Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.

The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.

Interplay between histone variants and chaperones in plants.

Histone chaperones and histone variants play crucial roles in DNA replication, gene transcription, and DNA repair in eukaryotes. Histone chaperones reversibly promote nucleosome assembly and disassembly by incorporating or evicting histones and histone variants to modulate chromatin accessibility, thereby altering the chromatin states and modulating DNA-related biological processes. Cofactors assist histone chaperones to target specific chromatin regions to regulate the exchange of histones and histone variants. In this review, we summarize recent progress in the interplay between histone variants and chaperones in plants. We discuss the structural basis of chaperone-histone complexes and the mechanisms of their cooperation in regulating gene transcription and plant development.

Loss of the histone chaperone UNC-85/ASF1 inhibits the epigenome-mediated longevity and modulates the activity of one-carbon metabolism.

Histone H3/H4 chaperone anti-silencing function 1 (ASF1) is a conserved factor mediating nucleosomal assembly and disassembly, playing crucial roles in processes such as replication, transcription, and DNA repair. Nevertheless, its involvement in aging has remained unclear. Here, we utilized the model organism Caenorhabditis elegans to demonstrate that the loss of UNC-85, the homolog of ASF1, leads to a shortened lifespan in a multicellular organism. Furthermore, we show that UNC-85 is required for epigenome-mediated longevity, as knockdown of the histone H3 lysine K4 methyltransferase ash-2 does not extend the lifespan of unc-85 mutants. In this context, we found that the longevity-promoting ash-2 RNA interference enhances UNC-85 activity by increasing its nuclear localization. Finally, our data indicate that the loss of UNC-85 increases the activity of one-carbon metabolism, and that downregulation of the one-carbon metabolism component dao-3/MTHFD2 partially rescues the short lifespan of unc-85 mutants. Together, these findings reveal UNC-85/ASF1 as a modulator of the central metabolic pathway and a factor regulating a pro-longevity response, thus shedding light on a mechanism of how nucleosomal maintenance associates with aging.

Interpreting the molecular mechanisms of RBBP4/7 and their roles in human diseases (Review).

Histone chaperones serve a pivotal role in maintaining human physiological processes. They interact with histones in a stable manner, ensuring the accurate and efficient execution of DNA replication, repair and transcription. Retinoblastoma binding protein (RBBP)4 and RBBP7 represent a crucial pair of histone chaperones, which not only govern the molecular behavior of histones H3 and H4, but also participate in the functions of several protein complexes, such as polycomb repressive complex 2 and nucleosome remodeling and deacetylase, thereby regulating the cell cycle, histone modifications, DNA damage and cell fate. A strong association has been indicated between RBBP4/7 and some major human diseases, such as cancer, age­related memory loss and infectious diseases. The present review assesses the molecular mechanisms of RBBP4/7 in regulating cellular biological processes, and focuses on the variations in RBBP4/7 expression and their potential mechanisms in various human diseases, thus providing new insights for their diagnosis and treatment.

Identification of ASF1A and HJURP by global H3-H4 histone chaperone analysis as a prognostic two-gene model in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignancy with poor prognosis. Abnormal expression of H3-H4 histone chaperones has been identified in many cancers and holds promise as a biomarker for diagnosis and prognosis. However, systemic analysis of H3-H4 histone chaperones in HCC is still lacking. Here, we investigated the expression of 19 known H3-H4 histone chaperones in HCC. Integrated analysis of multiple public databases indicated that these chaperones are highly expressed in HCC tumor tissues, which was further verified by immunohistochemistry (IHC) staining in offline samples. Additionally, survival analysis suggested that HCC patients with upregulated H3-H4 histone chaperones have poor prognosis. Using LASSO and Cox regression, we constructed a two-gene model (ASF1A, HJURP) that accurately predicts prognosis in ICGC-LIRI and GEO HCC data, which was further validated in HCC tissue microarrays with follow-up information. GSEA revealed that HCCs in the high-risk group were associated with enhanced cell cycle progression and DNA replication. Intriguingly, HCCs in the high-risk group exhibited increased immune infiltration and sensitivity to immune checkpoint therapy (ICT). In summary, H3-H4 histone chaperones play a critical role in HCC progression, and the two-gene (ASF1A, HJURP) risk model is effective for predicting survival outcomes and sensitivity to immunotherapy for HCC patients.

Set2 regulates Ccp1 and Swc2 to ensure centromeric stability by retargeting CENP-A.

Precise positioning of the histone-H3 variant, CENP-A, ensures centromere stability and faithful chromosomal segregation. Mislocalization of CENP-A to extra-centromeric loci results in aneuploidy and compromised cell viability associated with formation of ectopic kinetochores. The mechanism that retargets mislocalized CENP-A back to the centromere is unclarified. We show here that the downregulation of the histone H3 lysine 36 (H3K36) methyltransferase Set2 can preserve centromere localization of a temperature-sensitive mutant cnp1-1 Schizosaccharomyces pombe CENP-A (SpCENP-A) protein and reverse aneuploidy by redirecting mislocalized SpCENP-A back to centromere from ribosomal DNA (rDNA) loci, which serves as a sink for the delocalized SpCENP-A. Downregulation of set2 augments Swc2 (SWR1 complex DNA-binding module) expression and releases histone chaperone Ccp1 from the centromeric reservoir. Swc2 and Ccp1 are directed to the rDNA locus to excavate the SpCENP-Acnp1-1, which is relocalized to the centromere in a manner dependent on canonical SpCENP-A loaders, including Mis16, Mis17 and Mis18, thereby conferring cell survival and safeguarding chromosome segregation fidelity. Chromosome missegregation is a severe genetic instability event that compromises cell viability. This mechanism thus promotes CENP-A presence at the centromere to maintain genomic stability.

Coordination of histone chaperones for parental histone segregation and epigenetic inheritance.

Chromatin-based epigenetic memory relies on the accurate distribution of parental histone H3-H4 tetramers to newly replicated DNA strands. Mcm2, a subunit of the replicative helicase, and Dpb3/4, subunits of DNA polymerase ε, govern parental histone H3-H4 deposition to the lagging and leading strands, respectively. However, their contribution to epigenetic inheritance remains controversial. Here, using fission yeast heterochromatin inheritance systems that eliminate interference from initiation pathways, we show that a Mcm2 histone binding mutation severely disrupts heterochromatin inheritance, while mutations in Dpb3/4 cause only moderate defects. Surprisingly, simultaneous mutations of Mcm2 and Dpb3/4 stabilize heterochromatin inheritance. eSPAN (enrichment and sequencing of protein-associated nascent DNA) analyses confirmed the conservation of Mcm2 and Dpb3/4 functions in parental histone H3-H4 segregation, with their combined absence showing a more symmetric distribution of parental histone H3-H4 than either single mutation alone. Furthermore, the FACT histone chaperone regulates parental histone transfer to both strands and collaborates with Mcm2 and Dpb3/4 to maintain parental histone H3-H4 density and faithful heterochromatin inheritance. These results underscore the importance of both symmetric distribution of parental histones and their density at daughter strands for epigenetic inheritance and unveil distinctive properties of parental histone chaperones during DNA replication.

Activation of the FOXM1/ASF1B/PRDX3 axis confers hyperproliferative and antioxidative stress reactivity to gastric cancer.

Nucleosome assembly during DNA replication is dependent on histone chaperones. Recent studies suggest that dysregulated histone chaperones contribute to cancer progression, including gastric cancer (GC). Further studies are required to explore the prognostic and therapeutic implications of histone chaperones and their mechanisms of action in GC progression. Here we identified histone chaperone ASF1B as a potential biomarker for GC proliferation and prognosis. ASF1B was significantly upregulated in GC, which was associated with poor prognosis. In vitro and in vivo experiments demonstrated that the inhibition of ASF1B suppressed the malignant characteristics of GC, while overexpression of ASF1B had the opposite effect. Mechanistically, transcription factor FOXM1 directly bound to the ASF1B-promoter region, thereby regulating its transcription. Treatment with thiostrepton, a FOXM1 inhibitor, not only suppressed ASF1B expression, but also inhibited GC progression. Furthermore, ASF1B regulated the mitochondrial protein peroxiredoxin 3 (PRDX3) transcription in a FOXM1-dependent manner. The crucial role of ASF1B-regulated PRDX3 in GC cell proliferation and oxidative stress balance was also elucidated. In summary, our study suggests that the FOXM1-ASF1B-PRDX3 axis is a potential therapeutic target for treating GC.

The histone chaperone SPT2 regulates chromatin structure and function in Metazoa.

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

Intrinsic disorder of a nucleoplasmin-like histone chaperone specifies its discrete nuclear and nucleolar functions.

Nucleoplasmin (NPM) histone chaperones regulate distinct processes in the nucleus and nucleolus. While intrinsically disordered regions (IDRs) are hallmarks of NPMs, it is not clear whether all NPM functions require these unstructured features. We assessed the importance of IDRs in a yeast NPM-like protein and found that regulation of rDNA copy number and genetic interactions with the nucleolar RNA surveillance machinery require the highly conserved FKBP prolyl isomerase domain, but not the NPM domain or IDRs. By contrast, transcriptional repression in the nucleus requires IDRs. Furthermore, multiple lysines in polyacidic serine/lysine motifs of IDRs are required for both lysine polyphosphorylation and NPM-mediated transcriptional repression. These results demonstrate that this NPM-like protein relies on IDRs only for some of its chromatin-related functions.

PP2A inhibitor SET promotes mTORC1 and Bmi1 signaling through Akt activation and maintains the colony-formation ability of cancer cells.

Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colony-formation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling.

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

BACKGROUND: Classical 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. RESULTS: We 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. CONCLUSIONS: There 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 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 in vitro binds to histones with low nanomolar affinity. In vitro 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.

Energy landscape quantifications of histone H3.3 recognition by chaperone DAXX reveal an uncoupled binding specificity and affinity.

Histone variant H3.3 differs from the canonical histone H3.1 by only five amino acids, yet its chaperone death domain-associated protein (DAXX) can specifically recognize H3.3 over H3.1, despite having a large DAXX-interacting surface on the H3.3-H4 heterodimer common to that on the H3.1-H4 complex. This observation gives rise to the question of, from the binding energy point view, how high binding specificity may be achieved with small differences of the overall binding energy for protein-protein interactions in general. Here we investigate the mechanism of coupling of binding specificity and affinity in protein-protein interactions using the DAXX-H3.3-H4 complex as a model. Using a multi-scale method, we found that the hydrophobic interactions between DAXX and the H3.3-specific region contributed to their initial binding process. And the structural flexibility of the interacting partners contributed to the binding affinity after their encounter. By quantifying the free energy landscape, we revealed that the interaction between the specific residues of H3.3 and DAXX decreased the encounter barrier height while the folding of H3.3-H4 and DAXX increased the depth of the free energy basin of the final binding state. The encounter barrier height, which is not coupled to the thermodynamic stability of the final binding state, had a marked effect on the initial binding rate of flexible histones and chaperones. Based on the energy landscape theory, we found that the intrinsic binding energy funnel of this uncoupled recognition process was affected by the structural flexibility and the flexibility modulated the degree of coupling between binding specificity and affinity. Our work offers a biophysical explanation of the specific recognition between the histones and their chaperones, and also extends the use of energy landscape theory for understanding molecular recognitions in general.

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/H4 and 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.

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.

The Histone Chaperones SET/TAF-1ß 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-Iß 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.

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

Insights into Spt6: a histone chaperone that functions in transcription, DNA replication, and genome stability.

Transcription elongation requires elaborate coordination between the transcriptional machinery and chromatin regulatory factors to successfully produce RNA while preserving the epigenetic landscape. Recent structural and genomic studies have highlighted that suppressor of Ty 6 (Spt6), a conserved histone chaperone and transcription elongation factor, sits at the crux of the transcription elongation process. Other recent studies have revealed that Spt6 also promotes DNA replication and genome integrity. Here, we review recent studies of Spt6 that have provided new insights into the mechanisms by which Spt6 controls transcription and have revealed the breadth of Spt6 functions in eukaryotic cells.

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.