organization and regulation of ATAD2. Here, we present the 3.1 Å cryo-EM structure of human ATAD2 in the ATP state, showing a shallow hexameric spiral that binds a peptide substrate at the central pore. The spiral conformation is locked by an N-terminal linker domain (LD) that wedges between the seam subunits, thus limiting ATP-dependent symmetry breaking of the AAA+ ring. In contrast, structures of the ATAD2-histone H3/H4 complex show the LD undocked from the seam, suggesting that H3/H4 binding unlocks the AAA+ spiral by allosterically releasing the LD. These findings, together with the discovery of an inter-subunit signaling mechanism, reveal a unique regulatory mechanism for ATAD2 and lay the foundation for developing new ATAD2 inhibitors.

Histone and Histone Acetylation-Related Alterations of Gene Expression in Uninvolved Psoriatic Skin and Their Effects on Cell Proliferation, Differentiation, and Immune Responses.

Psoriasis is a chronic immune-mediated skin disease in which the symptom-free, uninvolved skin carries alterations in gene expression, serving as a basis for lesion formation. Histones and histone acetylation-related processes are key regulators of gene expression, controlling cell proliferation and immune responses. Dysregulation of these processes is likely to play an important role in the pathogenesis of psoriasis. To gain a complete overview of these potential alterations, we performed a meta-analysis of a psoriatic uninvolved skin dataset containing differentially expressed transcripts from nearly 300 individuals and screened for histones and histone acetylation-related molecules. We identified altered expression of the replication-dependent histones HIST2H2AA3 and HIST2H4A and the replication-independent histones H2AFY, H2AFZ, and H3F3A/B. Eight histone chaperones were also identified. Among the histone acetyltransferases, ELP3 and KAT5 and members of the ATAC, NSL, and SAGA acetyltransferase complexes are affected in uninvolved skin. Histone deacetylation-related alterations were found to affect eight HDACs and members of the NCOR/SMRT, NURD, SIN3, and SHIP HDAC complexes. In this article, we discuss how histone and histone acetylation-related expression changes may affect proliferation and differentiation, as well as innate, macrophage-mediated, and T cell-mediated pro- and anti-inflammatory responses, which are known to play a central role in the development of psoriasis.

High-throughput screening of the Saccharomyces cerevisiae genome for 2-amino-3-methylimidazo [4,5-f] quinoline resistance identifies colon cancer-associated genes.

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for 5 or 10 generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. We identified 424 ORFs, including 337 genes of known function, in duplicate screens of the "humanized" collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and "humanized" libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). These studies underscore the role of ribosomal protein genes in conferring IQ resistance, and illuminate DNA repair pathways for conferring resistance to activated IQ.

Characterization of histone chaperone MCM2 as a key regulator in arsenic-induced depletion of H3.3 at genomic loci

Arsenic exposure is associated with an increased risk of many cancers, and epigenetic mechanisms play a crucial role in arsenic-mediated carcinogenesis. Our previous studies have shown that arsenic exposure induces polyadenylation of H3.1 mRNA and inhibits the deposition of H3.3 at critical gene regulatory elements. However, the precise underling mechanisms are not yet understood. To characterize the factors governing arsenic-induced inhibition of H3.3 assembly through H3.1 mRNA polyadenylation, we utilized mass spectrometry to identify the proteins, especially histone chaperones, with reduced binding affinity to H3.3 under conditions of arsenic exposure and polyadenylated H3.1 mRNA overexpression. Our findings reveal that the interaction between H3.3 and the histone chaperon protein MCM2 is diminished by both polyadenylated H3.1 mRNA overexpression and arsenic treatment in human lung epithelial BEAS-2B cells. The increased binding of MCM2 to H3.1, resulting from elevated H3.1 protein levels, appears to contribute to the reduced availability of MCM2 for H3.3. To further investigate the role of MCM2 in H3.3 deposition during arsenic exposure and H3.1 mRNA polyadenylation, we overexpressed MCM2 in BEAS-2B cells overexpressing polyadenylated H3.1 or exposed to arsenic. Our results demonstrate that MCM2 overexpression attenuates H3.3 depletion at several genomic loci, suggesting its involvement in the arsenic-induced displacement of H3.3 mediated by H3.1 mRNA polyadenylation. These findings suggest that changes in the association between histone chaperone MCM2 and H3.3 due to polyadenylation of H3.1 mRNA may play a pivotal role in arsenic-induced carcinogenesis.

OTUD5 limits replication fork instability by organizing chromatin remodelers.

Proper regulation of replication fork progression is important for genomic maintenance. Subverting the transcription-induced conflicts is crucial in preserving the integrity of replication forks. Various chromatin remodelers, such as histone chaperone and histone deacetylases are known to modulate replication stress, but how these factors are organized or collaborate are not well understood. Here we found a new role of the OTUD5 deubiquitinase in limiting replication stress. We found that OTUD5 is recruited to replication forks, and its depletion causes replication fork stress. Through its C-terminal disordered tail, OTUD5 assembles a complex containing FACT, HDAC1and HDAC2 at replication forks. A cell line engineered to specifically uncouple FACT interaction with OTUD5 exhibits increases in FACT loading onto chromatin, R-loop formation, and replication fork stress. OTUD5 mediates these processes by recruiting and stabilizing HDAC1 and HDAC2, which decreases H4K16 acetylation and FACT recruitment. Finally, proteomic analysis revealed that the cells with deficient OTUD5-FACT interaction activates the Fanconi Anemia pathway for survival. Altogether, this study identified a new interaction network among OTUD5-FACT-HDAC1/2 that limits transcription-induced replication stress.

SET-mediated epigenetic dysregulation of p53 impairs trichloroethylene-induced DNA damage response

Trichloroethylene (TCE) was a widely used industrial solvent, and now has become a major environmental pollutant. Exposure to TCE has been found to result in significant damage to the liver, leading to hepatic toxicity. In our previous study, we discovered that a histone chaperon called SET plays a crucial role in mediating the DNA damage and apoptosis caused by TCE in hepatic cells. However, the precise function of SET in the response to DNA damage is still not fully understood. In this study, we evaluated TCE-induced DNA damage of hepatic L-02 cells with SET-knockdown, then analyzed alterations of H3K79me3 and p53 in hepatic cells and carcinogenic mice livers. Results suggested that SET interferes with DNA response via mediating down-regulation of p53 and partially suppressing H3K79me3 under treatment of TCE. To further verify the regulatory cascade, H3K79me3 was reduced and p53 was knocked down in L-02 cells respectively, and extent of DNA damage was evaluated. Reduced H3K79me3 was found leading to down-regulation of p53 which further exacerbated TCE-induced DNA injury. These findings demonstrated that SET-H3K79me3-p53 served as an epigenetic regulatory axis involved in TCE-induced DNA damage response.

The longevity and reversibility of quiescence in Schizosaccharomyces pombe are dependent upon the HIRA histone chaperone

ABSTRACT Quiescence (G0) is a reversible non-dividing state that facilitates cellular survival in adverse conditions. Here, we demonstrate that the HIRA histone chaperone complex is required for the reversibility and longevity of nitrogen starvation-induced quiescence in Schizosaccharomyces pombe. The HIRA protein, Hip1 is not required for entry into G0 or the induction of autophagy. Although hip1Δ cells retain metabolic activity in G0, they rapidly lose the ability to resume proliferation. After a short period in G0 (1 day), hip1Δ mutants can resume cell growth in response to the restoration of a nitrogen source but do not efficiently reenter the vegetative cell cycle. This correlates with a failure to induce the expression of MBF transcription factor-dependent genes that are critical for S phase. In addition, hip1Δ G0 cells rapidly progress to a senescent state in which they can no longer re-initiate growth following nitrogen source restoration. Analysis of a conditional hip1 allele is consistent with these findings and indicates that HIRA is required for efficient exit from quiescence and prevents an irreversible cell cycle arrest.

The histone chaperone function of Daxx is dispensable for embryonic development

Daxx functions as a histone chaperone for the histone H3 variant, H3.3, and is essential for embryonic development. Daxx interacts with Atrx to form a protein complex that deposits H3.3 into heterochromatic regions of the genome, including centromeres, telomeres, and repeat loci. To advance our understanding of histone chaperone activity in vivo, we developed two Daxx mutant alleles in the mouse germline, which abolish the interactions between Daxx and Atrx (DaxxY130A), and Daxx and H3.3 (DaxxS226A). We found that the interaction between Daxx and Atrx is dispensable for viability; mice are born at the expected Mendelian ratio and are fertile. The loss of Daxx-Atrx interaction, however, does cause dysregulated expression of endogenous retroviruses. In contrast, the interaction between Daxx and H3.3, while not required for embryonic development, is essential for postnatal viability. Transcriptome analysis of embryonic tissues demonstrates that this interaction is important for silencing endogenous retroviruses and for maintaining proper immune cell composition. Overall, these results clearly demonstrate that Daxx has both Atrx-dependent and independent functions in vivo, advancing our understanding of this epigenetic regulatory complex.

The Histone Chaperones SET/TAF-1β and NPM1 Exhibit Conserved Functionality in Nucleosome Remodeling and Histone Eviction in a Cytochrome c-Dependent Manner.

Chromatin homeostasis mediates essential processes in eukaryotes, where histone chaperones have emerged as major regulatory factors during DNA replication, repair, and transcription. The dynamic nature of these processes, however, has severely impeded their characterization at the molecular level. Here, fluorescence optical tweezers are applied to follow histone chaperone dynamics in real time. The molecular action of SET/template-activating factor-Iβ and nucleophosmin 1-representing the two most common histone chaperone folds-are examined using both nucleosomes and isolated histones. It is shown that these chaperones present binding specificity for fully dismantled nucleosomes and are able to recognize and disrupt non-native histone-DNA interactions. Furthermore, the histone eviction process and its modulation by cytochrome c are scrutinized. This approach shows that despite the different structures of these chaperones, they present conserved modes of action mediating nucleosome remodeling.

Imaging-based study demonstrates how the DEK nanoscale distribution differentially correlates with epigenetic marks in a breast cancer model

Epigenetic dysregulation of chromatin is one of the hallmarks of cancer development and progression, and it is continuously investigated as a potential general bio-marker of this complex disease. One of the nuclear factors involved in gene regulation is the unique DEK protein—a histone chaperon modulating chromatin topology. DEK expression levels increase significantly from normal to cancer cells, hence raising the possibility of using DEK as a tumor marker. Although DEK is known to be implicated in epigenetic and transcriptional regulation, the details of these interactions and their relevance in cancer development remain largely elusive. In this work, we investigated the spatial correlation between the nuclear distribution of DEK and chromatin patterns—alongside breast cancer progression—leveraging image cross-correlation spectroscopy (ICCS) coupled with Proximity Ligation Assay (PLA) analysis. We performed our study on the model based on three well-established human breast cell lines to consider this tumor's heterogeneity (MCF10A, MCF7, and MDA-MB-231 cells). Our results show that overexpression of DEK correlates with the overall higher level of spatial proximity between DEK and histone marks corresponding to gene promoters regions (H3K9ac, H3K4me3), although it does not correlate with spatial proximity between DEK and gene enhancers (H3K27ac). Additionally, we observed that colocalizing fractions of DEK and histone marks are lower for the non-invasive cell subtype than for the highly invasive cell line (MDA-MB-231). Thus, this study suggests that the role of DEK on transcriptionally active chromatin regions varies depending on the subtype of the breast cancer cell line.

FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells

BackgroundThe FACT complex is a conserved histone chaperone with critical roles in transcription and histone deposition. FACT is essential in pluripotent and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block induction of pluripotent stem cells, yet the mechanism through which FACT regulates cell fate decisions remains unclear.ResultsTo explore the mechanism for FACT function, we generated AID-tagged murine embryonic cell lines for FACT subunit SPT16 and paired depletion with nascent transcription and chromatin accessibility analyses. We also analyzed SPT16 occupancy using CUT&RUN and found that SPT16 localizes to both promoter and enhancer elements, with a strong overlap in binding with OCT4, SOX2, and NANOG. Over a timecourse of SPT16 depletion, nucleosomes invade new loci, including promoters, regions bound by SPT16, OCT4, SOX2, and NANOG, and TSS-distal DNaseI hypersensitive sites. Simultaneously, transcription of Pou5f1 (encoding OCT4), Sox2, Nanog, and enhancer RNAs produced from these genes’ associated enhancers are downregulated.ConclusionsWe propose that FACT maintains cellular pluripotency through a precise nucleosome-based regulatory mechanism for appropriate expression of both coding and non-coding transcripts associated with pluripotency.

The HSV-1 ICP22 protein selectively impairs histone repositioning upon Pol II transcription downstream of genes

Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) when transcription termination is disrupted by the viral ICP27 protein. This is accompanied by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperone FACT is not sufficient to induce dOCRs in ΔICP22 infection but increases dOCR induction in wild-type HSV-1 infection. Interestingly, this is accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone repositioning downstream of genes in the wake of Pol II in HSV-1 infection.

Structure and Dynamics of Compact Dinucleosomes: Analysis by Electron Microscopy and spFRET.

Formation of compact dinucleosomes (CODIs) occurs after collision between adjacent nucleosomes at active regulatory DNA regions. Although CODIs are likely dynamic structures, their structural heterogeneity and dynamics were not systematically addressed. Here, single-particle Förster resonance energy transfer (spFRET) and electron microscopy were employed to study the structure and dynamics of CODIs. spFRET microscopy in solution and in gel revealed considerable uncoiling of nucleosomal DNA from the histone octamer in a fraction of CODIs, suggesting that at least one of the nucleosomes is destabilized in the presence of the adjacent closely positioned nucleosome. Accordingly, electron microscopy analysis suggests that up to 30 bp of nucleosomal DNA are involved in transient uncoiling/recoiling on the octamer. The more open and dynamic nucleosome structure in CODIs cannot be stabilized by histone chaperone Spt6. The data suggest that proper internucleosomal spacing is an important determinant of chromatin stability and support the possibility that CODIs could be intermediates of chromatin disruption.

Nucleosome retention by histone chaperones and remodelers occludes pervasive DNA-protein binding.

DNA packaging within chromatin depends on histone chaperones and remodelers that form and position nucleosomes. Cells express multiple such chromatin regulators with overlapping in-vitro activities. Defining specific in-vivo activities requires monitoring histone dynamics during regulator depletion, which has been technically challenging. We have recently generated histone-exchange sensors in Saccharomyces cerevisiae, which we now use to define the contributions of 15 regulators to histone dynamics genome-wide. While replication-independent exchange in unperturbed cells maps to promoters, regulator depletions primarily affected gene bodies. Depletion of Spt6, Spt16 or Chd1 sharply increased nucleosome replacement sequentially at the beginning, middle or end of highly expressed gene bodies. They further triggered re-localization of chaperones to affected gene body regions, which compensated for nucleosome loss during transcription complex passage, but concurred with extensive TF binding in gene bodies. We provide a unified quantitative screen highlighting regulator roles in retaining nucleosome binding during transcription and preserving genomic packaging.

Spontaneous histone exchange between nucleosomes

The nucleosome is the fundamental gene-packing unit in eukaryotes. Nucleosomes comprise ∼147bp DNA wrapped around an octameric histone protein core composed of two H2A-H2B dimers and one (H3-H4)2 tetramer. The strong yet flexible DNA-histone interactions are the physical basis of the dynamic regulation of genes packaged in chromatin. The dynamic nature of DNA-histone interactions also implies thatnucleosomes dissociate DNA-histone contacts both transiently and repeatedly. This kinetic instability may lead to spontaneous nucleosome disassembly or histone exchange between nucleosomes. At high nucleosome concentrations, nucleosome-nucleosome collisions and subsequent histone exchange would be a more likely event, where nucleosomes could act as their own histone chaperone. This spontaneous histone exchange could serve as a mechanism for maintaining overall chromatin stability, although it has never been reported. Here we employed three-color single-molecule FRET (smFRET) to demonstrate that histone H2A-H2B dimers are exchanged spontaneously between nucleosomes on a time scale of a few tens of seconds at a physiological nucleosome concentration. We show that the rate of histone exchange increases at a higher monovalent salt concentration, with histone-acetylated nucleosomes, and in the presence of histone chaperone Nap1, while it remains unchanged at a higher temperature, and decreases upon DNA methylation. These results support the notion of histone exchange via transient and repetitive partial disassembly of the nucleosome and corroborate spontaneous histone diffusion in a compact chromatin context, modulating the local concentrations of histone modifications and variants.