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The treatment of bladder cancer is limited by low drug efficacy and drug resistance. Hence, this study aimed to screen and identify potential drug precursors and investigate their mechanism of action. A set of camptothecin derivatives showing high anti-tumor potential was selected from early-stage research or literature and synthesized to construct a compound library. A total of 135 compounds were screened in T24 and J82 cells, revealing that FL118 significantly inhibited the proliferation of GC (gemcitabine + cisplatin)-sensitive/insensitive cells. FL118 exhibited excellent penetration and killing ability in organoids and three GC-insensitive patient-derived xenografts. Chemical proteomic and docking calculations were employed to identify binding proteins, indicating that FL118 can bind into H2A.X and its entwined DNA. The results of Cellular thermal shift assay and surface plasmon resonance (K d  = 3.77E-6) support the above findings. Fluorescence localization revealed widespread binding of FL118 within the cell nucleus. Furthermore, WB showed that FL118 increased cellular DNA damage, resulting in significant cell cycle inhibition. The binding of FL118 to H2A.X hindered the damage repair process, leading to apoptosis. Controllable adverse reactions were observed in mice treated with FL118. In conclusion, FL118 may be a superior anti-bladder cancer compound that acts as a molecular glue binding to both H2A.X and DNA. The resistance mediated by the DNA damage repair to DNA damage caused by GC regimen can be reversed by FL118. This distinct mechanism of FL118 has the potential to complement existing mainstream treatment approaches for bladder cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Epigenetic regulation, which is characterized by reversible and heritable genetic alterations without changing DNA sequences, has recently been increasingly studied in diseases. Histone variant regulation is an essential component of epigenetic regulation. The substitution of canonical histones by histone variants profoundly alters the local chromatin structure and modulates DNA accessibility to regulatory factors, thereby exerting a pivotal influence on gene regulation and DNA damage repair. Histone H2A variants, mainly including H2A.Z, H2A.B, macroH2A, and H2A.X, are the most abundant identified variants among all histone variants with the greatest sequence diversity. Harboring varied chromatin occupancy and structures, histone H2A variants perform distinct functions in gene transcription and DNA damage repair. They are implicated in multiple pathophysiological mechanisms and the emergence of different illnesses. Cancer, embryonic development abnormalities, neurological diseases, metabolic diseases, and heart diseases have all been linked to histone H2A variant alterations. This review focuses on the functions of H2A histone variants in mammals, including H2A.Z, H2A.B, macroH2A, and H2A.X, and their current roles in various diseases.

Background: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction., Results: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA., Conclusions: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm., (© 2023. The Author(s).)

Chromatin, the functional organization of DNA with histone proteins in eukaryotic nuclei, is the tightly-regulated template for several biological processes, such as transcription, replication, DNA damage repair, chromosome stability and sister chromatid segregation. In order to achieve a reversible control of local chromatin structure and DNA accessibility, various interconnected mechanisms have evolved. One of such processes includes the deposition of functionally-diverse variants of histone proteins into nucleosomes, the building blocks of chromatin. Among core histones, the family of H2A histone variants exhibits the largest number of members and highest sequence-divergence. In this short review, we report and discuss recent discoveries concerning the biological functions of the animal histone variants H2A.B, H2A.X and H2A.Z and how dysregulation or mutation of the latter impacts the development of disease., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

During mammalian preimplantation development, stimulation of zygotic genome activation (ZGA) and transposable elements (TEs) shapes totipotency profiling. A rare mouse embryonic stem cells (mESCs) subpopulation is capable of transiently entering a state resembling 2-cell stage embryos, with subtypes of TEs expressed and ZGA genes transiently activated. In this study, we found that deletion of H2A.X in mESCs led to a significant upregulation of ZGA genes and misregulated TEs. ChIP-seq analysis indicated a direct association of H2A.X at the Dux locus for silencing the Dux gene and its downstream ZGA genes in mESCs. We also demonstrated that histone variant H2A.X is highly enriched in human cleavage embryos when ZGA genes and TEs are active. Therefore, we propose that H2A.X plays an important role in regulating ZGA genes and TEs to establish totipotency.

Background: Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis., Results: We find in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specific genes as well as a nuclear condensation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specific genes observed in the absence of H2A.X., Conclusions: We relate these findings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifically suppressed., (© 2021. The Author(s).)

Abscisic acid (ABA) and the AP2/ERF (APETALA2/ETHYLENE-RESPONSIVE FACTOR)-type transcription factor called ABA INSENSITIVE 4 (ABI4) play pivotal roles in plant growth responses to environmental stress. An analysis of seedling development in Arabidopsis ABA hypersensitive mutants suggested that OXS3 (OXIDATIVE STRESS 3), OXS3b, O3L3 (OXS3 LIKE 3), O3L4, and O3L6 were negative regulators of ABI4 expression. We therefore characterized the roles of the OXS3 family members in ABA signaling. All the above five OXS3 proteins were found to interact with AFP1 (ABI FIVE BINDING PROTEIN 1) in yeast two hybrid assays. Seven OXS3 family members including two other members O3L1 and O3L5 were found to interact with histone H2A.X, although OXS3b, O3L3, and O3L5 showed weaker interactions. ChIP-qPCR analysis showed that the absence of some of these OXS3 family proteins was associated with increased occupancy of histone γ-H2A.X at the ABI4 promoter, which also corresponded with de-repression of ABI4 expression. Repression of ABI4 expression, however, required both AFP1 and OXS3, OXS3b or O3L6. We conclude that in the absence of stress, OXS3 family proteins regulate γ-H2A.X deposition at the ABI4 promoter and that together with AFP1, OXS3 family proteins function to prevent ABA-induced growth arrest by co-repressing ABI4 through decreased promoter occupancy of histone γ-H2A.X., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ovarian cancer remains the gynecological cancer with the highest mortality rate. In our study, we compare a number of proteins from different effector pathways to assess their usefulness in the diagnosis of ovarian cancer. The tissue expression of the tested proteins was assessed by two methods: qRT-PCR and an immunohistochemical analysis. A significantly higher level of mRNA expression was found in the ovarian cancer group for YAP and TEAD4 ( p = 0.004 and p = 0.003, respectively). There was no statistical significance in the expression of mRNA for SMAD3 , and there was borderline statistical significance for SMAD2 between the groups of ovarian cancer patients and other subgroups of patients with simple cysts and healthy ovarian tissue ( p = 0.726 and p = 0.046, respectively). Significantly higher levels of transferrin receptor ( CD71 ), H2A.X , and ADH1A gene expression were found in the ovarian cancer group compared to the control group for YAP , and TEAD4 showed strong nuclear and cytoplasmic staining in ovarian carcinoma and weak staining in non-carcinoma ovarian samples, ADH1A1 showed strong staining in the cytoplasm of carcinoma sections and a weak positive reaction in the non-carcinoma section, H2A.X showed strong positive nuclear staining in carcinoma sections and moderate positive staining in non-carcinoma samples, and CD71 showed moderate positive staining in carcinoma and non-carcinoma samples. YAP, TEAD4, and ADH1A proteins appear to be promising biomarkers in the diagnosis of ovarian cancer.

Objective: Resveratrol (RSV) is a multitarget drug that has demonstrated activity against Toxoplasma gondii in macrophage and human foreskin fibroblast (HFF) cell line infection models. However, the mechanism of action of RSV has not yet been determined. Thus, with the aim of identifying a possible mechanism of the anti-T. gondii activity of this compound, we analyzed the effects of RSV on histones H3 and H4 lysine 16 acetylation (H4K16). We also analyzed RSV-induced DNA damage to intracellular tachyzoites by using the DNA damage marker phosphorylated histone H2A.X (γH2AX)., Results: RSV inhibited intracellular T. gondii tachyzoite growth at concentrations below the toxic threshold for host cells. The IC 50 value after 24 h of treatment was 53 μM. RSV induced a reduction in H4K16 acetylation (H4K16ac), a marker associated with transcription, DNA replication and homologous recombination repair. A similar deacetylation effect was observed on histone H3. RSV also increased T. gondii H2A.X phosphorylation at the SQE motif (termed γH2A.X), which is a DNA damage-associated posttranslational modification. Our findings suggest a possible link between RSV and DNA damage or repair processes that is possibly associated with DNA replication stress.

Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: In case of population exposure by ionizing radiation, a fast and reliable dose assessment of exposed and non-exposed individuals is crucial important. In initial triage, physicians have to take fast decisions whom to treat with adequate medical care. In addition, worries about significant exposure can be taken away from hundreds to thousands non- or low exposed individuals. Studies have shown that the γH2A.X radiation-induced foci assay is a promising test for fast triage decisions. However, in a large-scale scenario most biodosimetry laboratories will quickly reach their capacity limit. The aim of this study was to evaluate the benefit of inexperienced experimenters to speed up the foci assay and manual foci scoring., Materials and Methods: The participants of two training courses performed the radiation-induced foci assay (γH2A.X) under the guidance of experts and scored foci (γH2A.X and 53BP1) on sham-irradiated and irradiated blood samples (0.05-1.5 Gy). The outcome of laboratory experiments and manual foci scoring by 26 operators with basic experience in laboratory work was statistically analyzed in comparison to the results from experts., Results: Inexperienced operators prepared slides with significant dose-effects (0, 0.1 and 1.0 Gy) for semi-automatic microscopic analyses. Manual foci scoring by inexperienced scorer resulted in a dose-effect curve for γH2A.X, 53BP1 and co-localized foci. In addition, inexperienced scorers were able to distinguish low irradiation doses from unirradiated cells. While 53BP1 foci scoring was in accordance to the expert counting, differences between beginners and expert increased for γH2A.X or co-localized foci., Conclusions: In case of a large-scale radiation event, inexperienced staff is useful to support laboratories in slide preparation for semi-automatic foci counting as well as γH2A.X and 53BP1 manual foci scoring for triage-mode biodosimetry. Slides can be clearly classified in the non-, low- or high-exposed category.

Natural compounds isolated from plants are excellent starting points in drug design and have been widely studied as anticancer agents; they hence find use in a considerable proportion of anticancer drugs. The genus Plectranthus (Lamiaceae) comprises a large and widespread group of species with various applications in traditional medicine. Therefore, the aim of the present study was to determine the effectiveness of treatment with four abietane diterpenoids isolated from P. madagascariensis , 6,7-dehydroroyleanone, 7β,6β-dihydroxyroyleanone, 7α-acetoxy-6β-hydroxyroyleanone and parvifloron D, in initiating apoptosis in a glioma cell line. The pure compounds were found to exhibit anticancer effects in primary H7PX glioma cells line by inducing apoptosis G2/M cell cycle arrest and double-strand breaks, indicated by increased levels of phosphorylated H2A.X and decreasing mitochondrial membrane potential; they also influenced the expression of pro- and anti-apoptotic genes ( P. ecklonii , 6,7-dehydroroyleanone, 7β,6β-dihydroxyroyleanone, 7α-acetoxy-6β-hydroxyroyleanone and parvifloron D, in initiating apoptosis in a glioma cell line. The pure compounds were found to exhibit anticancer effects in primary H7PX glioma cells line by inducing apoptosis G2/M cell cycle arrest and double-strand breaks, indicated by increased levels of phosphorylated H2A.X and decreasing mitochondrial membrane potential; they also influenced the expression of pro- and anti-apoptotic genes ( Bax , Bcl-2, TP53 , or Cas-3 ). Our findings indicate that these compounds may offer potential as beneficial antitumor drugs but further in vivo studies are needed., Competing Interests: The authors declare no conflict of interest.

Objective: To investigate Livin-mediated regulation of H2A.X Y142 phosphorylation via a novel kinase activity and its effect on autophagy in colon cancer cells. Methods: The interaction between Livin and H2A.X was tested by immunoprecipitation. H2A.X-/- HCT116 cells were transfected with human influenza hemagglutinin (HA)-tagged WT or Y142F phospho-dead mutantH2A.X plasmids. GST-tagged recombinant Livin protein was used to perform in vitro pull-down experiment and kinase assay. H2A.X-/-Livin+/+ SW480 cells were co-transfected with H2A.X WT /H2A.X Y142F plasmid and LC3 EGFP-tagged plasmid to explore whether H2A.X Y142F was involved in Livin-mediated autophagy induced by starvation in colon cancer cells. Results: Co-immunoprecipitation studies confirmed that Livin interacted with H2A.X and that it was phosphorylation dependent. In vitro kinase assay confirmed that Livin could phosphorylate H2A.X. Knockdown of Livin (Livin-/-) in SW480 cells or HCT116 cells canceled the starvation-induced autophagy in colon cancer cells; H2A.X-/-Livin+/+ SW480 cells transfected with H2A.X WT activated autophagy induced by starvation while cells transfected with H2A.X Y142F had no significant difference; Livin-H2A.X Y142F axis activated autophagy in colon cancer cells through transcriptionally regulating ATG5 and ATG7 . Conclusion: Livin promotes autophagy in colon cancer cells via regulating the phosphorylation of H2A.X Y142 ., (Copyright © 2019 Ge, Liu, Cui and Li.)

Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Embryonic stem cells (ESCs) display an abbreviated cell cycle, resulting in a short doubling time and rapid proliferation. The histone variant H2A.X is critical for proliferation of stem cells, although mechanistic insights have remained obscure. Here, we show that H2A.X defines the rate of mouse ESC proliferation independently of the DNA damage response pathway, and it associates with three major chromatin-modifying complexes. Our functional and biochemical analyses demonstrate that H2A.X-associated factors mediate the H2A.X-dependent effect on ESC proliferation and involve the nucleolar remodeling complex (NoRC). A specific H2A.X deposition at rDNA promoters determines the chromatin recruitment of the NoRC, histone modifications, the rRNA transcription, and the rate of proliferation. Collectively, our results suggest that NoRC assembly by H2A.X deposition at rRNA promoters silences transcription, and this represents an important regulatory component for ESC proliferation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Histone variants are chromatin components that replace replication-coupled histones in a fraction of nucleosomes and confer particular characteristics to chromatin. H2A variants represent the most numerous and diverse group among histone protein families. In the nucleosomal structure, H2A-H2B dimers can be removed and exchanged more easily than the stable H3-H4 core. The unstructured N-terminal histone tails of all histones, but also the C-terminal tails of H2A histones protrude out of the compact structure of the nucleosome core. These accessible tails are the preferential target sites for a large number of post-translational modifications (PTMs). While some PTMs are shared between replication-coupled H2A and H2A variants, many modifications are limited to a specific histone variant. The present review focuses on the H2A variants H2A.Z, H2A.X, and macroH2A, and summarizes their functions in chromatin and how these are linked to cancer development and progression. H2A.Z primarily acts as an oncogene and macroH2A and H2A.X as tumour suppressors. We further focus on the regulation by PTMs, which helps to understand a degree of context dependency., Competing Interests: The authors declare no conflict of interest.

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACβ > PP2ACα > PP4C > PP6C), NRVM (PP2ACβ > PP2ACα = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACα > PP2ACβ > PP6C > PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACα, PP2ACβ, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (γH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H 2 O 2 increased levels of γH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium.

DNA double-strand break (DSB) repair depends on the ataxia telangiectasia mutated (ATM) kinase that phosphorylates the conserved C-terminal SQ motif present in the histone variant H2A.X [1-7]. In constitutive heterochromatin of mammals, DSB repair is delayed and relies on phosphorylation of the proteins HP1 and KAP1 by ATM [2, 8-14]. However, KAP1 is not conserved in plants and the HP1-related protein Like-HP1 (LHP1) is not localized at constitutive heterochromatin [15], suggesting that in plants, alternative mechanisms could be responsible for repair of DSBs in heterochromatin. In Arabidopsis, constitutive heterochromatin is marked by H3K9 methylation and the plant-specific histone variants H2A.W, which are distinguished by their C-terminal motif KSPKK and required for heterochromatin compaction [16-18]. We report that the Arabidopsis histone variant H2A.W.7 is confined to heterochromatin and carries a SQ motif that is phosphorylated by ATM. In response to DNA damage, phosphorylation of H2A.W.7 takes place in heterochromatin, while H2A.X phosphorylation takes place primarily in euchromatin. We propose that H2A.W.7 evolved in addition to H2A.X to facilitate DNA damage response in highly condensed heterochromatin, thus playing a role similar to KAP1 and HP1 phosphorylation in mammals. These data support the idea of the functional diversification of histone variants and their role in spatial compartmentalization of chromatin-related functions in eukaryotes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Double-stranded DNA breaks induce serine phosphorylation of histone H2A.X, producing γ-H2A.X foci that are then recognized by DNA damage response pathway proteins. Formation of γ-H2A.X is therefore critical for the repair of DNA double-stranded breaks and maintenance of genomic stability, and defects in the recognition or repair of double-stranded breaks can result in tumorigenesis. However, key details regarding the formation of γ-H2A.X and its possible role in tumorigenesis remain elusive. Here, we report a previously unknown phosphorylation site on H2A.X, Tyr39. Phosphorylation at this site is induced by ionizing radiation and is a prerequisite for γ-H2A.X formation. Increased phosphorylation of H2A.X at Tyr39 was observed in multiple cancer cell lines, and we found that H2AX Tyr39 phosphorylation positively correlated with histological grade, tumor size and tumor node metastasis stage, and negatively correlated with survival. We also identified a potential role for eyes absent 2 (EYA2) in regulating H2A.X Tyr39 phosphorylation. Our study supports an important role for H2AX Tyr39 phosphorylation in γ-H2A.X formation and cancer progression., (© 2016 Federation of European Biochemical Societies.)

In eukaryotic cells, DNA is packaged inside the nucleus together with histones to form nucleosomes. Each histone molecule contains two of each core histone subunits H2A, H2B, H3 and H4. Among core histones, the H2A family is of great interest due to the high diversity of specialized variants. These variants have shown important role in critical cellular processes. Epigenetic mechanism in oomycetes is barely known. Phytophthora infestans is a severe pathogen and a model species in oomycetes. In this study, we studied the sequence and expression pattern of H2A variants of P. infestans through genome search, sequences alignment, phylogenetic analysis and realtime qPCR detection. P. infestans contains conserved genes encoding histone variants H2A.X.1, H2A.X.2 and H2A.Z, and these genes have specific expression patterns during development and infection stages. Our datasets provide useful inputs to help explore the epigenetic mechanisms of oomycetes.

SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent γH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. We systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange., (© 2016. Published by The Company of Biologists Ltd.)

The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem-loop (SL) structure. This SL structure interacts with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3'hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3' end of RD-histone mRNAs together with SLBP., (© 2015 Brooks et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Studies in different species, including human, mice, bovine, and swine, demonstrated that early-cleaving embryos have higher capacity to develop to the blastocyst stage and produce better quality embryos with superior capacity to establish pregnancy than late-cleaving embryos. It has also been shown that experimentally induced DNA damage delays embryo cleavage kinetics and reduces blastocyst formation. To gain additional insights into the effects of genome damage on embryo cleavage kinetics and development, the present study compared the occurrence of DNA double-strand breaks (DSBs) with the expression profile of genes involved in DNA repair and cell cycle control between early- and late-cleaving embryos. Porcine oocytes matured in vitro were activated, and then early-cleaving (before 24 h) and late-cleaving (between 24 and 48 h) embryos were identified and cultured separately. Developing embryos, on Days 3, 5, and 7, were used to evaluate the total cell number and presence of DSBs (by counting the number of immunofluorescent foci for phosphorylated histone H2A.x [H2AX139ph] and RAD51 proteins) and to quantify transcripts of genes involved in DNA repair and cell cycle control by quantitative RT-PCR. Early-cleaving embryos had fewer DSBs, lower transcript levels for genes encoding DNA repair and cell cycle checkpoint proteins, and more cells than late-cleaving embryos. Interestingly, at the blastocyst stage, embryos that developed from early- and late-cleaving groups had similar number of DSBs as well as transcript levels of genes induced by DNA damage. This indicates that only embryos with less DNA damage and/or superior capacity for DNA repair are able to progress to the blastocyst stage. Collectively, findings in this study revealed a negative correlation between the occurrence of DSBs and embryo cleavage kinetics and embryo developmental capacity to the blastocyst stage., (© 2015 by the Society for the Study of Reproduction, Inc.)