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A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

Background: Cerebral arteriovenous malformations (cAVMs) represent tangles of abnormal vasculature without intervening capillaries. High-pressure vascular channels due to abnormal arterial and venous shunts can lead to rupture. Multiple pathways are involved in the pathobiology of cAVMs including inflammation and genetic factors such as KRAS mutations. Neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs), plays a multifunctional role in infection, inflammation, thrombosis, intracranial aneurysms, and tumor progression. However, the relationship between NETs and the pathobiology of cAVMs remains unknown. We tested whether NETs play a role in the pathobiology of cAVMs., Methods: We analyzed samples from patients who had undergone surgery for cAVM and immunohistochemically investigated expression of citrullinated histone H3 (CitH3) as a marker of NETs. CitH3 expression was compared among samples from cAVM patients, epilepsy patients, and normal human brain tissue. Expressions of thrombotic and inflammatory markers were also examined immunohistochemically in samples from cAVM patients., Results: Expression of CitH3 derived from neutrophils was observed intravascularly in all cAVM samples but not other samples. Nidi of AVMs showed migration of many Iba-I-positive cells adjacent to the endothelium and endothelial COX2 expression, accompanied by expression of IL-6 and IL-8 in the endothelium and intravascular neutrophils. Unexpectedly, expression of CitH3 was not necessarily localized to the vascular wall and thrombus., Conclusions: Our results offer the first evidence of intravascular expression of NETs, which might be associated with vascular inflammation in cAVMs., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Proliferating cells coordinate histone and DNA synthesis to maintain correct stoichiometry for chromatin assembly. Histone mRNA levels must be repressed when DNA replication is inhibited to prevent toxicity and genome instability due to free non-chromatinized histone proteins. In mammalian cells, replication stress triggers degradation of histone mRNAs, but it is unclear if this mechanism is conserved from other species. The aim of this study was to identify the histone mRNA decay pathway in the yeast Saccharomyces cerevisiae and determine the mechanism by which DNA replication stress represses histone mRNAs. Using reverse transcription-quantitative PCR and chromatin immunoprecipitation-quantitative PCR, we show here that histone mRNAs can be degraded by both 5' → 3' and 3' → 5' pathways; however, replication stress does not trigger decay of histone mRNA in yeast. Rather, replication stress inhibits transcription of histone genes by removing the histone gene-specific transcription factors Spt10p and Spt21p from histone promoters, leading to disassembly of the preinitiation complexes and eviction of RNA Pol II from histone genes by a mechanism facilitated by checkpoint kinase Rad53p and histone chaperone Asf1p. In contrast, replication stress does not remove SCB-binding factor transcription complex, another activator of histone genes, from the histone promoters, suggesting that Spt10p and Spt21p have unique roles in the transcriptional downregulation of histone genes during replication stress. Together, our data show that, unlike in mammalian cells, replication stress in yeast does not trigger decay of histone mRNAs but inhibits histone transcription., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as 'Trim away' (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The current model of replication-dependent (RD) histone biosynthesis posits that RD histone gene expression is coupled to DNA replication, occurring only in S phase of the cell cycle once DNA synthesis has begun. However, several key factors in the RD histone biosynthesis pathway are up-regulated by E2F or phosphorylated by CDK2, suggesting these processes may instead begin much earlier, at the point of cell-cycle commitment. In this study, we use both fixed- and live-cell imaging of human cells to address this question, revealing a hybrid model in which RD histone biosynthesis is first initiated in G1, followed by a strong increase in histone production in S phase of the cell cycle. This suggests a mechanism by which cells that have committed to the cell cycle build up an initial small pool of RD histones to be available for the start of DNA replication, before producing most of the necessary histones required in S phase. Thus, a clear distinction exists at completion of mitosis between cells that are born with the intention of proceeding through the cell cycle and replicating their DNA and cells that have chosen to exit the cell cycle and have no immediate need for histone synthesis., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The perinuclear theca (PT) of the eutherian sperm head is a cytoskeletal-like structure that houses proteins involved in important cellular processes during spermiogenesis and fertilization. Building upon our novel discovery of non-nuclear histones in the bovine PT, we sought to investigate whether this PT localization was a conserved feature of eutherian sperm. Employing cell fractionation, immunodetection, mass spectrometry, qPCR, and intracytoplasmic sperm injections (ICSI), we examined the localization, developmental origin, and functional potential of histones from the murid PT. Immunodetection localized histones to the post-acrosomal sheath (PAS) and the perforatorium (PERF) of the PT but showed an absence in the sperm nucleus. MS/MS analysis of selectively extracted PT histones indicated that predominately core histones (i.e., H3, H3.3, H2B, H2A, H2AX, and H4) populate the murid PT. These core histones appear to be de novo -synthesized in round spermatids and assembled via the manchette during spermatid elongation. Mouse ICSI results suggest that early embryonic development is delayed in the absence of PT-derived core histones. Here, we provide evidence that core histones are de novo -synthesized prior to PT assembly and deposited in PT sub-compartments for subsequent involvement in chromatin remodeling of the male pronucleus post-fertilization.

ADP-ribosylation of nuclear proteins is a critical feature of various DNA damage repair pathways. Histones, particularly H3 and H2B, are major targets of ADP-ribosylation and are primarily modified on serine with a single ADP-ribose unit following DNA damage. While the overall impact of PARP1-dependent poly-ADP-ribosylation is heavily investigated, very little is known about the specific roles of histone ADP-ribosylation. Here, we report the development of an efficient and modular semisynthetic route to full-length ADP-ribosylated histones H3 and H2B, chemically installed at specific serine residues. The modified histones were used to generate various chemically defined ADP-ribosylated chromatin substrates, which were employed in biophysical assays. These studies revealed that ADP-ribosylation of serine-6 of histone H2B (H2BS6ADPr) inhibits chromatin folding and higher-order organization; notably, this effect was enhanced by ADP-ribosylation of H3S10. In addition, ADP-ribosylated nucleosomes were utilized in biochemical experiments employing a panel of lysine methyltransferase enzymes, revealing a context-dependent inhibition of histone H3K9 methylation. The availability of designer ADP-ribosylated chromatin described here is expected to facilitate further biochemical and structural studies regarding the roles of histone ADP-ribosylation in the DNA damage response.

Activated macrophages have been implicated in lung injury and fibrosis induced by the cytotoxic alkylating agent, nitrogen mustard (NM). Herein, we determined if macrophage activation is associated with histone modifications and altered miRNA expression. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in increases in phosphorylation of H2A.X in lung macrophages at 1 d and 3 d post-exposure. This DNA damage response was accompanied by methylation of histone (H) 3 lysine (K) 4 and acetylation of H3K9, marks of transcriptional activation, and methylation of H3K36 and H3K9, marks associated with transcriptional repression. Increases in histone acetyl transferase and histone deacetylase were also observed in macrophages 1 d and 28 d post-NM exposure. PCR array analysis of miRNAs (miR)s involved in inflammation and fibrosis revealed unique and overlapping expression profiles in macrophages isolated 1, 3, 7, and 28 d post-NM. An IPA Core Analysis of predicted mRNA targets of differentially expressed miRNAs identified significant enrichment of Diseases and Functions related to cell cycle arrest, apoptosis, cell movement, cell adhesion, lipid metabolism, and inflammation 1 d and 28 d post NM. miRNA-mRNA interaction network analysis revealed highly connected miRNAs representing key upstream regulators of mRNAs involved in significantly enriched pathways including miR-34c-5p and miR-27a-3p at 1 d post NM and miR-125b-5p, miR-16-5p, miR-30c-5p, miR-19b-3p and miR-148b-3p at 28 d post NM. Collectively, these data show that NM promotes histone remodeling and alterations in miRNA expression linked to lung macrophage responses during inflammatory injury and fibrosis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Biochemical reactions typically depend on the concentrations of the molecules involved, and cell survival therefore critically depends on the concentration of proteins. To maintain constant protein concentrations during cell growth, global mRNA and protein synthesis rates are tightly linked to cell volume. While such regulation is appropriate for most proteins, certain cellular structures do not scale with cell volume. The most striking example of this is the genomic DNA, which doubles during the cell cycle and increases with ploidy, but is independent of cell volume. Here, we show that the amount of histone proteins is coupled to the DNA content, even though mRNA and protein synthesis globally increase with cell volume. As a consequence, and in contrast to the global trend, histone concentrations decrease with cell volume but increase with ploidy. We find that this distinct coordination of histone homeostasis and genome content is already achieved at the transcript level, and is an intrinsic property of histone promoters that does not require direct feedback mechanisms. Mathematical modeling and histone promoter truncations reveal a simple and generalizable mechanism to control the cell volume- and ploidy-dependence of a given gene through the balance of the initiation and elongation rates., (© 2021. The Author(s).)

Enhancer of zeste homolog 2 (EZH2), a component of the polycomb repressive complex 2 that catalyzes trimethylation of H3K27 (H3K27me3), has been shown to promote tumor development and progression. Expression of EZH2 is associated with cell cycle regulation and cell proliferation in various neoplasms. Oral verrucous hyperplasia (OVH) and Oral verrucous carcinoma (OVC) are rare entities and share several clinical and histopathologic features. Problems distinguishing these lesions are added by a lack of adjacent normal tissue of the biopsy samples and poorly oriented tissue sections. The aim of this study was to investigate the expression of EZH2 and H3K27me3 in OVH and OVC and comparing the expression with normal oral mucosa and oral squamous cell carcinoma (OSCC). Seventy-eight samples, including 25 cases of OVC, 8 cases of OVH, 35 cases of OSCC and 10 cases of normal oral mucosa, were retrieved and submitted for immunohistochemical staining. The results demonstrated that the mean labeling indices (LIs) of EZH2 and H3K27me3 expression were highest in OSCC, followed by the OVC, OVH, and normal mucosa. Statistical differences in EZH2 LI were observed among these lesions whereas H3K27me3 LI was significantly different among OSCC, OVH and normal mucosa. EZH2 LI was found to have a sensitivity of 72.00% and specificity of 87.50% in distinguishing OVH from OVC, and a sensitivity of 57.14% and specificity of 84.00% in distinguishing OVC from OSCC. A positive correlation between EZH2 and H3K27me3 expression was significantly found in OVC but not in OVH and OSCC. These findings highlight the involvement of epigenetic regulation by EZH2-mediated H3K27me3 in the pathogenesis of OVH and OVC, and EZH2 expression indicates disease progression of these verrucous lesions. Diagnostic test analysis further suggests that EZH2 may be used as an additional test for differentiating OVH from OVC in questionable cases.

Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. Here, we address this question in Drosophila , which encodes for a single germline-specific dBigH1 linker histone. We show that during female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. We find that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, we discuss the situation in testes, where Brat is not expressed, but dBigH1 is translationally silenced too.

Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenoteratozoospermia. We have identified protease serine 50 (PRSS50) as having a crucial role in sperm development, because Prss50-null mice presented with impaired fertility and sperm tail abnormalities. PRSS50 could also be involved in centrosome function because these mice showed a threefold increase in acephalic sperm (head-tail junction defect), sperm with multiple heads (spermatid division defect) and sperm with multiple tails, including novel two conjoined sperm (complete or partial parts of several flagellum on the same plasma membrane). Our data support that, in the testis, as in tumorigenesis, PRSS50 activates NFκB target genes, such as the centromere protein leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1), which is required for heterochromatin maintenance. Prss50-null testes have increased IκκB, and reduced LRWD1 and histone expression. Low levels of de-repressed histone markers, such as H3K9me3, in the Prss50-null mouse testis may cause increases in post-meiosis proteins, such as AKAP4, affecting sperm formation. We provide important insights into the complex mechanisms of sperm development, the importance of testis proteases in fertility and a novel mechanism for MMAF., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Incorporation of different H3 histone isoforms/variants have been reported to differentially regulate gene expression via alteration in chromatin organization during diverse cellular processes. However, the differential expression of highly conserved histone H3.2 genes, H3C14 and H3C13 in human cancer has not been delineated. In this study, we investigated the expression of H3.2 genes in primary human gastric, brain, breast, colon, liver, and head and neck cancer tissues and tumor cell lines. The data showed overexpression of H3.2 transcripts in tumor samples and cell lines with respect to normal counterparts. Furthermore, TCGA data of individual and TCGA PANCAN cohort also showed significant up-regulation of H3.2 genes. Further, overexpressed H3C14 gene coding for H3.2 protein was regulated by FOXC1 transcription factor and G4-cassette in gastric cancer cell lines. Elevated expression of FOXC1 protein and transcripts were also observed in human gastric cancer samples and cell lines. Further, FOXC1 protein was predominantly localized in the nuclei of neoplastic gastric cells compared to normal counterpart. In continuation, studies with EGF induction, FOXC1 knockdown, and ChIP-qPCR for the first time identified a novel axis, EGFR-FOXC1-H3C14 for regulation of H3C14 gene overexpression in gastric cancer. Therefore, the changes the epigenomic landscape due to incorporation of differential expression H3 variant contributes to change in gene expression pattern and thereby contributing to pathogenesis of cancer., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Irreparable double-strand breaks (DSBs) in response to ionizing radiation (IR) trigger prolonged DNA damage response (DDR) and induce premature senescence. Profound chromatin reorganization with formation of senescence-associated heterochromatin foci (SAHF) is an essential epigenetic mechanism for controlling the senescence-associated secretory phenotype (SASP). To decipher molecular mechanisms provoking continuous DDR leading to premature senescence, radiation-induced DSBs (53BP1-foci) and dynamics of histone variant H2A.J incorporation were analyzed together with chromatin re-modeling in human fibroblasts after IR exposure. High-resolution imaging by transmission electron microscopy revealed that persisting 53BP1-foci developed into DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), consistently located at the periphery of SAHFs. Quantitative immunogold-analysis by electron microscopy revealed that H2A.J, steadily co-localizing with 53BP1, is increasingly incorporated into DNA-SCARS during senescence progression. Strikingly, shRNA-mediated H2A.J depletion in fibroblasts modified senescence-associated chromatin re-structuring and abolished SASP, thereby shutting down the production of inflammatory mediators. These findings provide mechanistic insights into biological phenomena of SASP and suggest that H2A.J inhibition could ablate SASP, without affecting the senescence-associated growth arrest.

Aims: Phosphohistone H3 (PhH3) has been proposed as a novel proliferation marker in breast cancer. This study compares the interobserver agreement for assessment of the mitotic activity index (MAI), Ki67 expression, and PhH3 in a cohort of oestrogen receptor (ER)-positive breast cancer patients., Methods and Results: Tumour samples of 159 luminal breast cancer patients were collected. MAI and PhH3 scores were assessed by three breast cancer pathologists. Ki67 scores were assessed separately by two of the three pathologists. PhH3-positive cells were counted in an area of 2 mm 2 , with a threshold of ≥13 positive cells being used to discriminate between low-proliferative and high-proliferative tumours. Ki67 expression was assessed with the global scoring method. Ki67 percentages of <20% were considered to be low. The intraclass correlation coefficient (ICC) and Cohen's κ statistics were used to evaluate interobserver agreement. The impact on histological grading of replacing the MAI with PhH3 was assessed. Counting PhH3-positive cells was highly reproducible among all three observers (ICC of 0.86). The κ scores for the categorical PhH3 count (κ = 0.78, κ = 0.68, and κ = 0.80) reflected substantial agreement among all observers, whereas agreement for the MAI (κ = 0.38, κ = 0.52, and κ = 0.26) and Ki67 (κ = 0.55) was fair to moderate. When PhH3 was used to determine the histological grade, agreement in grading increased (PhH3, κ = 0.52, κ = 0.48, and κ = 0.52; MAI, κ = 0.43, κ = 0.35, and κ = 0.32), and the proportion of grade III tumours increased (14%, 18%, and 27%)., Conclusion: PhH3 seems to outperform Ki67 and the MAI as a reproducible means to measure tumour proliferation in luminal-type breast cancer. Variation in the assessment of histological grade might be reduced by using PhH3, but would result in an increase in the proportion of high-grade cancers., (© 2020 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Purpose: Breast cancer is the leading cause of cancer death in females. Histone modifications have been shown to have an influence on the gene expression. This study focusses on the histone modifications H3K9ac and H3K4me3 in breast cancer and their impact on survival METHODS: H3K4me3 and H3K9ac expression was immunohistochemically examined in 235 tissue samples., Results: Positive estrogen receptor status was correlated with a higher IRS of the nuclear (p = 0.033), and of the cytoplasmic H3K4me3 staining (p = 0.009). H3K9ac intensity was associated to the Her2 status (p = 0.045) and to poor prognosis in cells with positive Ki67 status (p = 0.013). A high intensity of nuclear H3K4me3 staining was found to be correlated with a lower 10-year-survival (p = 0.026) and with lower breast cancer-specific survival (p = 0.004). High percentage score (> 190) of H3K9ac expression was correlated to worse breast cancer-specific survival (p = 0.005). Shorter progression-free survival was found in patients with nuclear (p = 0.013) and cytoplasmic H3K4me3expression (p = 0.024) and H3K9ac expression (p = 0.023)., Conclusion: This analysis provides new evidence of histone modifications in breast cancer. High H3K4me3 and H3K9ac expression was correlated with survival rates. Further investigation of histone modifications in breast cancer could lead to a more profound understanding of the molecular mechanisms of cancer development and could result in new therapeutic strategies.

Deoxynivalenol (DON) is a mycotoxin produced by multipleFusariumspecies that often contaminates cereals and threatens human and animal health. A wide range of cytotoxic effects, such as the induction of DNA damage, an increase in mitochondrial permeability and the inhibition of macromolecule synthesis, have been reported. However, the effects of DON on cell migration-a fundamental process in living cells critical for normal development, immune responses, and disease processes-and the mechanism underlying these effects are still unclear. Here, we showed that DONsignificantly inhibited the migration of MRC-5, CCD-18Co, HCT116 and WM793 cells at 50 ng/ml, 50 ng/ml, 400 ng/ml and 250 ng/ml, respectively, which maintained cell viability at 90%. Further analysis showed that DON inhibited the expression of tumour endothelial marker 8 (TEM8), a key gene in cell migration. Furthermore, we showed that DON inhibited the expression of TEM8 through increasing the level of H3K27me3 in the TEM8 promoter. Finally, overexpression of TEM8 or treating by H3K27me3-specific inhibitor GSK126 attenuated the inhibitory effect of DON on cell migration. In summary, low doses of DON at approximately dietary exposure significantly inhibited cell migration by downregulating the expression of TEM8 in a manner mediated by H3K27me3, which may generate increasing concerns for the risk of DON exposure., 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 © 2020 Elsevier Inc. All rights reserved.)

Introduction: Meningiomas comprises of a wide variety of histological entities with heterogeneous biological behaviour and prognosis. The plethora of genetic data are yet to produce relevant biomarkers for routine use. In contrast, epigenetic alterations are less elucidated., Material and Methods: The expression of the key molecules involved in the two principal epigenetic systems, i.e. DNA methylation (DNA methyltransferases [DNMT-1, -3A and -3B]) and histone modification (Enhancer of Zeste homolog-2 [EZH2] and trimethyl histone-3 [H3K27me3]) were assessed in 149 cases of meningiomas (grade I - 102, grade II - 47) by immunohistochemistry., Results: Immunopositivity for EZH2 (38.3% vs. 6.0%) and negativity for H3K27me3 (10.6% vs. 1.0%) were significantly more common in grade II tumours. Both were associated with significantly higher proliferative activity. The majority of the cases of both grades showed expression of all three DNMTs. However, high expression of DNMT-1 was significantly more common in grade II tumours (87.8% vs. 66.2%). Expression of EZH2 and loss of H3K27me3 were associated with significantly shorter progression-free survival (hazard ratio [HR] = 4.07 and 0.24, respectively)., Conclusions: The key epigenetic regulators play important roles in the pathobiology of meningiomas. EZH2 positivity and H3K27me3 negativity are associated with aggressive tumour-biology and poor prognosis. Both these markers can easily be assessed by immunohistochemistry and can be incorporated in routine practice.

Nucleosomes, the basic unit of chromatin, contain a protein core of histone proteins, which are heavily posttranslationally modified. These modifications form a combinatorial language which defines the functional state of the underlying genome. As each histone type exists in two copies in a nucleosome, the modification patterns can differ between the individual histones, resulting in asymmetry and increasing combinatorial complexity. To systematically explore the regulation of chromatin regulatory enzymes (writers, erasers, or readers), chemically defined nucleosomes are required. We have developed strategies to chemically modify histones and control nucleosome assembly, thereby enabling the reconstitution of asymmetric histone modification patterns. Here, we report a detailed protocol for the modular assembly of such nucleosomes. Employing a three-segment ligation strategy for the semisynthesis of H3, coupled with the use of the protease cleavable "lnc-tag," we provide an efficient and traceless method for the controlled semisynthesis and reconstitution of asymmetrically modified nucleosomes.

Histone acetylation plays a major role in epigenetic regulation of gene expression. Monocyte-derived macrophages express functional NADPH oxidase 5 (Nox5) that contributes to oxidative stress in atherogenesis. The mechanisms of Nox5 regulation are not entirely elucidated. The aim of this study was to investigate the expression pattern of key histone acetyltransferase subtypes (p300, HAT1) in human atherosclerosis and to determine their role in mediating the upregulation of Nox5 in macrophages under inflammatory conditions. Human nonatherosclerotic and atherosclerotic tissue samples were collected in order to determine the expression of p300 and HAT1 isoforms, H3K27ac, and Nox5. In vitro determinations were done on human macrophages exposed to lipopolysaccharide in the absence or presence of histone acetyltransferase inhibitors. Western blot, immunohistochemistry, immunofluorescence, real-time PCR, transfection, and chromatin immunoprecipitation assay were employed. The protein levels of p300 and HAT1 isoforms, H3K27ac, and Nox5 were found significantly elevated in human atherosclerotic specimens. Immunohistochemistry/immunofluorescence staining revealed that p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins were colocalized in the area of CD45 + /CD68 + immune cells and lipid-rich deposits within human atherosclerotic plaques. Lipopolysaccharide induced the levels of HAT1, H3K27ac, H3K9ac, and Nox5 and the recruitment of p300 and HAT1 at the sites of active transcription within Nox5 gene promoter in cultured human macrophages. Pharmacological inhibition of histone acetyltransferase significantly reduced the Nox5 gene and protein expression in lipopolysaccharide-challenged macrophages. The overexpression of p300 or HAT1 enhanced the Nox5 gene promoter activity. The histone acetyltransferase system is altered in human atherosclerosis. Under inflammatory conditions, HAT subtypes control Nox5 overexpression in cultured human macrophages. The data suggest the existence of a new epigenetic mechanism underlying oxidative stress in atherosclerosis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 Mihaela-Loredana Vlad et al.)

Histone H2AX undergoes phosphorylation as an answer to DNA double-strand breaks, which in turn are part of the oncogenic procedure. The detection of gamma-H2AX can potentially serve as a biomarker for transformation of normal tissue to premalignant and consequently to malignant tissues. The aim of this study was to evaluate the clinical significance of gamma-H2AX expression in breast cancer. Gamma-H2AX expression in tissues from 110 breast cancer patients was analyzed by immunohistochemistry and correlated with clinicopathological variables. Greater tumor size, higher grade, and the number of affected lymph nodes are significantly associated with greater values of gamma-H2AX. In addition, gamma-H2AX differs significantly among patients' International Federation of Gynecology and Obstetrics stage. Higher values of estrogen receptor and progesterone receptor are significantly associated with lower gamma-H2AX values. In conclusion, a positive association between gamma-H2AX expression and infaust histopathological parameters was observed.

H2AX phosphorylation is a novel marker of DNA double-stranded breaks. In the present study, we assessed the γ-H2AX expression, its association with other clinicopathologic characteristics, and the prognosis in a cohort of 97 patients with breast cancer. Ninety-seven specimens of tumor tissue and 77 adjacent normal tissues from patients with breast cancer were examined. All patients underwent modified radical mastectomy or local tumor resection without lymph node dissection. γ-H2AX expression was assessed by standard immunohistochemistry. Patients were followed after surgery for a mean duration of 70.1 ± 18.7 months (range, 6-93 months). The γ-H2AX staining was positive in 27 (27.8%) patients. The positive rates of H2AX were 26.0% and 2.6% in tumor tissue and adjacent normal tissues, respectively. γ-H2AX positive status was negatively associated with TNM staging, with 24 positive cases (32.4%) in TNM staging I-II, while no positive cases in TNM staging III-IV (P = 0.026). Sixteen patients (16.5%) died during the follow-up. No significant association between γ-H2AX expression and patient survival was detected. The unadjusted HR (hazard ratio) for γ-H2AX positive was 0.84 (95% CI: 0.27, 2.60). In TNM staging subgroup analysis, death only occurred in γ-H2AX negative patients. Our study is the first study to demonstrate that expression of γ-H2AX is associated with TNM staging. Due to the small sample and limited follow-up time, we did not observe a significant association between γ-H2AX and patient survival. γ-H2AX expression could be a potential biomarker for cancer diagnosis and prediction, and further studies are in need., (© 2019 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.)

Insoluble radioactive microparticles emitted by the incident at the Fukushima nuclear power plant have drawn keen interests from the viewpoint of radiation protection. Cs-bearing particles have been assumed to adhere in the long term to trachea after aspirated into respiratory system, leading to heterogeneous dose distribution within healthy tissue around the particles. However, the biological effects posed by an insoluble radioactive particle remain unclear. Here, we show cumulative DNA damage in normal human lung cells proximal and distal to the particle (β-ray and γ-ray-dominant areas, respectively) under localized chronic exposure in comparison with uniform exposure. We put a Cs-bearing particle into a microcapillary tip and placed it onto a glass-base dish containing fibroblast or epithelial cells cultured in vitro. A Monte Carlo simulation with PHITS code provides the radial distribution of absorbed dose-rate around the particle, and subsequently we observed a significant change in nuclear γ-H2AX foci after 24 h or 48 h exposure to the particle. The nuclear foci in the cells distal to the particle increased even under low-dose-rate exposure compared with uniform exposure to 137 Cs γ-rays, which was suppressed by a treatment with a scavenger of reactive oxygen species. In contrast, such focus formation was less manifested in the exposed cells proximal to the particle compared with uniform exposure. These data suggest that the localized exposure to a Cs-bearing particle leads to not only disadvantage to distal cells but also advantage to proximal cells. This study is the first to provide quantitative evaluation for the spatial distribution of DNA double strand breaks after the heterogeneous chronic exposure to a Cs-bearing particle in comparison with uniform Cs exposure.

Green tea polyphenols (GTPs) and their major constituent, epigallocatechin-3-gallate (EGCG), have been reported to demonstrate many interesting biological activities, including anticancer properties. Recent studies on prostate cancer provide strong evidence that epigenetic mechanisms are major players in the regulation of matrix metalloproteinases (MMPs) and their binding partner tissue inhibitor of MMPs (TIMPs) involved in prostate cancer progression. Here we demonstrate that GTP/EGCG mediate epigenetic reactivation of TIMP-3 that plays a key role in suppressing invasiveness and cancer progression. Treatment of human prostate cancer DUPRO and LNCaP cells with 10 µg/mL GTP and 20 µM EGCG induced TIMP-3 mRNA and protein expression. This transcriptional activation of TIMP-3 was associated with the decrease in the expression of both enhancers of zeste homolog 2 (EZH2) and its catalytic product trimethylation of histone H3 at lysine 27 (H3K27me3) repressive marks at the TIMP-3 promoter with an accompanying increase in histone H3K9/18 acetylation. In addition, GTP/EGCG treatment significantly reduced class I histone deacetylase (HDAC) activity/expression and EZH2 and H3K27me3 levels in prostate cancer cells. EGCG/GTP exposure also reduced MMP-2/MMP-9 gelatinolytic activity and abrogated invasion and migration capabilities in these cells. Silencing of EZH2 and class I HDACs strikingly increased the expression of TIMP-3 independent of DNA methylation. Furthermore, clinical trials performed on patients undergoing prostatectomy consuming 800 mg EGCG (Polyphenon E) up to 6 weeks and grade-matched controls demonstrate an increase in plasma TIMP-3 levels. A marked reduction in class I HDACs activity/expression and EZH2 and H3K27me3 levels were noted in GTP-supplemented prostate tissue. Our findings highlight that TIMP-3 induction, as a key epigenetic event modulated by green tea in restoring the MMP:TIMP balance suppresses prostate cancer progression., (© 2019 Wiley Periodicals, Inc.)

Replication-dependent histone (RDH) mRNAs have a nonpolyadenylated 3'-UTR that ends in a highly conserved stem-loop structure. Nonetheless, a subset of RDH mRNAs has a poly(A) tail under physiological conditions. The biological meaning of poly(A)-containing (+) RDH mRNAs and details of their biosynthesis remain elusive. Here, using HeLa cells and Western blotting, qRT-PCR, and biotinylated RNA pulldown assays, we show that poly(A) + RDH mRNAs are post-transcriptionally regulated via adenylate- and uridylate-rich element-mediated mRNA decay (AMD). We observed that the rapid degradation of poly(A) + RDH mRNA is driven by butyrate response factor 1 (BRF1; also known as ZFP36 ring finger protein-like 1) under normal conditions. Conversely, cellular stresses such as UV C irradiation promoted BRF1 degradation, increased the association of Hu antigen R (HuR; also known as ELAV-like RNA-binding protein 1) with the 3'-UTR of poly(A) + RDH mRNAs, and eventually stabilized the poly(A) + RDH mRNAs. Collectively, our results provide evidence that AMD surveils poly(A) + RDH mRNAs via BRF1-mediated degradation under physiological conditions., (© 2019 Ryu and Kim.)

Mequindox (MEQ) is a synthetic antibacterial agent. Recent studies showed that MEQ and its primary metabolites exhibit strong genotoxicity to mammalian cells, and MEQ induced carcinogenicity in mice. These findings suggest that chronic exposure to MEQ could lead to an increased risk of cancer later in life. In the present study, four groups of Wistar rats (55 rats/sex/group) were fed with diets containing MEQ (0, 25, 55, and 110 mg/kg) for 2 years. The results showed that the hematological system, liver, kidneys, and adrenal glands, as well as the developmental and reproductive systems, were the main targets for MEQ. Liver toxicity mediated by MEQ was associated with apoptosis and the nuclear factor κB (NF-κB) signaling pathway. In addition, MEQ increased the incidence of tumors in rats. Phosphorylated histone H2AX (γ-H2AX) is identified as a biomarker of cellular response to DNA double-strand breaks (DSB). Our data demonstrated that γ-H2AX expression was significantly increased in tumors. Thus, high levels of DSB might be responsible for carcinogenesis in rats, and further investigation is absolutely required to clarify the exact molecular mechanisms for carcinogenicity caused by MEQ in vivo., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Diethylstilbestrol (DES), an estrogen agonist, increases prolactin (PRL) cells through transdifferentiation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells to PRL cells as well as proliferation of PRL cells in adult male mouse pituitary. Since hyperacetylation of histone H3 is implicated in the regulation of activation of various genes, we examined the effect of DES on the state of histone H3 acetylation. DES significantly reduced the immunohistochemical signal for acetylated histone H3 at lysine 9 (H3K9ac) in PRL, LH and FSH cells, but not for H3K18ac or H3K23ac. DES-treated mice were injected intraperitoneally with HDAC inhibitors (HDACi), sodium phenylbutyrate (NaPB) or valproic acid (VPA), to mimic the acetylation level of histone H3. As expected, HDACi treatment restored the level of H3K9ac expression in these cells, and also inhibited DES-induced increase in PRL cells. Furthermore, NaPB and VPA also abrogated the effects of DES on the population density of both LH and FSH cells. Similarly, the numbers of proliferating and apoptotic cells in the pituitary in NaPB- or VPA-treated mice were comparable to those of the control mice. Considered together, these results indicated that the acetylation level of histone H3 plays an important role in DES-induced transdifferentiation of LH to PRL cells as well as proliferation of PRL cells.

Histone ubiquitylation and deubiquitylation processes and the mechanisms of their regulation are closely relevant to the field of epigenetics. Recently, the deubiquitylating enzyme USP51 was reported to selectively cleave ubiquitylation on histone H2A at K13 or K15 (i.e., H2AK13Ub and H2AK15Ub), but not at K119 (i.e., H2AK119Ub), in nucleosomes in vivo. To elucidate the mechanism for the selectivity of USP51, we constructed structurally well-defined in vitro protein systems with a ubiquitin modification at precise sites. A total chemical protein synthesis procedure was developed, wherein hydrazide-based native chemical ligation was used to efficiently generate five ubiquitylated histones (H2AK13Ub, H2AK15Ub, H2AK119Ub, H2BK34Ub, and H2BK120Ub). These synthetic ubiquitylated histones were assembled into nucleosomes and subjected to in vitro USP51 deubiquitylation assays. Surprisingly, USP51 did not show preference between H2AK13/15Ub and H2AK119Ub, in contrast to previous in vivo observations. Accordingly, an understanding of the selectivity of USP51 may require consideration of other factors, such as alternative pre-existing histone modifications, competitive reader proteins, or different nucleosome quality among the in vivo extraction nucleosome and the in vitro reconstitution one. Further experiments established that USP51 in vitro could deubiquitylate a nucleosome carrying H2BK120Ub, but not H2BK34Ub. Molecular dynamics simulations suggested that USP51-catalyzed hydrolysis of ubiquitylated nucleosomes was affected by steric hindrance of the isopeptide bond., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To investigate the genotoxicity of gaseous toxicants carbon monoxide (CO) and 1,3-butadiene in vitro, a novel combination technology-the in vitro γH2AX high content screening assay combined with air-liquid interface system (ALIS) was established. The results showed that this new technology was available and effective. Based on the joint technology, genotoxicity of CO and 1,3-butadiene was evaluated further in this study. The results showed that treatment concentrations (0, 20%,40%, 80% and 100%, v/v) and exposure time (15, 30, 45, 60 and 90 min) of CO both had no statistically significant effects on the induction of γH2AX (p > 0.05). However, 1,3-butadiene can induce significant γH2AX (p < 0.01) in A549 cells in a dose/time-dependent manner both in the absence and presence of rat liver S9. When the concentrations of 1,3-butadiene were more than 80%, a higher γH2AX level could be induced than the 1.5-fold of vehicle controls after 1 h of treatment. Overall, this new technology can be used as a complementary tool to evaluate the genotoxicity of air-borne toxicants in vitro based on the in vitro γH2AX high content screening assay combined with ALIS. Based on the joint technology, CO was not genotoxic in A549 cells, while 1,3-butadiene showed significant genotoxicity in the dose/time-dependency on the induction of γH2AX.

Enhancer of zeste homolog 2 (EZH2), an H3K27-specific histone methyltransferase, has been shown to be frequently overexpressed in various human cancers including lymphoma. Here we investigate the expression and functionality of EZH2 and H3K27me3 in extranodal NK/T-cell lymphoma, nasal type (ENKTL). Results of NanoString analysis revealed that EZH2 and related histone H3 families were up-regulated genes in ENKTL tissues. Results of immunohistochemistry demonstrated that EZH2 and trimethylation of Lys-27 in histone (H3K27me3) were highly expressed in 55.2% and 78.0% of patients with ENKTL, respectively. EZH2 overexpression was significantly associated with higher tumor cell proliferation (r = 0.582, P = .000), advanced stage (P = .012), and predicted poorer overall survival (P = .016) in ENKTL. H3K27me3-positive expression was correlated with lower tumor cell proliferation (r = -0.623, P = .036), earlier stage (P = .043), and predicted better overall survival (P = .020). In addition, EZH2 and H3K27me3 showed inverse correlations (r = -0.652, P = .002) in clinical samples by immunohistochemistry. Furthermore, inhibition of EZH2 by 3-deazaneplanocin A significantly suppressed tumor cell growth. Interestingly, pharmacologic suppression of the JAK3/STAT3 pathway effectively reduced EZH2 and enhanced H3K27me3 in NK/T tumor cell lines. Our data suggest that EZH2 and H3K27me3 are important prognostic markers and potential therapeutic targets in ENKTL., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The dysregulation of microRNAs (miRNAs) is associated with the pathogenesis of non-small cell lung cancer (NSCLC). However, the mechanisms by which miR-516a-5p contributes to NSCLC remain unclear. The association between miR-516a-5p expression and the clinicopathological characteristics and prognosis in patients with NSCLC was analyzed by The Cancer Genome Atlas (TCGA) data set. The targets of miR-516a-5p were identified by bioinformatic analysis and luciferase report assay. MTT and soft agar assays were conducted to investigate the function of miR-516a-5p in NSCLC cells. We found that the expression of miR-516a-5p was decreased in NSCLC tissues and associated with the age, pathological stage, and tumor size, acting as an independent prognostic factor of tumor recurrence in patients with NSCLC. Restoration of miR-516a-5p inhibited the cell viability and anchorage-independent growth of NSCLC cells, but its inhibitor had the opposite effects. Histone cluster 3 H2A (HIST3H2A) was further identified as a direct target of miR-516a-5p and displayed a negative correlation with miR-516a-5p expression in NSCLC tissues. Overexpression of HIST3H2A reversed the anti-proliferation effects induced by miR-516a-5p and acted as an independent prognostic factor of poor survival in patients with NSCLC. Altogether, our findings demonstrate that miR-516a-5p may function as a tumor suppressive factor in NSCLC cells by targeting HIST3H2A and might represent a potential indicator of tumor recurrence in patients with NSCLC.

Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo β-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target., Competing Interests: IP, PG, CR, JB, MM, SD, CS No competing interests declared, NP Reviewing editor, eLife, (© 2018, Pettinati et al.)

Maintenance of epigenetic integrity relies on coordinated recycling and partitioning of parental histones and deposition of newly synthesized histones during DNA replication. This process depends upon a poorly characterized network of histone chaperones, remodelers, and binding proteins. Here we implicate the POLE3-POLE4 subcomplex of the leading-strand polymerase, Polε, in replication-coupled nucleosome assembly through its ability to selectively bind to histones H3-H4. Using hydrogen/deuterium exchange mass spectrometry and physical mapping, we define minimal domains necessary for interaction between POLE3-POLE4 and histones H3-H4. Biochemical analyses establish that POLE3-POLE4 is a histone chaperone that promotes tetrasome formation and DNA supercoiling in vitro. In cells, POLE3-POLE4 binds both newly synthesized and parental histones, and its depletion hinders helicase unwinding and chromatin PCNA unloading and compromises coordinated parental histone retention and new histone deposition. Collectively, our study reveals that POLE3-POLE4 possesses intrinsic H3-H4 chaperone activity, which facilitates faithful nucleosome dynamics at the replication fork., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

Background: Excessive intake of a high-calorie diet has been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Several studies have investigated the effect of NAFLD on liver regeneration, but the effects of simple steatosis have been found to be inconsistent. We aimed to assess whether the initial phase of diet-induced lipid accumulation, induced by a diet containing moderate levels of fat, impairs liver regeneration after partial hepatectomy (PHx) of mice., Methods: Male ddY mice are prone to obesity, even when fed a relatively low-fat diet (FD). A model of early simple steatosis was created by feeding a 10% FD for 6 weeks to male ddY mice. Liver regeneration rate, DNA synthesis in hepatocytes, and DNA damage were then assessed., Results: FD-fed mice had a slightly higher body mass (44.5 ± 2.6 grams vs. 48.1 ± 3.6 grams, P < 0.05), but not liver mass (2.55 ± 0.37 grams vs. 2.69 ± 0.26 grams). Lipid droplets appeared in FD-fed mouse hepatocytes and Oil Red O staining was five times as intense as in control mice. In FD-fed mice, liver regeneration rate after two-thirds PHx was lower and impaired DNA replication was also observed. FD induced the expression of the cyclin-dependent kinase inhibitor p27KIP, but not p21CIP. Moreover, greater histone 2A.X phosphorylation was observed, indicating that FD caused DNA damage., Conclusions: Even short-term feeding of a moderately high FD to male ddY mice results in lipid accumulation in hepatocytes, DNA damage, and greater expression of p27KIP, implying lower DNA synthesis.

Coordination of cell growth with division is essential for proper cell function. In budding yeast, although some molecular mechanisms responsible for cell size control during G1 have been elucidated, the mechanism by which cell size homeostasis is established remains to be discovered. Here, we developed a new technique based on quantification of histone levels to monitor cell cycle progression in individual cells with unprecedented accuracy. Our analysis establishes the existence of a mechanism controlling bud size in G2/M that prevents premature onset of anaphase, and controls the overall size variability. While most G1 mutants do not display impaired size homeostasis, mutants in which cyclin B-Cdk regulation is altered display large size variability. Our study thus demonstrates that size homeostasis is not controlled by a G1-specific mechanism alone but is likely to be an emergent property resulting from the integration of several mechanisms that coordinate cell and bud growth with division., Competing Interests: CG, OT, AM, NM, GC No competing interests declared, (© 2018, Garmendia-Torres et al.)

Formation of the 3' ends of mature mRNAs requires recognition of the correct site within the last exon, cleavage of the nascent pre-mRNA, and, for most mRNAs, addition of a poly(A) tail. Several factors are involved in recognition of the correct 3'-end site. The cleavage stimulation factor (CstF) has three subunits, CstF-50 (gene symbol Cstf1), CstF-64 (Cstf2), and CstF-77 (Cstf3). Of these, CstF-64 is the RNA-binding subunit that interacts with the pre-mRNA downstream of the cleavage site. In male germ cells where CstF-64 is not expressed, a paralog, τCstF-64 (gene symbol Cstf2t) assumes its functions. Accordingly, Cstf2t knockout (Cstf2t -/- ) mice exhibit male infertility due to defective development of spermatocytes and spermatids. To discover differentially expressed genes responsive to τCstF-64, we performed RNA-Seq in seminiferous tubules from wild-type and Cstf2t -/- mice, and found that several histone and histone-like mRNAs were reduced in Cstf2t -/- mice. We further observed delayed accumulation of the testis-specific histone, H1fnt (formerly, H1t2 or Hanp1) in Cstf2t -/- mice. High-throughput sequence analysis of polyadenylation sites (A-seq) indicated reduced use of polyadenylation sites within a cluster downstream of H1fnt in knockout mice. However, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) was not consistent with a direct role of τCstF-64 in polyadenylation of H1fnt. These findings together suggest that the τCstF-64 may control other reproductive functions that are not directly linked to the formation of 3' ends of mature polyadenylated mRNAs during male germ cell formation., (© 2018 American Society of Andrology and European Academy of Andrology.)

The innate immune system has numerous mechanisms to fight against pathogens, including the formation of neutrophil extracellular traps (NETs). By spreading out chromatin, antimicrobial peptides and enzymes, neutrophils efficiently trap pathogens like bacteria and facilitate their elimination. During this process, high concentrations of extracellular histones can be reached. Several researchers have demonstrated that the cytotoxic characteristics of these histones can trigger diseases like sepsis. Interestingly, the carbohydrate polysialic acid (polySia) can bind histones and reduce histone-mediated cytotoxicity in a chain length-dependent manner. In the present study, we examined the chain length of polySia in plasma and tested its ability to decrease the cytotoxic characteristics of extracellular histones. Remarkably, we detected polySia not only in the soluble fraction of plasma, but also on enriched extracellular vesicles (EVs). Chain length analysis revealed that polySia chains originating from human plasma can consists of more than 40 sialic acid residues and show a cytoprotective effect against extracellular histones. Intriguingly, polySia is not only present in human plasma but also in fish and other branches of vertebrates. Thus, polySia is a physiological element in plasma and may represent a natural buffer for extracellular histones.

Rainbow trout cell cultures were exposed to three genotoxicants and examined for effects on γH2AX and p53 levels by western blotting and on cell viability using the indicator dyes Alamar Blue (AB) for energy metabolism and 5'-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for plasma membrane integrity. Bleomycin induced γH2AX and p53 in a dose- and time-dependent manner and had little cytotoxic effect. However, induction was first seen at 0.3 μM for γH2AX but not until 16.5 μM for p53. Methyl methanesulfonate (MMS) increased H2AX phosphorylation but diminished p53 levels as the dose was increased from 908 μM up to 2724 μM. Over this dose range cell viability was progressively lost. 4-nitroquinoline N-oxide (NQO) induced both γH2AX and p53, beginning at 62.5 nM, which was also the concentration at which cell viability began to decline. As the NQO concentration increased further, elevated γH2AX was detected at up to 2.0 μM, while p53 was elevated up to 1.0 μM. Therefore, H2AX phosphorylation was superior to p53 levels as a marker of DNA damage caused by genotoxicants that act by introducing double-stranded DNA breaks (bleomycin), alkyl groups (MMS), and quinoline adducts (NQO)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Head and neck high grade malignant peripheral nerve sheath tumors (HN-MPNSTs) are rare highly aggressive soft tissue sarcomas that show overlapping morphologic and immunophenotypic features with melanoma and other high grade sarcomas, resulting in diagnostic challenges, particularly in sporadic settings. Recent discoveries have implicated loss of function mutations in the polycomb repressive complex 2 (PRC2) components, including EED or SUZ12 genes, as one of the leading pathogenetic mechanisms in high grade MPNST. MPNSTs with PRC2 loss are associated with complete loss of trimethylation at lysine 27 of histone H3 (H3K27me3), which emerged as a reliable immunohistochemical marker in the diagnosis of sporadic and radiation induced MPNST. As the diagnosis of MPNST in the HN is particularly challenging to distinguish from melanoma and other sarcoma types, we carried out a clinicopathologic analysis on HN-MPNST patients managed at our institution over a 20-year period (1997-2016), using the latest diagnostic criteria including H3K27me3 staining and other molecular investigations. The overall survival of HN-MPNST was compared with other HN soft tissue sarcomas. The diagnosis of HN-MPNST was confirmed in 13 patients (seven males and six females), with a mean age of 31 years; with 3 (23%) patients being of pediatric age. The most common site was the neck soft tissue (77%). Two-thirds of patients (n = 9) had stigmata of NF1, three had prior radiotherapy and only one developed a de novo MPNST. All except one tumor (86%) tested showed loss of H3K27me3 expression, including all non-NF1 patients. The 2 and 5-year DSS rates were 50 and 30%. The 2-year DFS rate was 21%. Adverse predictors on DSS included adult age (p = 0.011), prior-history of RT (p = 0.003) and recurrence (p = 0.003). Compared to other molecularly confirmed subsets of HN sarcomas (Ewing and Ewing-like sarcoma, rhabdomyosarcoma and synovial sarcoma), HN-MPNST had the worst overall survival (p < 0.0001). We conclude that HN-MPNSTs are highly aggressive sarcomas associated with an unfavorable outcome and the utility of H3K27me3 IHC stains in the evaluation of MPNST is a reliable ancillary diagnostic adjunct.

Dronedarone is used to treat patients with cardiac arrhythmias and has been reported to be associated with liver injury. Our previous mechanistic work demonstrated that DNA damage-induced apoptosis contributes to the cytotoxicity of dronedarone. In this study, we examined further the underlying mechanisms and found that after a 24-h treatment of HepG2 cells, dronedarone caused cytotoxicity, G1-phase cell cycle arrest, suppression of topoisomerase II, and DNA damage in a concentration-dependent manner. We also investigated the role of cytochrome P450s (CYPs)-mediated metabolism in the dronedarone-induced toxicity using our previously established HepG2 cell lines expressing individually 14 human CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). We demonstrated that CYP3A4, 3A5, and 2D6 were the major enzymes that metabolize dronedarone, and that CYP3A7, 2E1, 2C19, 2C18, 1A1, and 2B6 also metabolize dronedarone, but to a lesser extent. Our data showed that the cytotoxicity of dronedarone was decreased in CYP3A4-, 3A5-, or 2D6-overexpressing cells compared to the control HepG2 cells, indicating that the parent dronedarone has higher potency than the metabolites to induce cytotoxicity in these cells. In contrast, cytotoxicity was increased in CYP1A1-overexpressing cells, demonstrating that CYP1A1 exerts an opposite effect in dronedarone's toxicity, comparing to CYP3A4, 3A5, or 2D6. We also studied the involvement of topoisomerase II in dronedarone-induced toxicity, and demonstrated that the overexpression of topoisomerase II caused an increase in cell viability and a decrease in γ-H2A.X induction, suggesting that suppression of topoisomerase II may be one of the mechanisms involved in dronedarone-induced liver toxicity.

Intrahepatic cholangiocarcinoma (ICC) is a fatal, malignant tumor of the liver; effective diagnostic biomarkers and therapeutic targets for ICC have not been identified yet. High expression of H2A histone family member Z (H2A.Z) is a high-risk factor for poor prognosis in patients with breast cancer and primary hepatocellular cancer. However, the significance of H2A.Z and its expression in ICC remains unknown. The present study demonstrated that H2A.Z is overexpressed in ICC and expression of H2A.Z correlated with poor prognosis in patients with ICC. H2A.Z regulated cell proliferation in vitro and in vivo via H2A.Z/S-phase kinase-associated protein 2/p27/p21 signaling. Inhibition of H2A.Z reduced cell proliferation and induced apoptosis in ICC. In addition, downregulation of H2AZ reduced tumor metastasis by repressing epithelial-mesenchymal transition and enhanced the antitumor effects of cisplatin in the treatment of ICC. Overall, H2A.Z promoted cell proliferation and epithelial-mesenchymal transition in ICC, suggesting that H2A.Z may be a novel biomarker and therapeutic target for ICC.

Background: Increasing cannabis consumption among adolescents, studies that link its early use with mental illnesses, and the political debate on cannabis legalization together call for an urgent need to study molecular underpinnings of adolescent brain vulnerability. The emerging role of epigenetic mechanisms in psychiatric diseases led us to hypothesize that epigenetic alterations could play a role in causes and subsequent development of the depressive/psychotic-like phenotype induced by adolescent, but not adult, Δ9-tetrahydrocannabinol (THC) exposure in female rats., Methods: We performed a time-course analysis of histone modifications, chromatin remodelling enzymes and gene expression in the prefrontal cortex of female rats after adolescent and adult THC exposure. We also administered a specific epigenetic drug (chaetocin) with THC to investigate its impact on THC-induced behavioural alterations., Results: Adolescent THC exposure induced alterations of selective histone modifications (mainly H3K9me3), impacting the expression of genes closely associated with synaptic plasticity. Changes in both histone modifications and gene expression were more widespread and intense after adolescent treatment, suggesting specific adolescent susceptibility. Adolescent THC exposure significantly increased Suv39H1 levels, which could account for the enhanced H3K9me3. Pharmacological blockade of H3K9me3 during adolescent THC treatment prevented THC-induced cognitive deficits, suggesting the relevant role played by H3K9me3 in THC-induced effects., Limitations: Only female rats were investigated, and the expression studies were limited to a specific subset of genes., Conclusion: Through a mechanism involving SUV39H1, THC modifies histone modifications and, thereby, expression of plasticity genes. This pathway appears to be relevant for the development of cognitive deficits.

Malarial infection in naive individuals induces a robust innate immune response. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed training) or hyporespond (tolerance) to subsequent immune challenge. Previous work in both mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with Plasmodium falciparum -infected RBCs or the malaria crystal hemozoin induced human adherent PBMCs to hyperrespond to subsequent ligation of TLR2. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that the induction of trained immunity by malaria and its ligands may occur via a previously unrecognized mechanism(s)., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Given the potential biological functions of spermatogonial stem cells (SSCs) in spermatogenesis and in delivering parental genetic information to the next generation, how these cells respond to environmental toxins and carcinogens should be investigated. We examined the toxic effect of hexavalent chromium (Cr(VI)) on global histone modifications and apoptotic signaling pathways in SSCs. We determined the effect of melatonin, one of the most powerful endogenous free radical scavengers and wide-spectrum antioxidants, in protecting SSCs from Cr(VI)-induced apoptosis and global histone modification by Western blot analysis. In addition, we examined the in vivo effect of melatonin on Cr(VI)-induced histological changes of seminiferous tubules in mouse testes. We also evaluated the fertility of male mice by monitoring litter size following intraperitoneal injection of these chemicals. Our study demonstrated the Cr(VI)-induced global increases in H3K9me3 and H3K27me3 and activated the apoptotic signaling pathway. Pretreatment of SSCs with melatonin alleviated Cr(VI)-induced apoptosis and the global increase of H3K9me3. Exposure to melatonin also attenuated the Cr(VI)-induced increase of the abundance of histone methyltransferase ESET. Furthermore, exogenous administration of melatonin protected mice against Cr(VI)-induced changes in testicular histology and germ cell apoptosis, which helped maintain normal spermatogenesis and male fertility. Our study revealed a potential new therapeutic approach for male reproductive injury caused by Cr(VI)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/aim: Lung squamous cell carcinoma often arises from precancerous lesions where alterations in tumor suppressor genes and subsequent chromosomal instability are often observed due to carcinogen exposure. These tumors are often immunogenic; as such, immune checkpoint inhibitors are a promising therapeutic option. We hypothesized that the DNA damage response in tumor cells induces an immune response, thereby up-regulating programmed death-ligand 1 (PD-L1) expression on tumor cells, which in turn sensitizes them to anti-PD-1 therapy., Patients and Methods: An immunohistochemical analysis was performed in 41 consecutive lung squamous cell carcinoma patients who underwent surgery at our institution between April 2013 and March 2014., Results: The analysis revealed a high PD-L1 expression in 15 patients (37%) (p=0.028). The PD-L1 expression was positively associated with the nuclear γH2AX expression (p=0.02), that was confirmed by immunofluorescent staining., Conclusion: Our findings demonstrate that nuclear γH2AX expression is positively associated with the PD-L1 expression in lung squamous cell carcinoma., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: Multimodal treatments that include preoperative platinum-based chemotherapy are fundamental to the treatment of advanced non-small cell lung cancer (NSCLC). This study aimed to investigate the predictive value of DNA repair protein expression in surgically resected NSCLCs in terms of prognosis and responses to platinum-containing chemotherapy., Methods: This retrospective study included 136 patients with NSCLC who were treated with preoperative platinum-based chemotherapy, followed by curative lung resection. ATM, RAD51, LKB1, H2AX, and SIRT1 expression levels were analyzed in resected tumor specimens via immunostaining and were used to classify patients and compare survival and responses to chemotherapy., Results: SIRT1 expression correlated significantly with improved responses to platinum-based chemotherapy (odds ratio, 2.28; p = 0.024), progression-free survival (hazard ratio [HR], 0.74; p = 0.036), overall survival (HR, 0.63; p = 0.006), and tumor-bearing survival (HR, 0.62; p = 0.014). After adjusting for clinical variables, the HR of SIRT1 expression remained significant for overall survival (HR, 0.59; p = 0.039) but not for progression-free survival (HR, 0.74; p = 0.183). No prognostic stratification was observed for the other 4 markers., Conclusion: Patients with SIRT1-expressing NSCLC had superior responses to chemotherapy and longer survival durations than those with SIRT1-negative cancer., (© 2018 S. Karger AG, Basel.)

Distinct histone variants mark chromatin domains in the nucleus. To understand how these marks are established and maintained, one has to decipher how the dynamic distribution of these variants is orchestrated. These dynamics are associated with all DNA-based processes such as DNA replication, repair, transcription, heterochromatin formation and chromosome segregation. Key factors, known as histone chaperones, have been involved in escorting histones, thereby contributing to the chromatin landscape of given cell types. SNAP-tag-based imaging system enables the distinction between old and newly deposited histones, and has proved to be a powerful method for the visualization of histone variant dynamics on a cell-by-cell basis. This approach enables the tracking of specific variants in vivo and defining their timing and mode of deposition throughout the cell cycle and in different nuclear territories. Here, we provide a detailed protocol to exploit the SNAP-tag technology to assess the dynamics of newly synthesized and old histones. We then show that combining the SNAP-tagging of histones with the knockdown of candidate factors, represents an effective approach to decipher the role of key actors in guiding histone dynamics. Here, we specifically illustrate how this strategy was used to identify the essential role of the chaperone HIRA in deposition of newly synthesized histone variant H3.3.

Topoisomerase IIβ (topoIIβ) is a nuclear enzyme specifically expressed in neurons, and plays an important role in the development of the cerebellum. To date, the expression of topoIIβ protein in medulloblastoma (MB) has not been investigated. In this study, 16 MB specimens including 10 classical subtypes of MB and 6 desmoplastic subtypes of MB (DMB), along with 5 normal cerebellum samples, were obtained from clinics. With immunohistochemical staining, prominently expressed topoIIβ was seen in normal cerebellar tissues, while there was no or less pronounced staining in classical MB cells. Interestingly, on comparing topoIIβ expression in different regions of DMB samples, relatively high levels of topoIIβ were revealed within nodules composed of differentiated neurocytic cells, which are known to predict a favorable clinical outcome for MB. We also examined the expression of two epigenetic factors, H3K27me3 and JMJD3 in the different tissues. Very high levels of H3K27me3 were found in all MB samples, except the intranodules of DMB, where JMJD3 expression was more prominent. Furthermore, a negative correlation between topoIIβ and H3K27me3 in MB was revealed in this study. Thus, our data primarily indicate that topoIIβ can be used to estimate neuronal differentiation in MB, and may serve as a target for improving the survival rates for this condition. We speculate that H3K27me3 repression of topoIIβ at the transcriptional level may occur, although this needs to be verified using larger numbers of MB samples in future experiments., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Objectives: Accurate diagnosis of malignant peripheral nerve sheath tumor (MPNST) is often challenging on fine-needle aspiration (FNA) or core needle biopsy. Recurrent mutations in EED and SUZ12, which encode subunits of polycomb repressive complex 2 (PRC2), have been identified in 70% to 92% of MPNSTs; PRC2 inactivation leads to loss of trimethylation of lysine 27 of histone H3 (H3K27me3). We evaluated the utility of H3K27me3 immunohistochemistry for distinguishing MPNST from its cytomorphologic mimics., Methods: H3K27me3 immunohistochemistry was performed on 180 cases of spindle cell neoplasms sampled by FNA (n = 66) and needle biopsy (n = 114), and loss of nuclear staining was scored. Tumor types included MPNST, dedifferentiated liposarcoma, schwannoma, solitary fibrous tumor, leiomyosarcoma, melanoma, synovial sarcoma, sarcomatoid carcinoma, gastrointestinal stromal tumor, desmoid fibromatosis, low-grade fibromyxoid sarcoma, and unclassified spindle cell sarcoma/undifferentiated pleomorphic sarcoma., Results: Complete loss of H3K27me3 was observed in 54% (13/24) of MPNSTs. In contrast, only two (of 156) histologic mimics showed complete loss of H3K27me3. Partial loss of H3K27me3 was present in a subset of cases (26/180), including both MPNST and non-MPNSTs., Conclusions: Complete loss of H3K27me3 is a highly specific (98.7%) marker of MPNST that can distinguish MPNST from cytomorphologic mimics in FNA cell block and small biopsy specimens., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)