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Spn1/Iws1 is an essential eukaryotic transcription elongation factor that is conserved from yeast to humans as an integral member of the RNA polymerase II elongation complex. Several studies have shown that Spn1 functions as a histone chaperone to control transcription, RNA splicing, genome stability, and histone modifications. However, the precise role of Spn1 is not understood, and there is little understanding of why it is essential for viability. To address these issues, we have isolated 8 suppressor mutations that bypass the essential requirement for Spn1 in Saccharomyces cerevisiae. Unexpectedly, the suppressors identify several functionally distinct complexes and activities, including the histone chaperone FACT, the histone methyltransferase Set2, the Rpd3S histone deacetylase complex, the histone acetyltransferase Rtt109, the nucleosome remodeler Chd1, and a member of the SAGA coactivator complex, Sgf73. The identification of these distinct groups suggests that there are multiple ways in which Spn1 bypass can occur, including changes in histone acetylation and alterations in other histone chaperones. Thus, Spn1 may function to overcome repressive chromatin by multiple mechanisms during transcription. Our results suggest that bypassing a subset of these functions allows viability in the absence of Spn1., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Yeast Hmo1 is a high mobility group B (HMGB) protein that participates in the transcription of ribosomal protein genes and rDNA, and also stimulates the activities of some ATP-dependent remodelers. Hmo1 binds both DNA and nucleosomes and has been proposed to be a functional yeast analog of mammalian linker histones. We used EMSA and single particle Förster resonance energy transfer (spFRET) microscopy to characterize the effects of Hmo1 on nucleosomes alone and with the histone chaperone FACT. Hmo1 induced a significant increase in the distance between the DNA gyres across the nucleosomal core, and also caused the separation of linker segments. This was opposite to the effect of the linker histone H1, which enhanced the proximity of linkers. Similar to Nhp6, another HMGB factor, Hmo1, was able to support large-scale, ATP-independent, reversible unfolding of nucleosomes by FACT in the spFRET assay and partially support FACT function in vivo. However, unlike Hmo1, Nhp6 alone does not affect nucleosome structure. These results suggest physiological roles for Hmo1 that are distinct from Nhp6 and possibly from other HMGB factors and linker histones, such as H1.

The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly., (© 2022 Jiang et al.)

DNA and core histones are hierarchically packaged into a complex organization called chromatin. The nucleosome assembly protein (NAP) family of histone chaperones is involved in the deposition of histone complexes H2A/H2B and H3/H4 onto DNA and prevents nonspecific aggregation of histones. Testis-specific Y-encoded protein (TSPY)-like protein 5 (TSPYL5) is a member of the TSPY-like protein family, which has been previously reported to interact with ubiquitin-specific protease USP7 and regulate cell proliferation and is thus implicated in various cancers, but its interaction with chromatin has not been investigated. In this study, we characterized the chromatin association of TSPYL5 and found that it preferentially binds histone H3/H4 via its C-terminal NAP-like domain both in vitro and ex vivo. We identified the critical residues involved in the TSPYL5-H3/H4 interaction and further quantified the binding affinity of TSPYL5 toward H3/H4 using biolayer interferometry. We then determined the binding stoichiometry of the TSPYL5-H3/H4 complex in vitro using a chemical cross-linking assay and size-exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that a TSPYL5 dimer binds to either two histone H3/H4 dimers or a single tetramer. We further demonstrated that TSPYL5 has a specific affinity toward longer DNA fragments and that the same histone-binding residues are also critically involved in its DNA binding. Finally, employing histone deposition and supercoiling assays, we confirmed that TSPYL5 is a histone chaperone responsible for histone H3/H4 deposition and nucleosome assembly. We conclude that TSPYL5 is likely a new member of the NAP histone chaperone family., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) remains a major public health concern, associated with a high rate of morbidity and mortality. Several factors have been implicated in its occurrence and development, which includes histone chaperones. The role of spty2d1 (spt2)-a novel histone chaperone protein-has rarely been investigated in CRC. Therefore, we demonstrated in this study that spt2 undergoes different genetic alterations in colorectal adenocarcinoma datasets and that it was associated with the proliferation of colon carcinoma. Spt2 silencing can reduce the ability of proliferation and increase the rate of apoptosis of LoVo cells. Regarding the overall survival associated with spt2, only the quartile disease-free survival of colon adenocarcinoma (COAD) was found to be statistically significant, while that of rectum adenocarcinoma (READ) was not. The positive (+++) expression of spt2 was correlated with a deeper invasion depth in colorectal adenocarcinoma, and this effect was more pronounced in COAD. These data collectively suggest that spt2 can influence the progression and prognosis in some subtypes of colorectal adenocarcinoma. Therefore, we propose spt2 as a potential target for application in enhancing the overall therapeutic efficacy in some specific subtypes of colorectal adenocarcinoma., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The SET-CAN/NUP214 fusion gene resulting from chromosomal del(9)(q34.11q34.13) or t(9;9) (q34;q34) has been found in T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML) and myeloid sarcoma (MS). Furthermore, the SET-CAN/NUP214 fusion gene has been found in the T-ALL cell line LOUCY and the AML line MEGAL. The common features of these cases are insensitivity to chemotherapy and poor prognosis. We reviewed the characteristics and prognostic significance of the SET-CAN/NUP214 fusion gene in hematological malignancies., Methods: This systematic literature search was conducted using the PubMed, Web of Science, Embase, and Cochrane Library databases. With the inclusion and exclusion criteria, we summarized all of the papers and performed a statistical analyses., Results: In general, the SET-CAN/NUP214 fusion gene is very rare in adult acute leukemia, more frequently found in T-ALL than in other types of leukemia, and more often in males. Flow cytometry data indicated that the markers CD34, CD33, CD13, and CD7 were common in SET-CAN/NUP214 positive acute leukemia, including ALL. Fluorescence in situ hybridization and arrays are important methods for detecting the fusion gene in newly diagnosed patients and can detect chromosomal del(9)(q34) will be detected. The chromosomal karyotype may be normal or complex, and, in terms of survival analysis, transplantation results in a better prognosis than chemotherapy alone., Conclusions and Implications of Key Findings: The presence of SET-CAN/NUP214 fusion gene may be a Minimal Residual Disease of early recurrence, and it might be a poor indicator of outcome., Limitations: The mechanism, clinical characteristics, therapy and prognosis of the SET-CAN/NUP214 fusion gene in hematological malignancies require further research., Competing Interests: The authors of this work have no conflict of interest to declare., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLF AD ) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLF AD -histone octamer complex shows that APLF AD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLF AD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted.

Hepatocellular carcinoma (HCC) is the most prevalent primary cancer of the liver and it is the fourth most common cause of cancer related death worldwide. In Egypt, liver cancer constitutes the most common cause of mortality-related cancer. This study aimed to evaluate the immunohistochemical expression of SET oncoprotein in HCC tissues in comparison with its expression in non tumorous liver tissues and to correlate its expression with clinicopathological parameters. This study investigated 100 cases of HCC (including tumorous and non tumorous tissues). One hundred percent of tumorous and non-tumorous tissues were positive for SET expression. The mean and median values of H -score for SET expression were higher in tumorous than non tumorous tissues ( P = .03). Higher SET expression was significantly correlated with larger tumor size ( P = .012), positive lymphovascular invasion ( P = .028), and shorter overall survival ( P < .001). SET expression in tumor tissues is the most independent factor to affect the overall survival of HCC patients. SET plays a role in hepatocarcinogenesis proved by the increase of SET expression from non-tumorous to tumorous tissues. Also, SET can be used as a prognostic indicator and a novel target therapy in HCC patients.

Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6's function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Oncohistone mutations are crucial drivers for tumorigenesis, but how a living organism governs the loss-of-function oncohistone remains unclear. We generated a histone H2B triple knockout (3KO) strain in Caenorhabditis elegans, which decreased the embryonic H2B, disrupted cell divisions, and caused animal sterility. By performing genetic suppressor screens, we uncovered that mutations defective in the histone H3-H4 chaperone UNC-85 restored H2B 3KO fertility by decreasing chromatin H3-H4 levels. RNA interference of other H3-H4 chaperones or H3 or H4 histones also rescued H2B 3KO sterility. We showed that blocking H3-H4 chaperones recovered cell division in C. elegans carrying the oncohistone H2BE74K mutation that distorts the H2B-H4 interface and induces nucleosome instability. Our results indicate that reducing chromatin H3-H4 rescues the dysfunctional H2B in vivo and suggest that inhibiting H3-H4 chaperones may provide an effective therapeutic strategy for treating cancers resulting from loss-of-function H2B oncohistone., Competing Interests: The authors have declared that no competing interests exist.

Replication timing regulatory factor 1 (RIF1) acts downstream of p53-binding protein 53BP1 to inhibit the resection of DNA broken ends, which plays critical roles in determining the DNA double-strand break repair pathway choice between nonhomologous end joining and homologous recombination (HR). However, the mechanism by which this choice is made is not yet clear. In this study, we identified that histone chaperone protein ASF1 associates with RIF1 and regulates RIF1-dependent functions in the DNA damage response. Similar to loss of RIF1, we found that loss of ASF1 resulted in resistance to poly (ADP-ribose) polymerase (PARP) inhibition in BRCA1-deficient cells with restored HR and decreased telomere fusion in telomeric repeat-binding protein 2 (TRF2)-depleted cells. Moreover, we showed that these functions of ASF1 are dependent on its interaction with RIF1 but not on its histone chaperone activity. Thus, our study supports a new role for ASF1 in dictating double-strand break repair choice. Considering that the status of 53BP1-RIF1 axis is important in determining the outcome of PARP inhibitor-based therapy in BRCA1- or HR-deficient cancers, the identification of ASF1 function in this critical pathway uncovers an interesting connection between these S-phase events, which may reveal new strategies to overcome PARP inhibitor resistance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The lack of a consensus DNA sequence defining replication origins in mammals has led researchers to consider chromatin as a means to specify these regions. However, to date, there is no mechanistic understanding of how this could be achieved and maintained given that nucleosome disruption occurs with each fork passage and with transcription. Here, by genome-wide mapping of the de novo deposition of the histone variants H3.1 and H3.3 in human cells during S phase, we identified how their dual deposition mode ensures a stable marking with H3.3 flanked on both sides by H3.1. These H3.1/H3.3 boundaries correspond to the initiation zones of early origins. Loss of the H3.3 chaperone HIRA leads to the concomitant disruption of H3.1/H3.3 boundaries and initiation zones. We propose that the HIRA-dependent deposition of H3.3 preserves H3.1/H3.3 boundaries by protecting them from H3.1 invasion linked to fork progression, contributing to a chromatin-based definition of early replication zones., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Eukaryotic genomes are pervasively transcribed by RNA polymerase II (RNAPII), and transcription of long non-coding RNAs often overlaps with coding gene promoters. This might lead to coding gene repression in a process named Transcription Interference (TI). In Saccharomyces cerevisiae, TI is mainly driven by antisense non-coding transcription and occurs through re-shaping of promoter Nucleosome-Depleted Regions (NDRs). In this study, we developed a genetic screen to identify new players involved in Antisense-Mediated Transcription Interference (AMTI). Among the candidates, we found the HIR histone chaperone complex known to be involved in de novo histone deposition. Using genome-wide approaches, we reveal that HIR-dependent histone deposition represses the promoters of SAGA-dependent genes via antisense non-coding transcription. However, while antisense transcription is enriched at promoters of SAGA-dependent genes, this feature is not sufficient to define the mode of gene regulation. We further show that the balance between HIR-dependent nucleosome incorporation and transcription factor binding at promoters directs transcription into a SAGA- or TFIID-dependent regulation. This study sheds light on a new connection between antisense non-coding transcription and the nature of coding transcription initiation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Pleiotropic Drug Resistance (PDR) network is central to the drug response in fungi, and its overactivation is associated with drug resistance. However, gene regulation of the PDR network is not well understood. Here, we show that the histone chaperone Rtt106 and the chromatin remodeller SWI/SNF control expression of the PDR network genes and confer drug resistance. In Saccharomyces cerevisiae, Rtt106 specifically localises to PDR network gene promoters dependent on transcription factor Pdr3, but not Pdr1, and is essential for Pdr3-mediated basal expression of the PDR network genes, while SWI/SNF is essential for both basal and drug-induced expression. Also in the pathogenic fungus Candida glabrata, Rtt106 and SWI/SNF regulate drug-induced PDR gene expression. Consistently, loss of Rtt106 or SWI/SNF sensitises drug-resistant S. cerevisiae mutants and C. glabrata to antifungal drugs. Since they cooperatively drive PDR network gene expression, Rtt106 and SWI/SNF represent potential therapeutic targets to combat antifungal resistance., (© 2022. The Author(s).)

Histone chaperones are key regulators of chromatin structure and function. Their frequent mis-regulation in various cancers can impact tumor initiation and progression. Here, we focus on H3-H4 histone chaperones to highlight recent studies concerning their roles in several cancers thereby expanding on previous reports illustrating their functions as tumor-promoting and/or as useful biomarkers for clinical applications. In particular, we discuss how imperfect compensation between H3-H4 histone chaperones favors tumor progression by stimulating the Epithelial mesenchymal transition (EMT) or the Alternative lengthening of telomeres (ALT) pathway. Finally, we present initial studies pointing towards therapies that target H3-H4 histone chaperones for cancer treatment., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Histone cell cycle regulator (HIRA) complex is an important histone chaperone that mediates the deposition of the H3.3 histone variant onto chromatin independently from DNA synthesis. However, it is still unknown whether it participates in the expression control of retrotransposons and cell fate determination., Methods: We screened the role of HIRA complex members in repressing the expression of retrotransposons by shRNA depletion in embryonic stem cells (ESCs) followed by RT-qPCR. RNA-seq was used to study the expression profiles after depletion of individual HIRA member. RT-qPCR and western blot were used to determine overexpression of HIRA complex members. Chromatin immunoprecipitation (ChIP)-qPCR was used to find the binding of H3.3, HIRA members to chromatin. Co-immunoprecipitation was used to identify the interaction between Hira mutant and Ubn2. ChIP-qPCR was used to identify H3.3 deposition change and western blot of chromatin extract was used to validate the epigenetic change. Bioinformatics analysis was applied for the analysis of available ChIP-seq data., Results: We revealed that Hira, Ubn2, and Ubn1 were the main repressors of 2-cell marker retrotransposon MERVL among HIRA complex members. Surprisingly, Ubn2 and Hira targeted different groups of retrotransposons and retrotransposon-derived long noncoding RNAs (lncRNAs), despite that they partially shared target genes. Furthermore, Ubn2 prevented ESCs to gain a 2-cell like state or activate trophectodermal genes upon differentiation. Mechanistically, Ubn2 and Hira suppressed retrotransposons by regulating the deposition of histone H3.3. Decreased H3.3 deposition, that was associated with the loss of Ubn2 or Hira, caused the reduction of H3K9me2 and H3K9me3, which are known repressive marks of retrotransposons., Conclusions: Overall, our findings shed light on the distinct roles of HIRA complex members in controlling retrotransposons and cell fate conversion in ESCs., (© 2022. The Author(s).)

Successful reproduction requires an oocyte competent to sustain early embryo development. By the end of oogenesis, the oocyte has entered a transcriptionally silenced state, the mechanisms and significance of which remain poorly understood. Histone H3.3, a histone H3 variant, has unique cell cycle-independent functions in chromatin structure and gene expression. Here, we have characterised the H3.3 chaperone Hira/Cabin1/Ubn1 complex, showing that loss of function of any of these subunits causes early embryogenesis failure in mouse. Transcriptome and nascent RNA analyses revealed that transcription is aberrantly silenced in mutant oocytes. Histone marks, including H3K4me3 and H3K9me3, are reduced and chromatin accessibility is impaired in Hira/Cabin1 mutants. Misregulated genes in mutant oocytes include Zscan4d, a two-cell specific gene involved in zygote genome activation. Overexpression of Zscan4 in the oocyte partially recapitulates the phenotypes of Hira mutants and Zscan4 knockdown in Cabin1 mutant oocytes partially restored their developmental potential, illustrating that temporal and spatial expression of Zscan4 is fine-tuned at the oocyte-to-embryo transition. Thus, the H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and embryogenesis, through modulating chromatin condensation and transcriptional quiescence., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

SETD2 is a histone H3 lysine 36 (H3K36) trimethyltransferase that is mutated with high prevalence (13%) in clear cell renal cell carcinoma (ccRCC). Genomic profiling of primary ccRCC tumors reveals a positive correlation between SETD2 mutations and metastasis. However, whether and how SETD2 loss promotes metastasis remains unclear. In this study, we used a SETD2-mutant (SETD2 MT ) metastatic ccRCC human-derived cell line and xenograft models and showed that H3K36me3 restoration greatly reduced distant metastases of ccRCC in mice in a matrix metalloproteinase 1 (MMP1)-dependent manner. An integrated multiomics analysis using assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) established a tumor suppressor model in which loss of SETD2-mediated H3K36me3 activates enhancers to drive oncogenic transcriptional output through regulation of chromatin accessibility. Furthermore, we uncovered mechanism-based therapeutic strategies for SETD2-deficient cancer through the targeting of specific histone chaperone complexes, including ASF1A/ASF1B and SPT16. Overall, SETD2 loss creates a permissive epigenetic landscape for cooperating oncogenic drivers to amplify transcriptional output, providing unique therapeutic opportunities., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The identification of FACT as a histone chaperone enabling transcription through chromatin in vitro has strongly shaped how its roles are envisioned. However, FACT has been implicated in essentially all aspects of chromatin biology, from transcription to DNA replication, DNA repair, and chromosome segregation. In this review, we focus on recent literature describing the role and mechanisms of FACT during transcription. We highlight the prime importance of FACT in preserving chromatin integrity during transcription and challenge its role as an elongation factor. We also review evidence for FACT's role as a cell-type/gene-specific regulator of gene expression and briefly summarize current efforts at using FACT inhibition as an anti-cancer strategy.

The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in the linker between the catalytic core and the C-terminal domain (CTD) heptad repeats. This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in a distinct pattern of Spt6 accumulation at these sites. Splicing efficiency was also diminished, as was apparent progression through introns that encode snoRNAs. Chromatin-mediated repression was impaired, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the interaction modulates the availability or activity of the core elongation and histone chaperone functions of Spt6, contributing to coordination between RNAPII and its accessory factors as varying local conditions call for dynamic responses., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Histone modification and nucleosome assembly are mainly regulated by various histone-modifying enzymes and chaperones. The roles of histone-modification enzymes have been well analyzed, but the molecular mechanism of histone chaperones in histone modification and nucleosome assembly is incompletely understood. We previously found that the histone chaperone Nrp1 is localized in the micronucleus (MIC) and the macronucleus (MAC) and involved in the chromatin stability and nuclear division of Tetrahymena thermophila . In the present work, we found that truncated C-terminal mutant HA-Nrp1 TrC abnormally localizes in the cytoplasm. The truncated-signal-peptide mutants HA-Nrp1 TrNLS1 and HA-Nrp1 TrNLS2 are localized in the MIC and MAC. Overexpression of Nrp1 TrNLS1 inhibited cellular proliferation and disrupted micronuclear mitosis during the vegetative growth stage. During sexual development, Nrp1 TrNLS1 overexpression led to abnormal bouquet structures and meiosis arrest. Furthermore, Histone H3 was not transported into the nucleus; instead, it formed an abnormal speckled cytoplastic distribution in the Nrp1 TrNLS1 mutants. The acetylation level of H3K56 in the mutants also decreased, leading to significant changes in the transcription of the genome of the Nrp1 TrNLS1 mutants. The histone chaperone Nrp1 regulates the H3 nuclear import and acetylation modification of H3K56 and affects chromatin stability and genome transcription in Tetrahymena .

Background & Aims: Upon hepatitis B virus (HBV) infection, partially double-stranded viral DNA converts into a covalently closed circular chromatinized episomal structure (cccDNA). This form represents the long-lived genomic reservoir responsible for viral persistence in the infected liver. Although the involvement of host cell DNA damage response in cccDNA formation has been established, this work investigated the yet-to-be-identified histone dynamics on cccDNA during early phases of infection in human hepatocytes., Methods: Detailed studies of host chromatin-associated factors were performed in cell culture models of natural infection (ie, Na + -taurocholate cotransporting polypeptide (NTCP)-overexpressing HepG2 cells, HepG2 hNTCP ) and primary human hepatocytes infected with HBV, by cccDNA-specific chromatin immunoprecipitation and loss-of-function experiments during early kinetics of viral minichromosome establishment and onset of viral transcription., Results: Our results show that cccDNA formation requires the deposition of the histone variant H3.3 via the histone regulator A (HIRA)-dependent pathway. This occurs simultaneously with repair of the cccDNA precursor and independently from de novo viral protein expression. Moreover, H3.3 in its S31 phosphorylated form appears to be the preferential H3 variant found on transcriptionally active cccDNA in infected cultured cells and human livers. HIRA depletion after cccDNA pool establishment showed that HIRA recruitment is required for viral transcription and RNA production., Conclusions: Altogether, we show a crucial role for HIRA in the interplay between HBV genome and host cellular machinery to ensure the formation and active transcription of the viral minichromosome in infected hepatocytes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromatin alterations brought by histone variants and modifications potentially regulate gene transcription from tumor initiation to progression. Histone H3.3 variant is one such epigenetic player important for disease progression and development. Though many studies have implicated H3.3 role in cancer progression and metastasis, its regulation, importance of specific modifications and chaperones have been not understood yet. We report DNA methylation mediated downregulation of histone H3 variant H3.3 in HCC and a concomitant increase in the level of the H3.2 variant. The loss of H3.3 in cancer tissues correlates with a decrease in the histone modifications associated with active transcription like H3K9/K14/K27Ac and H3K4Me3. The ectopic overexpression of H3.3 and H3.2 did not affect global PTMs and cell physiology, probably owing to the deregulation of specific histone chaperones CAF-1 (for H3.2) and HIRA (for H3.3) as observed in HCC tissues. Notably, knockdown of P150, a subunit of CAF-1 leads to a cell cycle arrest in S-phase in a neoplastic rat liver cell line, possibly due to the decrease in the histone levels necessary for DNA packaging. Remarkably, modulation of H3.3 in pre-neoplastic rat liver cells lead to an increase in cell proliferation and a decreased transcription of tumor suppressor genes, recapitulating the tumor cell phenotype. Our data suggests, inhibition of DNA methylation and histone deacetylation leads to the restoration of histone H3 variant expression in tumor cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

Histone chaperoning ensures genomic integrity during routine processes such as DNA replication and transcription as well as DNA repair upon damage. Here, we identify a nuclear J domain protein, Dnj4, in the fungal pathogen Cryptococcus neoformans and demonstrate that it interacts with histones 3 and 4, suggesting a role as a histone chaperone. In support of this idea, a dnj4Δ deletion mutant had elevated levels of DNA damage and was hypersensitive to DNA-damaging agents. The transcriptional response to DNA damage was also impaired in the dnj4Δ mutant. Genes related to DNA damage and iron homeostasis were upregulated in the wild-type strain in response to hydroxyurea treatment; however, their upregulation was either absent from or reduced in the dnj4Δ mutant. Accordingly, excess iron rescued the mutant's growth in response to DNA-damaging agents. Iron homeostasis is crucial for virulence in C. neoformans; however, Dnj4 was found to be dispensable for disease in a mouse model of cryptococcosis. Finally, we confirmed a conserved role for Dnj4 as a histone chaperone by expressing it in Saccharomyces cerevisiae and showing that it disrupted endogenous histone chaperoning. Altogether, this study highlights the importance of a JDP cochaperone in maintaining genome integrity in C. neoformans. IMPORTANCE DNA replication, gene expression, and genomic repair all require precise coordination of the many proteins that interact with DNA. This includes the histones as well as their chaperones. In this study, we show that a histone chaperone, Dnj4, is required for genome integrity and for the response to DNA damage. The gene encoding this protein in Cryptococcus neoformans lacks an ortholog in Saccharomyces cerevisiae; however, it is conserved in humans in which its ortholog is essential. Since it is not essential in C. neoformans, we were able to generate deletion mutants to characterize the roles of Dnj4. We also expressed Dnj4 in S. cerevisiae, in which it was able to bind S. cerevisiae histones and interfere with existing histone chaperoning machinery. Therefore, we show a conserved role for Dnj4 in histone chaperoning that suggests that C. neoformans is useful to better understand aspects of this important biological process.

The H3.3 histone variant and its chaperone HIRA are involved in active transcription, but their detailed roles in regulating somatic hypermutation (SHM) of immunoglobulin variable regions in human B cells are not yet fully understood. In this study, we show that the knockout (KO) of HIRA significantly decreased SHM and changed the mutation pattern of the variable region of the immunoglobulin heavy chain (IgH) in the human Ramos B cell line without changing the levels of activation-induced deaminase and other major proteins known to be involved in SHM. Except for H3K79me2/3 and Spt5, many factors related to active transcription, including H3.3, were substantively decreased in HIRA KO cells, and this was accompanied by decreased nascent transcription in the IgH locus. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the IgH locus was also reduced in the HIRA KO. Somewhat surprisingly, HIRA loss increased the chromatin accessibility of the IgH V region locus. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit both the trimethylation of H3.3K36 and the recruitment of ZMYND11 significantly reduced SHM in Ramos cells, while the H3.3K79M did not. Consistent with the HIRA KO, the H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on the IgH variable and downstream switching regions. Our results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and extend our knowledge of how transcription-associated chromatin structure and accessibility contribute to SHM in human B cells., Competing Interests: The authors declare no competing interest.

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The ATRX ATP-dependent chromatin remodelling/helicase protein associates with the DAXX histone chaperone to deposit histone H3.3 over repetitive DNA regions. Because ATRX-protein interactions impart functions, such as histone deposition, we used proximity-dependent biotinylation (BioID) to identify proximal associations for ATRX. The proteomic screen captured known interactors, such as DAXX, NBS1, and PML, but also identified a range of new associating proteins. To gauge the scope of their roles, we examined three novel ATRX-associating proteins that likely differed in function, and for which little data were available. We found CCDC71 to associate with ATRX, but also HP1 and NAP1, suggesting a role in chromatin maintenance. Contrastingly, FAM207A associated with proteins involved in ribosome biosynthesis and localized to the nucleolus. ATRX proximal associations with the SLF2 DNA damage response factor help inhibit telomere exchanges. We further screened for the proteomic changes at telomeres when ATRX, SLF2, or both proteins were deleted. The loss caused important changes in the abundance of chromatin remodelling, DNA replication, and DNA repair factors at telomeres. Interestingly, several of these have previously been implicated in alternative lengthening of telomeres. Altogether, this study expands the repertoire of ATRX-associating proteins and functions., Competing Interests: The authors have declared that no competing interests exist.

Background: MicroRNAs (miRNAs) have been reported to play significant roles in non-small-cell lung cancer (NSCLC). However, the roles of microRNA (miR)-1915-3p in NSCLC remain unclear. In this study, we aimed to explore the biological functions of miR-1915-3p in NSCLC., Methods: The expression of miR-1915-3p and SET nuclear proto-oncogene (SET) in NSCLC tissues were examined by quantitative real-time PCR (qRT-PCR). Migratory and invasive abilities of lung cancer were tested by wound healing and transwell invasion assay. The direct target genes of miR-1915-3p were measured by dual-luciferase reporter assay and western blot. Finally, the regulation between METTL3/YTHDF2/KLF4 axis and miR-1915-3p were evaluated by qRT-PCR, promoter reporter assay and chromatin immunoprecipitation (CHIP)., Results: miR-1915-3p was downregulated in NSCLC tissues and cell lines, and inversely associated with clinical TNM stage and overall survival. Functional assays showed that miR-1915-3p significantly suppressed migration, invasion and epithelial-mesenchymal transition (EMT) in NSCLC cells. Furthermore, miR-1915-3p directly bound to the 3'untranslated region (3'UTR) of SET and modulated the expression of SET. SET inhibition could recapitulate the inhibitory effects on cell migration, invasion and EMT of miR-1915-3p, and restoration of SET expression could abrogate these effects induced by miR-1915-3p through JNK/Jun and NF-κB signaling pathways. What's more, miR-1915-3p expression was regulated by METTL3/YTHDF2 m6A axis through transcription factor KLF4., Conclusions: These findings demonstrate that miR-1915-3p function as a tumor suppressor by targeting SET and may have an anti-metastatic therapeutic potential for lung cancer treatment., (© 2021. The Author(s).)

Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks., Competing Interests: Declaration of interests A.G.L. is a founder, CSO, shareholder, and managing director of Eisbach Bio GmbH, a biotechnology company developing cancer medicines. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Medulloblastoma (MB) is a malignant pediatric brain tumor with a poor prognosis. Post-surgical radiation and cisplatin-based chemotherapy have been a mainstay of treatment, which often leads to substantial neurocognitive impairments and morbidity, highlighting the need for a novel therapeutic target to enhance the sensitivity of MB tumors to cytotoxic therapies. We performed a comprehensive study using a cohort of 71 MB patients' samples and pediatric MB cell lines and found that MB tumors have elevated levels of nucleosome remodeling FACT (FAcilitates Chromatin Transcription) complex and DNA repair enzyme AP-endonuclease1 (APE1). FACT interacts with APE1 and facilitates recruitment and acetylation of APE1 to promote repair of radiation and cisplatin-induced DNA damage. Further, levels of FACT and acetylated APE1 both are correlate strongly with MB patients' survival. Targeting FACT complex with CBL0137 inhibits DNA repair and alters expression of a subset of genes, and significantly improves the potency of cisplatin and radiation in vitro and in MB xenograft. Notably, combination of CBL0137 and cisplatin significantly suppressed MB tumor growth in an intracranial orthotopic xenograft model. We conclude that FACT complex promotes chemo-radiation resistance in MB, and FACT inhibitor CBL0137 can be used as a chemo-radiation sensitizer to augment treatment efficacy and reduce therapy-related toxicity in high-risk pediatric patients., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The nucleosome, a basic unit of chromatin found in all eukaryotes, is thought to be assembled through the orchestrated activity of several histone chaperones and chromatin assembly factors in a stepwise manner, proceeding from tetrasome assembly, to H2A/H2B deposition, and finally to formation of the mature nucleosome. In this study, we demonstrate chaperone-mediated assembly of both tetrasomes and nucleosomes on the well-defined Widom 601 positioning sequence using a co-expression/reconstitution wheat germ cell-free system. The purified tetrasomes and nucleosomes were positioned around the center of a given sequence. The heights and diameters were measured by atomic force microscopy. Together with the reported unmodified native histones produced by the wheat germ cell-free platform, our method is expected to be useful for downstream applications in the field of chromatin research., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

SET-Nup214 is a recurrent fusion gene that is mainly observed in T-cell acute lymphoblastic leukemia (T-ALL). Dysregulation of homeobox (Hox) genes is frequently observed in patients with leukemia. Consistent with this, HoxA genes are upregulated in the SET-Nup214 + T-ALL cell line and patients. Although SET-Nup214 has been reported to be recruited to the promoter regions of HoxA genes, the detailed mechanisms of how SET-Nup214 specifically binds to HoxA gene promoters and regulates HoxA gene expression are not known. In this study, we demonstrated that SET-Nup214 interacts with MLL via the SET acidic region of SET-Nup214. SET-Nup214 and MLL cooperatively enhance the promoter activity of the HoxA10 gene. Neither the SET region alone nor the Nup214 region alone sufficiently enhanced the HoxA10 gene promoter. Our results indicated that the SET portion of the SET-Nup214-fusion protein is important for interactions with MLL and transcription enhancement of the HoxA10 gene. Thus, our study will contribute to the understanding of how SET-Nup214 and MLL disturb the expression of HoxA10 gene in leukemia., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating cotranscriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII-interacting factors between wildtype cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Telomere, the terminus of linear chromosome in eukaryotes, is composed of specific repeat DNA which is mainly synthesized by a protein complex called telomerase. The maintenance of telomere DNA is important for unlimited proliferative capacity of cancer cells. The telomerase activity is controlled by the expression level of telomerase reverse transcriptase (TERT), a catalytic unit of telomerase, in some species including human. Therefore, to reveal the regulatory mechanisms of the transcription of TERT gene is important for understanding the tumor development. We found that template activating factor-I (TAF-I), a multifunctional nuclear protein, is involved in the transcriptional activation of TERT for the maintenance of telomere DNA in HeLa cells. TAF-I maintains the histone H3 modifications involved in transcriptional activation and hypomethylated cytosines in CpG dinucleotides around the transcription start site (TSS) in the TERT gene locus. Collectively, TAF-I is involved in the maintenance of telomere DNA through the regulation of TERT transcription, then consequently the occurrence and/or recurrence of cancer cells., (© 2021. The Author(s).)

The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails, and binding partially disassembled nucleosomes. Here, we systematically test these and other scenarios in Saccharomyces cerevisiae and find that FACT binds transcribed chromatin, not RNAPII. Through a combination of high-resolution genome-wide mapping, single-molecule tracking, and mathematical modeling, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

During RNA polymerase II (RNA Pol II) transcription, the chromatin structure undergoes dynamic changes, including opening and closing of the nucleosome to enhance transcription elongation and fidelity. These changes are mediated by transcription elongation factors, including Spt6, the FACT complex, and the Set2-Rpd3S HDAC pathway. These factors not only contribute to RNA Pol II elongation, reset the repressive chromatin structures after RNA Pol II has passed, thereby inhibiting aberrant transcription initiation from the internal cryptic promoters within gene bodies. Notably, the internal cryptic promoters of infrequently transcribed genes are sensitive to such chromatin-based regulation but those of hyperactive genes are not. To determine why, the weak core promoters of genes that generate cryptic transcripts in cells lacking transcription elongation factors (e.g. STE11) were replaced with those from more active genes. Interestingly, as core promoter activity increased, activation of internal cryptic promoter dropped. This associated with loss of active histone modifications at the internal cryptic promoter. Moreover, environmental changes and transcription elongation factor mutations that downregulated the core promoters of highly active genes concomitantly increased their cryptic transcription. We therefore propose that the chromatin-based regulation of internal cryptic promoters is mediated by core promoter strength as well as transcription elongation factors., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Histone variants contribute to the complexity of the chromatin landscape and play an integral role in defining DNA domains and regulating gene expression. The histone H3 variant H3.3 is incorporated into genic elements independent of DNA replication by its chaperone HIRA. Here we demonstrate that Hira is required for the self-renewal of adult hematopoietic stem cells (HSCs) and to restrain erythroid differentiation. Deletion of Hira led to rapid depletion of HSCs while differentiated hematopoietic cells remained largely unaffected. Depletion of HSCs after Hira deletion was accompanied by increased expression of bivalent and erythroid genes, which was exacerbated upon cell division and paralleled increased erythroid differentiation. Assessing H3.3 occupancy identified a subset of polycomb-repressed chromatin in HSCs that depends on HIRA to maintain the inaccessible, H3.3-occupied state for gene repression. HIRA-dependent H3.3 incorporation thus defines distinct repressive chromatin that represses erythroid differentiation of HSCs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcription factors (TFs) harboring broad-complex, tramtrack, and bric-a-brac (BTB) domains play important roles in development and disease. These BTB domains are thought to recruit transcriptional modulators to target DNA regions. However, a systematic molecular understanding of the mechanism of action of this TF family is lacking. Here, we identify the zinc finger BTB-TF Zbtb2 from a genetic screen for regulators of exit from pluripotency and demonstrate that its absence perturbs embryonic stem cell differentiation and the gene expression dynamics underlying peri-implantation development. We show that ZBTB2 binds the chromatin remodeler Ep400 to mediate downstream transcription. Independently, the BTB domain directly interacts with nucleosome remodeling and deacetylase and histone chaperone histone regulator A. Nucleosome remodeling and deacetylase recruitment is a common feature of BTB TFs, and based on phylogenetic analysis, we propose that this is a conserved evolutionary property. Binding to UBN2, in contrast, is specific to ZBTB2 and requires a C-terminal extension of the BTB domain. Taken together, this study identifies a BTB-domain TF that recruits chromatin modifiers and a histone chaperone during a developmental cell state transition and defines unique and shared molecular functions of the BTB-domain TF family., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In cancer cells, tumor suppressor proteins loss-of-function are usually the result of genetic mutations. Protein Phosphatase 2A is a tumor suppressor that inactivates several signaling pathways through removal of phosphate residues important for other proteins stability and/or activation. Different from other tumor suppressors, PP2A is, in many cancer types, inactivated by endogenous inhibitors. In physiological conditions, these inhibitors are important to balance PP2A activity. However, in cancer cells, overexpression of these inhibitors can keep PP2A inactive, resulting in sustained activation of mitogenic signaling pathways and transcription factors, metabolic reprogramming, with the resulting cancer progression and the resistance to anti-cancer therapies. One of these endogenous inhibitors is the protein SET (SE Translocation). SET is a multifunctional protein, which high expression has been associated with several types of cancer, as well as other diseases such as Alzheimer's disease. Disruption of the interaction between SET and PP2A to rescue the activity of PP2A may represent a new therapeutic strategy and opportunity for cancer treatment. This review brings up-to-date advances on the interactions between SET and PP2A and their biological consequences. Moreover, we review reported inhibitors of SET-PP2A interaction under investigation as therapeutic opportunities for the treatment of cancers., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Histone chaperones facilitate DNA replication and repair by promoting chromatin assembly, disassembly and histone exchange. Following histones synthesis and nucleosome assembly, the histones undergo posttranslational modification by different enzymes and are deposited onto chromatins by various histone chaperones. In Tetrahymena thermophila, histones from macronucleus (MAC) and micronucleus (MIC) have been comprehensively investigated, but the function of histone chaperones remains unclear. Histone chaperone Nrp1 in Tetrahymena contains four conserved tetratricopepeptide repeat (TPR) domains and one C-terminal nuclear localization signal. TPR2 is typically interrupted by a large acidic motif. Immunofluorescence staining showed that Nrp1 is located in the MAC and MICs, but disappeared in the apoptotic parental MAC and the degraded MICs during the conjugation stage. Nrp1 was also colocalized with α-tubulin around the spindle structure. NRP1 knockdown inhibited cellular proliferation and led to the loss of chromosome, abnormal macronuclear amitosis, and disorganized micronuclear mitosis during the vegetative growth stage. During sexual developmental stage, the gametic nuclei failed to be selected and abnormally degraded in NRP1 knockdown mutants. Affinity purification combined with mass spectrometry analysis indicated that Nrp1 is co-purified with core histones, heat shock proteins, histone chaperones, and DNA damage repair proteins. The physical direct interaction of Nrp1 and Asf1 was also confirmed by pull-down analysis in vitro. The results show that histone chaperone Nrp1 is involved in micronuclear mitosis and macronuclear amitosis in the vegetative growth stage and maintains gametic nuclei formation during the sexual developmental stage. Nrp1 is required for chromatin stability and nuclear division in Tetrahymena thermophila., (© 2021. The Author(s).)

In the nuclear compartment of yeast, NuB4 core complex consists of three proteins, Hat1, Hat2, and Hif1, and interacts with a number of other factors. In particular, it was shown that NuB4 complex physically interacts with Hsm3p. Early we demonstrated that the gene HSM3 participates in the control of replicative and reparative spontaneous mutagenesis, and that hsm3Δ mutants increase the frequency of mutations induced by different mutagens. It was previously believed that the HSM3 gene controlled only some minor repair processes in the cell, but later it was suggested that it had a chaperone function with its participation in proteasome assembly. In this work, we analyzed the properties of three hsm3Δ , hif1Δ , and hat1Δ mutants. The results obtained showed that the Hsm3 protein may be a functional subunit of NuB4 complex. It has been shown that hsm3 - and hif1 -dependent UV-induced mutagenesis is completely suppressed by inactivation of the Polη polymerase. We showed a significant role of Polη for hsm3 -dependent mutagenesis at non-bipyrimidine sites (NBP sites). The efficiency of expression of RNR (RiboNucleotid Reducase) genes after UV irradiation in hsm3Δ and hif1Δ mutants was several times lower than in wild-type cells. Thus, we have presented evidence that significant increase in the dNTP levels suppress hsm3 - and hif1 -dependent mutagenesis and Polη is responsible for hsm3 - and hif1 -dependent mutagenesis.

Transcription restart after a genotoxic challenge is a fundamental yet poorly understood process. Here, we dissect the interplay between transcription and chromatin restoration after DNA damage by focusing on the human histone chaperone complex HIRA, which is required for transcription recovery post UV. We demonstrate that HIRA is recruited to UV-damaged chromatin via the ubiquitin-dependent segregase VCP to deposit new H3.3 histones. However, this local activity of HIRA is dispensable for transcription recovery. Instead, we reveal a genome-wide function of HIRA in transcription restart that is independent of new H3.3 and not restricted to UV-damaged loci. HIRA coordinates with ASF1B to control transcription restart by two independent pathways: by stabilising the associated subunit UBN2 and by reducing the expression of the transcription repressor ATF3. Thus, HIRA primes UV-damaged chromatin for transcription restart at least in part by relieving transcription inhibition rather than by depositing new H3.3 as an activating bookmark.

In this issue of Molecular Cell, Hammond et al. (2021) and Piette et al. (2021) identify the essential heat shock co-chaperone DNAJC9 as a new bona fide histone chaperone, linking ATP-dependent molecular chaperones to the histone supply and deposition pathways., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.

The 22q11.2 deletion syndrome (22q11DS) is associated with a wide spectrum of cognitive and psychiatric symptoms. Despite the considerable work performed over the past 20 years, the genetic etiology of the neurodevelopmental phenotype remains speculative. Here, we report de novo heterozygous truncating variants in the HIRA (Histone cell cycle regulation defective, S. Cerevisiae, homolog of, A) gene associated with a neurodevelopmental disorder in two unrelated patients. HIRA is located within the commonly deleted region of the 22q11DS and encodes a histone chaperone that regulates neural progenitor proliferation and neurogenesis, and that belongs to the WD40 Repeat (WDR) protein family involved in brain development and neuronal connectivity. To address the specific impact of HIRA haploinsufficiency in the neurodevelopmental phenotype of 22q11DS, we combined Hira knock-down strategies in developing mouse primary hippocampal neurons, and the direct study of brains from heterozygous Hira +/- mice. Our in vitro analyses revealed that Hira gene is mostly expressed during neuritogenesis and early dendritogenesis stages in mouse total brain and in developing primary hippocampal neurons. Moreover, shRNA knock-down experiments showed that a twofold decrease of endogenous Hira expression level resulted in an impaired dendritic growth and branching in primary developing hippocampal neuronal cultures. In parallel, in vivo analyses demonstrated that Hira +/- mice displayed subtle neuroanatomical defects including a reduced size of the hippocampus, the fornix and the corpus callosum. Our results suggest that HIRA haploinsufficiency would likely contribute to the complex pathophysiology of the neurodevelopmental phenotype of 22q11DS by impairing key processes in neurogenesis and by causing neuroanatomical defects during cerebral development.

Elucidating the mechanisms underpinning fertilisation is essential to optimising IVF procedures. One of the critical steps involves paternal chromatin reprogramming, in which compacted sperm chromatin packed by protamines is removed by oocyte factors and new histones, including histone H3.3, are incorporated. HIRA is the main H3.3 chaperone governing this protamine-to-histone exchange. Failure of this step results in abnormally fertilised zygotes containing only one pronucleus (1PN), in contrast to normal two-pronuclei (2PN) zygotes. 1PN zygotes are frequently observed in IVF treatments, but the genotype-phenotype correlation remains elusive. We investigated the maternal functions of two other molecules of the HIRA complex, Cabin1 and Ubn1, in mouse. Loss-of-function Cabin1 and Ubn1 mouse models were developed: their zygotes displayed an abnormal 1PN zygote phenotype. We then studied human 1PN zygotes and found that the HIRA complex was absent in 1PN zygotes that lacked the male pronucleus. This shows that the role of the HIRA complex in male pronucleus formation potentially has coherence from mice to humans. Furthermore, rescue experiments in mouse showed that the abnormal 1PN phenotype derived from Hira mutants could be resolved by overexpression of HIRA. We have demonstrated that HIRA complex regulates male pronucleus formation in mice and is implicated in humans, that both CABIN1 and UBN1 components of the HIRA complex are equally essential for male pronucleus formation, and that rescue is feasible.

Nucleosomes are assembled or disassembled with the aid of histone chaperones in a cell. Viruses can exist either as minichromosomes/episomes or can integrate into the host genome and in both the cases the viral proteins interact and manipulate the cellular nucleosome assembly machinery to ensure their survival and propagation. Recent studies have provided insight into the mechanism and role of histone chaperones in nucleosome assembly and disassembly on the virus genome. Further, the interactions between viral proteins and histone chaperones have been implicated in the integration of the virus genome into the host genome. This review highlights the recent progress and future challenges in understanding the role of histone chaperones in viruses with DNA or RNA genome and their role in governing viral pathogenesis., (Copyright © 2021. Published by Elsevier B.V.)

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions., (© 2021 Viktorovskaya et al.; Published by Cold Spring Harbor Laboratory Press.)