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.

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.

Clinical implication of megestrol acetate in metastatic gastric cancer: a big data analysis from Health Insurance Review and Assessment (HIRA) database.

OBJECTIVE: Megestrol acetate (MA) is used to manage anorexia and cachexia in patients with advanced cancer. This study investigated the prescription patterns of MA in patients with metastatic gastric cancer, as well as evaluated its impact on survival outcomes and the incidence of venous thromboembolism (VTE). METHODS: A Health Insurance Review and Assessment (HIRA) service database was used to investigate differences in baseline characteristics, survival, and the incidence of VTE according to MA prescription patterns (i.e., prescription vs. no prescription) in patients diagnosed with metastatic gastric cancer from July 2014 to December 2015. RESULTS: A total of 1938 patients were included in this study. In total, 65% of the patients were prescribed MA. Older age, treatment in tertiary hospitals, and palliative chemotherapy were statistically significant predictive factors for MA prescription. Continuous prescription of MA was observed in 37% of patients. There was no statistically significant difference in survival between the MA and non-MA prescription groups on multivariate analysis. Among the 1427 patients included in the analysis for VTE incidence, 4.3% and 2.9% were diagnosed with VTE during the follow-up period in the MA and non-MA prescription groups, respectively. However, there was no statistically significant difference in VTE diagnosis between the groups on multivariate analysis. CONCLUSION: MA is commonly prescribed for metastatic gastric cancer, especially in elderly patients and those undergoing palliative chemotherapy, without significantly affecting survival or VTE risk.

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.

Identification and Characterization of HIRIP3 as a Histone H2A Chaperone.

HIRIP3 is a mammalian protein homologous to the yeast H2A.Z deposition chaperone Chz1. However, the structural basis underlying Chz's binding preference for H2A.Z over H2A, as well as the mechanism through which Chz1 modulates histone deposition or replacement, remains enigmatic. In this study, we aimed to characterize the function of HIRIP3 and to identify its interacting partners in HeLa cells. Our findings reveal that HIRIP3 is specifically associated in vivo with H2A-H2B dimers and CK2 kinase. While bacterially expressed HIRIP3 exhibited a similar binding affinity towards H2A and H2A.Z, the associated CK2 kinase showed a notable preference for H2A phosphorylation at serine 1. The recombinant HIRIP3 physically interacted with the H2A αC helix through an extended CHZ domain and played a crucial role in depositing the canonical core histones onto naked DNA. Our results demonstrate that mammalian HIRIP3 acts as an H2A histone chaperone, assisting in its selective phosphorylation by Ck2 kinase at serine 1 and facilitating its deposition onto chromatin.

A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae reveals novel insights into factors that stabilize chromatin structure on newly replicated DNA.

The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.

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.

HIRA vs. DAXX: the two axes shaping the histone H3.3 landscape.

H3.3, the most common replacement variant for histone H3, has emerged as an important player in chromatin dynamics for controlling gene expression and genome integrity. While replicative variants H3.1 and H3.2 are primarily incorporated into nucleosomes during DNA synthesis, H3.3 is under the control of H3.3-specific histone chaperones for spatiotemporal incorporation throughout the cell cycle. Over the years, there has been progress in understanding the mechanisms by which H3.3 affects domain structure and function. Furthermore, H3.3 distribution and relative abundance profoundly impact cellular identity and plasticity during normal development and pathogenesis. Recurrent mutations in H3.3 and its chaperones have been identified in neoplastic transformation and developmental disorders, providing new insights into chromatin biology and disease. Here, we review recent findings emphasizing how two distinct histone chaperones, HIRA and DAXX, take part in the spatial and temporal distribution of H3.3 in different chromatin domains and ultimately achieve dynamic control of chromatin organization and function. Elucidating the H3.3 deposition pathways from the available histone pool will open new avenues for understanding the mechanisms by which H3.3 epigenetically regulates gene expression and its impact on cellular integrity and pathogenesis.

Regulation of replicative histone RNA metabolism by the histone chaperone ASF1.

In S phase, duplicating and assembling the whole genome into chromatin requires upregulation of replicative histone gene expression. Here, we explored how histone chaperones control histone production in human cells to ensure a proper link with chromatin assembly. Depletion of the ASF1 chaperone specifically decreases the pool of replicative histones both at the protein and RNA levels. The decrease in their overall expression, revealed by total RNA sequencing (RNA-seq), contrasted with the increase in nascent/newly synthesized RNAs observed by 4sU-labeled RNA-seq. Further inspection of replicative histone RNAs showed a 3' end processing defect with an increase of pre-mRNAs/unprocessed transcripts likely targeted to degradation. Collectively, these data argue for a production defect of replicative histone RNAs in ASF1-depleted cells. We discuss how this regulation of replicative histone RNA metabolism by ASF1 as a "chaperone checkpoint" fine-tunes the histone dosage to avoid unbalanced situations deleterious for cell survival.

A study on the 10-year trend of surgeries performed for lumbar disc herniation and comparative analysis of prescribed opioid analgesics and hospitalization duration: 2010-2019 HIRA NPS Data.

BACKGROUND: This study, utilizing the claims data from the Health Insurance Review and Assessment Service of Korea, aimed to examine the 10-year (2010-2019) trends in various types of lumbar spine surgeries performed on patients diagnosed with lumbar herniated intervertebral disc (HIVD), and the current status of opioid prescriptions, as well as the duration of postoperative hospital stays based on the type of surgery performed. METHOD: This retrospective cross-sectional study examined patients with one or more national health insurance claims carrying a primary or secondary diagnosis of HIVD (ICD-10 codes: M511, M518, M519) over a 10-year period (2010-2019). From the patients undergoing lumbar spine surgery, we selected those who did not require reoperation within 30 days following the initial lumbar surgery. Our final study sample comprised patients who underwent only one type of surgery. RESULTS: Among the patients diagnosed with HIVD and subsequently undergoing lumbar surgery between 2010 and 2019, a slight downward trend was observed in those undergoing open discectomy (OD); however, OD persistently accounted for the highest proportion over the 10 years. Percutaneous endoscopic lumbar discectomy (PELD) demonstrated a consistent upward trend from 2016 to 2018. When inspecting trends, we noted a consistent escalation over the decade in the postoperative opioid prescription rates of strong opioids (50.7% in 2010 to 77.8% in 2019) and tramadol (50.9% in 2010 to 76.8% in 2019). Analyzing these trends by surgery type, spinal fusion exhibited a slightly higher rate of opioid prescriptions than other lumbar surgeries. Regarding the length of postoperative hospital stays, patients undergoing PELD recorded the shortest stay (7.04 ± 6.78 days), while spinal fusion necessitated the longest (20.14 ± 12.18 days). CONCLUSION: This study analyzed the trends in types of lumbar spine surgeries, opioid analgesic prescriptions, and length of hospital stays over 10 years (2010-2019) among patients with HIVD in Korea. Our data and findings provide valuable evidence that may prove beneficial for clinicians and researchers involved in HIVD-related practices.

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.

Transcriptional regulation of FACT involves Coordination of chromatin accessibility and CTCF binding.

Histone chaperone FACT (facilitates chromatin transcription) is well known to promote chromatin recovery during transcription. However, the mechanism how FACT regulates genome-wide chromatin accessibility and transcription factor binding has not been fully elucidated. Through loss-of-function studies, we show here that FACT component Ssrp1 is required for DNA replication and DNA damage repair and is also essential for progression of cell phase transition and cell proliferation in mouse embryonic fibroblast cells. On the molecular level, absence of the Ssrp1 leads to increased chromatin accessibility, enhanced CTCF binding, and a remarkable change in dynamic range of gene expression. Our study thus unequivocally uncovers a unique mechanism by which FACT complex regulates transcription by coordinating genome-wide chromatin accessibility and CTCF binding.

Histone binding of ASF1 is required for fruiting body development but not for genome stability in the filamentous fungus Sordaria macrospora.

IMPORTANCE: Histone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication, and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone-binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the Δasf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the Δasf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.

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.

An Analysis of the Demographics and Clinical Characteristics of Transgender and Intersex Populations in Korea: A Retrospective Study Using HIRA Database.

BACKGROUND: Transgender and intersex populations have long remained under-documented in South Korea, largely due to the absence of comprehensive epidemiological data. With increasing societal acknowledgment, there's an urgent need to understand the demographics and health challenges faced by these communities. METHODS: This retrospective, large-scale data study included people who received the F64 codes from the Korean Health Insurance Review & Assessment Service between January 2007 and December 2021. Demographics, gender-affirmative treatments, and psychiatric related medications were examined. RESULTS: Between 2007 and 2021, 8,602 patients were diagnosed with "gender identity disorder" and 45 with "intersex." A steadily increasing annual prevalence was observed, peaking at 986 cases in 2021. The majority (79.8%) were aged between 10 and 30. Nearly half (53.2%) exhibited mental and behavioral disorders. Two-thirds had been prescribed anxiolytics or sedatives either before or after diagnosis. Merely 12.1% received hormone therapy covered by health insurance. CONCLUSION: This is the first large-scale study highlighting the demographics and clinical characteristics of the transgender and intersex populations in Korea. The study reveals a consistent growth of these communities over the past 15 years, with a significant proportion under 30 years of age facing mental and behavioral challenges. Findings underscore the need for targeted healthcare interventions, early psychological support, and comprehensive insurance coverage tailored to the specific needs of these individuals in Korea.

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.

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.

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.