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Heavy water, containing the heavy hydrogen isotope, is toxic to cells, although the underlying mechanism remains incompletely understood. In addition, certain enzymatic proton transfer reactions exhibit kinetic isotope effects attributed to hydrogen isotopes and their temperature dependencies, indicative of quantum tunneling phenomena. However, the correlation between the biological effects of heavy water and the kinetic isotope effects mediated by hydrogen isotopes remains elusive. In this study, we elucidated the kinetic isotope effects arising from hydrogen isotopes of water and their temperature dependencies in vitro, focusing on deacetylation, DNA cleavage, and protein cleavage, which are crucial enzymatic reactions mediated by hydrolysis. Intriguingly, the intracellular isotope effects of heavy water, related to the in vitro kinetic isotope effects, significantly impeded multiple DNA double-strand break repair mechanisms crucial for cell survival. Additionally, heavy water exposure enhanced histone acetylation and associated transcriptional activation in cells, consistent with the in vitro kinetic isotope effects observed in histone deacetylation reactions. Moreover, as observed for the in vitro kinetic isotope effects, the cytotoxic effect on cell proliferation induced by heavy water exhibited temperature-dependency. These findings reveal the substantial impact of heavy water-induced isotope effects on cellular functions governed by hydrolytic enzymatic reactions, potentially mediated by quantum-level mechanisms underlying kinetic isotope effects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yasuda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNMT1 is an essential DNA methyltransferase that catalyzes the transfer of methyl groups to CpG islands in DNA and generates a prominent epigenetic mark. The catalytic activity of DNMT1 relies on its conformational plasticity and ability to change conformation from an auto-inhibited to an activated state. Here, we present four cryo-EM reconstructions of apo DNMT1 and DNTM1: non-productive DNA, DNTM1: H3Ub2-peptide, DNTM1: productive DNA complexes. Our structures demonstrate the flexibility of DNMT1's N-terminal regulatory domains during the transition from an apo 'auto-inhibited' to a DNA-bound 'non-productive' and finally a DNA-bound 'productive' state of DNMT1. Furthermore, we address the regulation of DNMT1's methyltransferase activity by a DNMT1-selective small-molecule inhibitor and ubiquitinated histone H3. We observe that DNMT1 binds DNA in a 'non-productive' state despite the presence of the inhibitor and present the cryo-EM reconstruction of full-length DNMT1 in complex with a di-ubiquitinated H3 peptide analogue. Taken together, our results provide structural insights into the reaction cycle of DNMT1., Competing Interests: "N.C. has been employee of GlaxoSmithKline (GSK) when this work has been carried out. The remaining authors declare no competing interests.", (Copyright: © 2024 De et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chromatin exhibits non-random distribution within the nucleus being arranged into discrete domains that are spatially organized throughout the nuclear space. Both the spatial distribution and structural rearrangement of chromatin domains in the nucleus depend on epigenetic modifications of DNA and/or histones and structural elements such as the nuclear envelope. These components collectively contribute to the organization and rearrangement of chromatin domains, thereby influencing genome architecture and functional regulation. This study develops an innovative, user-friendly, ImageJ-based plugin, called IsoConcentraChromJ, aimed quantitatively delineating the spatial distribution of chromatin regions in concentric patterns. The IsoConcentraChromJ can be applied to quantitative chromatin analysis in both two- and three-dimensional spaces. After DNA and histone staining with fluorescent probes, high-resolution images of nuclei have been obtained using advanced fluorescence microscopy approaches, including confocal and stimulated emission depletion (STED) microscopy. IsoConcentraChromJ workflow comprises the following sequential steps: nucleus segmentation, thresholding, masking, normalization, and trisection with specified ratios for either 2D or 3D acquisitions. The effectiveness of the IsoConcentraChromJ has been validated and demonstrated using experimental datasets consisting in nuclei images of pre-adipocytes and mature adipocytes, encompassing both 2D and 3D imaging. The outcomes allow to characterize the nuclear architecture by calculating the ratios between specific concentric nuclear areas/volumes of acetylated chromatin with respect to total acetylated chromatin and/or total DNA. The novel IsoConcentrapChromJ plugin could represent a valuable resource for researchers investigating the rearrangement of chromatin architecture driven by epigenetic mechanisms using nuclear images obtained by different fluorescence microscopy methods., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Zeaiter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), are essential polycations with wide-ranging roles in cellular functions. PA levels decline with age, making exogenous PA supplementation, particularly Spd, an intriguing prospect. Previous research in honey bees demonstrated that millimolar Spd added to their diet increased lifespan and reinforced oxidative resilience. The present study is aimed to assess the anti-aging effects of spermidine supplementation at concentrations of 0.1 and 1 mM in honey bees, focusing on autophagy and associated epigenetic changes. Results showed a more pronounced effect at the lower Spd concentration, primarily in the abdomen. Spd induced site-specific histone 3 hypoacetylation at sites K18 and 27, hyperacetylation at K9, with no change at K14 in the entire body. Additionally, autophagy-related genes (ATG3, 5, 9, 13) and genes associated with epigenetic changes (HDAC1, HDAC3, SIRT1, KAT2A, KAT6B, P300, DNMT1A, DNMT1B) were upregulated in the abdomens of honey bees. In conclusion, our findings highlight profound epigenetic changes and autophagy promotion due to spermidine supplementation, contributing to increased honey bee longevity. Further research is needed to fully understand the precise mechanisms and the interplay between epigenetic alterations and autophagy in honey bees, underscoring the significance of autophagy as a geroprotective mechanism., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kojić et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Riccardi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Teresa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Histone variants are paralogs that replace canonical histones in nucleosomes, often imparting novel functions. However, how histone variants arise and evolve is poorly understood. Reconstruction of histone protein evolution is challenging due to large differences in evolutionary rates across gene lineages and sites. Here we used intron position data from 108 nematode genomes in combination with amino acid sequence data to find disparate evolutionary histories of the three H2A variants found in Caenorhabditis elegans: the ancient H2A.ZHTZ-1, the sperm-specific HTAS-1, and HIS-35, which differs from the canonical S-phase H2A by a single glycine-to-alanine C-terminal change. Although the H2A.ZHTZ-1 protein sequence is highly conserved, its gene exhibits recurrent intron gain and loss. This pattern suggests that specific intron sequences or positions may not be important to H2A.Z functionality. For HTAS-1 and HIS-35, we find variant-specific intron positions that are conserved across species. Patterns of intron position conservation indicate that the sperm-specific variant HTAS-1 arose more recently in the ancestor of a subset of Caenorhabditis species, while HIS-35 arose in the ancestor of Caenorhabditis and its sister group, including the genus Diploscapter. HIS-35 exhibits gene retention in some descendent lineages but gene loss in others, suggesting that histone variant use or functionality can be highly flexible. Surprisingly, we find the single amino acid differentiating HIS-35 from core H2A is ancestral and common across canonical Caenorhabditis H2A sequences. Thus, we speculate that the role of HIS-35 lies not in encoding a functionally distinct protein, but instead in enabling H2A expression across the cell cycle or in distinct tissues. This work illustrates how genes encoding such partially-redundant functions may be advantageous yet relatively replaceable over evolutionary timescales, consistent with the patchwork pattern of retention and loss of both genes. Our study shows the utility of intron positions for reconstructing evolutionary histories of gene families, particularly those undergoing idiosyncratic sequence evolution., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Singh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kato-Inui et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fukushima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although histone proteins are widely known for their intranuclear functions where they organize DNA, all five histone types can also be released into the extracellular space from damaged cells. Extracellular histones can interact with pattern recognition receptors of peripheral immune cells, including toll-like receptor 4 (TLR4), causing pro-inflammatory activation, which indicates they may act as damage-associated molecular patterns (DAMPs) in peripheral tissues. Very limited information is available about functions of extracellular histones in the central nervous system (CNS). To address this knowledge gap, we applied mixed histones (MH) to cultured cells modeling neurons, microglia, and astrocytes. Microglia are the professional CNS immunocytes, while astrocytes are the main support cells for neurons. Both these cell types are critical for neuroimmune responses and their dysregulated activity contributes to neurodegenerative diseases. We measured effects of extracellular MH on cell viability and select neuroimmune functions of microglia and astrocytes. MH were toxic to cultured primary murine neurons and also reduced viability of NSC-34 murine and SH-SY5Y human neuron-like cells in TLR4-dependent manner. MH did not affect the viability of resting or immune-stimulated BV-2 murine microglia or U118 MG human astrocytic cells. When applied to BV-2 cells, MH enhanced secretion of the potential neurotoxin glutamate, but did not modulate the release of nitric oxide (NO), tumor necrosis factor-α (TNF), C-X-C motif chemokine ligand 10 (CXCL10), or the overall cytotoxicity of lipopolysaccharide (LPS)- and/or interferon (IFN)-γ-stimulated BV-2 microglial cells towards NSC-34 neuron-like cells. We demonstrated, for the first time, that MH downregulated phagocytic activity of LPS-stimulated BV-2 microglia. However, MH also exhibited protective effect by ameliorating the cytotoxicity of LPS-stimulated U118 MG astrocytic cells towards SH-SY5Y neuron-like cells. Our data demonstrate extracellular MH could both damage neurons and alter neuroimmune functions of glial cells. These actions of MH could be targeted for treatment of neurodegenerative diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Da Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Some pregnant women have to experience non-obstetric surgery during pregnancy under general anesthesia. Our previous studies showed that maternal exposure to sevoflurane, isoflurane, propofol, and ketamine causes cognitive deficits in offspring. Histone acetylation has been implicated in synaptic plasticity. Propofol is commonly used in non-obstetric procedures on pregnant women. Previous studies in our laboratory showed that maternal propofol exposure in pregnancy impairs learning and memory in offspring by disturbing histone acetylation. The present study aims to investigate whether HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) could attenuate learning and memory deficits in offspring caused by maternal surgery under propofol anesthesia during mid-pregnancy. Maternal rats were exposed to propofol or underwent abdominal surgery under propofol anesthesia during middle pregnancy. The learning and memory abilities of the offspring rats were assessed using the Morris water maze (MWM) test. The protein levels of histone deacetylase 2 (HDAC2), phosphorylated cAMP response-element binding (p-CREB), brain-derived neurotrophic factor (BDNF), and phosphorylated tyrosine kinase B (p-TrkB) in the hippocampus of the offspring rats were evaluated by immunofluorescence staining and western blot. Hippocampal neuroapoptosis was detected by TUNEL staining. Our results showed that maternal propofol exposure during middle pregnancy impaired the water-maze learning and memory of the offspring rats, increased the protein level of HDAC2 and reduced the protein levels of p-CREB, BDNF and p-TrkB in the hippocampus of the offspring, and such effects were exacerbated by surgery. SAHA alleviated the cognitive dysfunction and rescued the changes in the protein levels of p-CREB, BDNF and p-TrkB induced by maternal propofol exposure alone or maternal propofol exposure plus surgery. Therefore, SAHA could be a potential and promising agent for treating the learning and memory deficits in offspring caused by maternal nonobstetric surgery under propofol anesthesia., Competing Interests: The authors declared no competing interests., (Copyright: © 2024 Feng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lactate, which is synthesized as an end product by lactate dehydrogenase A (LDHA) from pyruvate during anaerobic glycolysis, has attracted attention for its energy metabolism and oxidant effects. A novel histone modification-mediated gene regulation mechanism termed lactylation by lactate was recently discovered. The present study examined the involvement of histone lactylation in undifferentiated cells that underwent differentiation into osteoblasts. C2C12 cells cultured in medium with a high glucose content (4500 mg/L) showed increases in marker genes (Runx2, Sp7, Tnap) indicating BMP-2-induced osteoblast differentiation and ALP staining activity, as well as histone lactylation as compared to those cultured in medium with a low glucose content (900 mg/L). Furthermore, C2C12 cells stimulated with the LDH inhibitor oxamate had reduced levels of BMP-2-induced osteoblast differentiation and histone lactylation, while addition of lactate to C2C12 cells cultured in low glucose medium resulted in partial restoration of osteoblast differentiation and histone lactylation. These results indicate that lactate synthesized by LDHA during glucose metabolism is important for osteoblast differentiation of C2C12 cells induced by BMP-2. Additionally, silencing of p300, a possible modifier of histone lactylation, also inhibited osteoblast differentiation and reduced histone lactylation. Together, these findings suggest a role of histone lactylation in promotion of undifferentiated cells to undergo differentiation into osteoblasts., Competing Interests: NO authors have competing interests., (Copyright: © 2023 Minami et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Epigenetic modifications, particularly histone acetylation-deacetylation and its related enzymes, such as sirtuin 1 (SIRT1) deacetylase, may have substantial roles in the pathogenesis of obesity and its associated health issues. This study aimed to evaluate global histone acetylation status and SIRT1 gene expression in children and adolescents with obesity and their association with metabolic and anthropometric parameters., Methods: This study included 60 children and adolescents, 30 with obesity and 30 normal-weight. The evaluation consisted of the analysis of global histone acetylation levels and the expression of the SIRT1 gene in peripheral blood mononuclear cells, by specific antibody and real-time PCR, respectively. Additionally, insulin, fasting plasma glucose, lipid profile and tumor necrosis factor α (TNF-α) levels were measured. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR). Metabolic syndrome was determined based on the diagnostic criteria established by IDF., Results: Individuals with obesity, particularly those with insulin resistance, had significantly higher histone acetylation levels compared to control group. Histone acetylation was positively correlated with obesity indices, TNF-α, insulin, and HOMA-IR. Additionally, a significant decrease in SIRT1 gene expression was found among obese individuals, which was negatively correlated with the histone acetylation level. Furthermore, SIRT1 expression levels showed a negative correlation with various anthropometric and metabolic parameters., Conclusion: Histone acetylation was enhanced in children and adolescents with obesity, potentially resulting from down-regulation of SIRT1, and could play a role in the obesity-associated metabolic abnormalities and insulin resistance. Targeting global histone acetylation modulation might be considered as an epigenetic approach for early obesity management., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Taghizadeh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Globally, colorectal cancer (CRC) is a major health concern. Despite improvements in CRC treatment, mortality rates remain high. Genetic instability and epigenetic dysregulation of gene expression are instigators of CRC development that result in genotypic differences, leading to often variable and unpredictable treatment responses. Three miRNAs, miR-143, -145 and -133b, are most commonly downregulated in CRC and are proposed here as potential tumour suppressors. Although the downregulation of these miRNAs in CRC is largely unexplained, epigenetic silencing has been postulated to be a causative regulatory mechanism. Potential epigenetic modulation of miRNA expression, by means of histone acetylation and DNA methylation, was assessed in this study by treating early (SW1116) and late stage (DLD1) CRC cells with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-Aza-2'C) and the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), respectively. Subsequent quantification of miRNA expression revealed that while all the selected miRNAs were susceptible to DNA demethylation in early- and late-stage CRC cells, susceptibility to DNA demethylation was significantly pronounced in late-stage DLD1 cells. Conversely, although histone acetylation moderately affected miRNA expression in early-stage CRC, it had a marginal effect on the expression of miRNAs in late-stage CRC cells. Overall, this study provides further understanding of the contribution of epigenetics to the regulation of putative tumour suppressor miRNAs in CRC., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Omar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Gene duplication is an evolutionary mechanism that provides new genetic material. Since gene duplication is a major driver for molecular evolution, examining the fate of duplicated genes is an area of active research. The fate of duplicated genes can include loss, subfunctionalization, and neofunctionalization. In this manuscript, we chose to experimentally study the fate of duplicated genes using the Arabidopsis NUCLEAR FACTOR Y (NF-Y) transcription factor family. NF-Y transcription factors are heterotrimeric complexes, composed of NF-YA, NF-YB, and NF-YC. NF-YA subunits are responsible for nucleotide-specific binding to a CCAAT cis-regulatory element. NF-YB and NF-YC subunits make less specific, but essential complex-stabilizing contacts with the DNA flanking the core CCAAT pentamer. While ubiquitous in eukaryotes, each NF-Y family has expanded by duplication in the plant lineage. For example, the model plant Arabidopsis contains 10 each of the NF-Y subunits. Here we examine the fate of duplicated NF-YB proteins in Arabidopsis, which are composed of central histone fold domains (HFD) and less conserved flanking regions (N- and C-termini). Specifically, the principal question we wished to address in this manuscript was to what extent can the 10 Arabidopsis NF-YB paralogs functionally substitute the genes NF-YB2 and NF-YB3 in the promotion of photoperiodic flowering? Our results demonstrate that the conserved histone fold domains (HFD) may be under pressure for purifying (negative) selection, while the non-conserved N- and C-termini may be under pressure for diversifying (positive) selection, which explained each paralog's ability to substitute. In conclusion, our data demonstrate that the N- and C-termini may have allowed the duplicated genes to undergo functional diversification, allowing the retention of the duplicated genes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Siriwardana et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mammalian protein arginine methyltransferase 7 (PRMT7) has been shown to target substrates with motifs containing two arginine residues separated by one other residue (RXR motifs). In particular, the repression domain of human histone H2B (29-RKRSR-33) has been a key substrate in determining PRMT7 activity. We show that incubating human PRMT7 and [3H]-AdoMet with full-length Xenopus laevis histone H2B, containing the substitutions K30R and R31K (RKRSR to RRKSR), results in greatly reduced methylation activity. Using synthetic peptides, we have now focused on the enzymology behind this specificity. We show for the human and Xenopus peptide sequences 23-37 the difference in activity results from changes in the Vmax rather than the apparent binding affinity of the enzyme for the substrates. We then characterized six additional peptides containing a single arginine or a pair of arginine residues flanked by glycine and lysine residues. We have corroborated previous findings that peptides with an RXR motif have much higher activity than peptides that contain only one Arg residue. We show that these peptides have similar apparent km values but significant differences in their Vmax values. Finally, we have examined the effect of ionic strength on these peptides. We found the inclusion of salt had little effect on the Vmax value but a considerable increase in the apparent km value, suggesting that the inhibitory effect of ionic strength on PRMT7 activity occurs largely by decreasing apparent substrate-enzyme binding affinity. In summary, we find that even subtle substitutions in the RXR recognition motif can dramatically affect PRMT7 catalysis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bondoc et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The decidua undergoes proinflammatory activation in late pregnancy, promoting labor. Bromodomain and Extra-Terminal (BET) family proteins interact with acetylated histones and may control gene expression in inflammation. Here, we assessed whether BETs are involved in inflammatory gene regulation in human decidual cells. We have treated primary cultures of decidual stromal cells (DSCs) from term pregnancies with endotoxin (LPS) and measured the expression of a panel of pro-and anti-inflammatory genes. BET involvement was assessed using the selective BET inhibitors (+)-JQ1 and I-BET-762 or the negative control compound (-)-JQ1. Histone 3 and -4 acetylation and BETs binding at the target gene promoters were determined to assess whether these processes are involved in the actions of LPS, BETs, and BET inhibitors. LPS increased the expression of the proinflammatory (PTGS2, IL6, CXCL8/IL8, TNF) and the anti-inflammatory (IL10, IDO1) genes of the panel. The constitutively expressed inflammatory genes (PTGS1, PTGES) were unaffected. The BET inhibitors, but not the control compound, reduced the basal and LPS-induced expression of PTGS1, PTGS2, IL6, CXCL8/IL8, IL10, and IDO1. TNF expression was not changed by BET inhibition. The dominant BETs were Bromodomain-containing protein -2 (BRD2) and -4L (BRD4L) in DSCs. LPS increased histone 4 acetylation at the CXCL8/IL8 and TNF promoters and histone 3 and -4 acetylation at the IDO1 promoter, while (+)-JQ1 abrogated histone acetylation at several promoters. Overall, histone acetylation and promoter binding of BETs showed no consistent relationship with gene expression across the gene panel and the treatments. BET proteins, predominantly BRD2 and BRD4L, control critical pro- and anti-inflammatory genes in DSCs. TNF induction exemplifies a BET-independent pathway. Changing histone acetylation at the promoters is not a general obligatory requirement for inflammatory gene expression in response to LPS. BETs likely act at chromatin loci separate from the examined promoters. BET inhibitors may block decidual activation at labor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ajgaonkar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Various studies have described epigenetic inheritance through sperms. However, the detailed mechanisms remain unclear. In this study, we focused on DNA methylation in mice treated with valproic acid (VPA), an inducer of epigenomic changes, and analyzed the treatment effects on the sperm from the next generation of mice. The administration of 200 mg/kg/day VPA to mice for 4 weeks caused transient histone hyperacetylation in the testes and DNA methylation changes in the sperm, including promoter CpGs of genes related to brain function. Oocytes fertilized with VPA-treated mouse sperm showed methylation fluctuations at the morula stage. Pups that were fathered by these mice also showed behavioral changes in the light/dark transition test after maturation. Brain RNA-seq of these mice showed that the expression of genes related to neural functions were altered. Comparison of the sperm DNA methylation status of the next generation of mice with that of the parental generation revealed the disappearance of methylation changes observed in the sperm of the parental generation. These findings suggest that VPA-induced histone hyperacetylation may have brain function-related effects on the next generation through changes in sperm DNA methylation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sakai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aims: Accumulating data demonstrates that new adult cardiomyocytes (CMs) are generated throughout life from pre-existing CMs, although the absolute magnitude of CM self-renewal is very low. Modifying epigenetic histone modifications or activating the Hippo-Yap pathway have been shown to promote adult CM cycling and proliferation. Whether these interventions work through common pathways or act independently is unknown. For the first time we have determined whether lysine demethylase 4D (KDM4D)-mediated CM-specific H3K9 demethylation and Hippo pathways inhibition have additive or redundant roles in promoting CM cell cycle re-entry., Methods and Results: We found that activating Yap1 in cultured neonatal rat ventricular myocytes (NRVM) through overexpressing Hippo pathway inhibitor, miR-199, preferentially increased S-phase CMs, while H3K9me3 demethylase KDM4D preferentially increased G2/M markers in CMs. Together KDM4D and miR-199 further increased total cell number of NRVMs in culture. Inhibition of Hippo signaling via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1) also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducible activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle, while miR-199 and si-Sav1 preferentially up-regulated genes involved in G1/S phase. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc. Using transgenic mice over-expressing KDM4D together with Myc, we demonstrated that KDM4D/Myc significantly increased CM cell cycling but did not affect cardiac function., Conclusions: KDM4D effects on CM cell cycle activity are additive with the Hippo-Yap1 pathway and appear to preferentially regulate different cell cycle regulators. This may have important implications for strategies that target cardiac regeneration in treating heart disease., Competing Interests: The authors declare no competing financial interests., (Copyright: © 2023 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cataract, a disease that causes opacity of the lens, is the leading cause of blindness worldwide. Cataracts secondary to diabetes are common, even in young patients, so they are of significant clinical importance. Here, we used an ex vivo model of galactose-induced cataracts in the rat lens to investigate the therapeutic effects of histone acetyltransferase (HAT) inhibitors. Among the tested HAT inhibitors, TH1834 was the only one that could reverse most of the opacity once it had formed in the lens. Combination treatment with C646/CPTH2 and CBP30/CPTH2 also had therapeutic effects. In lens cross-sections, vacuoles were present in the tissue of the cortical equatorial region of untreated cataract samples. In treated cataract samples, lens tissue regenerated to fill the vacuoles. To identify the genes regulated by HAT inhibitors, qRT-PCR was performed on treated and untreated cataract samples to determine candidate genes. Expression of Acta1 and Stmn4, both of which are involved in the cytoskeleton, were altered significantly in C646+CPTH2 samples. Expression of Emd, a nuclear membrane protein, and Prtfdc1, which is involved in cancer cell proliferation, were altered significantly in CBP30+CPTH2 samples. Acta1, Acta2, Arrdc3, Hebp2, Hist2h2ab, Pmf1, Ppdpf, Rbm3, RGD1561694, Slc16a6, Slfn13, Tagln, Tgfb1i1, and Tuba1c in TH1834 samples were significantly altered. These genes were primarily related to regulation of cell proliferation, the cytoskeleton, and cell differentiation. Expression levels increased with the onset of cataracts and was suppressed in samples treated with HAT inhibitors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Nagaya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The histone deacetylase (HDAC) inhibitor vorinostat, used with gemcitabine and other therapies, has been effective in treatment of experimental models of pancreatic cancer. In this study, we demonstrated that M344, an HDAC inhibitor, is efficacious against pancreatic cancer in vitro and in vivo, alone or with gemcitabine. By 24 hours post-treatment, M344 augments the population of pancreatic cancer cells in G1, and at a later time point (48 hours) it increases apoptosis. M344 inhibits histone H3 deacetylation and slows pancreatic cancer cell proliferation better than vorinostat, and it does not decrease the viability of a non-malignant cell line more than vorinostat. M344 also elevates pancreatic cancer cell major histocompatibility complex (MHC) class I molecule expression, potentially increasing the susceptibility of pancreatic cancer cells to T cell lysis. Taken together, our findings support further investigation of M344 as a pancreatic cancer treatment., Competing Interests: The authors have declared that no competing interests exist.

Structural fluctuations of nucleosomes modulate the access to internal DNA in eukaryotic cells; clearly characterisation of this fundamental process is crucial to understanding gene regulation. Here we apply PhAST (Photochemical Analysis of Structural Transitions) to monitor at a base pair level, structural alterations induced all along the DNA upon histone binding or release. By offering the first reliable, detailed comparison of nucleosome assembly and disassembly in vitro, we reveal similarities and differences between the two processes. We identify multiple, sequential intermediate states characterised by specific PhAST signals whose localisation and amplitude reflect asymmetries of DNA/histone interactions with respect to the nucleosome pseudo dyad. These asymmetries involve not only the DNA extremities but also regions close to the pseudo dyad. Localisations of asymmetries develop in a consistent manner during both assembly and disassembly processes; they primarily reflect the DNA sequence effect on the efficiency of DNA-histone binding. More unexpectedly, the amplitude component of PhAST signals not only evolves as a function of intermediate states but does so differently between assembly and disassembly pathways. Our observation of differences between assembly and disassembly opens up new avenues to define the role of the DNA sequence in processes underlying the regulation of gene expression. Overall, we provide new insights into how the intrinsic properties of DNA are integrated into a holistic mechanism that controls chromatin structure., Competing Interests: The authors have declared that no competing interests exist.

Midazolam is a widely used short-acting benzodiazepine. However, midazolam is also criticized for its deliriogenic potential. Since delirium is associated with a malfunction of the neurotransmitter acetylcholine, midazolam appears to interfere with its proper metabolism, which can be triggered by epigenetic modifications. Consequently, we tested the hypothesis that midazolam indeed changes the expression and activity of cholinergic genes by acetylcholinesterase assay and qPCR. Furthermore, we investigated the occurrence of changes in the epigenetic landscape by methylation specific PCR, ChiP-Assay and histone ELISA. In an in-vitro model containing SH-SY5Y neuroblastoma cells, U343 glioblastoma cells, and human peripheral blood mononuclear cells, we found that midazolam altered the activity of acetylcholinesterase /buturylcholinesterase (AChE / BChE). Interestingly, the increased expression of the buturylcholinesterase evoked by midazolam was accompanied by a reduced methylation of the BCHE gene and the di-methylation of histone 3 lysine 4 and came along with an increased expression of the lysine specific demethylase KDM1A. Last, inflammatory cytokines were not induced by midazolam. In conclusion, we found a promising mechanistic link between midazolam treatment and delirium, due to a significant disruption in cholinesterase homeostasis. In addition, midazolam seems to provoke profound changes in the epigenetic landscape. Therefore, our results can contribute to a better understanding of the hitherto poorly understood interactions and risk factors of midazolam on delirium., Competing Interests: The authors have declared that no competing interests exist.

We have performed a comprehensive analysis of the involvement of histone H3 and H4 residues in the regulation of chronological lifespan in yeast and identify four structural groups in the nucleosome that influence lifespan. We also identify residues where substitution with an epigenetic mimic extends lifespan, providing evidence that a simple epigenetic switch, without possible additional background modifications, causes longevity. Residues where substitution result in the most pronounced lifespan extension are all on the exposed face of the nucleosome, with the exception of H3E50, which is present on the lateral surface, between two DNA gyres. Other residues that have a more modest effect on lifespan extension are concentrated at the extremities of the H3-H4 dimer, suggesting a role in stabilizing the dimer in its nucleosome frame. Residues that reduce lifespan are buried in the histone handshake motif, suggesting that these mutations destabilize the octamer structure. All residues exposed on the nucleosome disk face and that cause lifespan extension are known to interact with Sir3. We find that substitution of H4K16 and H4H18 cause Sir3 to redistribute from telomeres and silent mating loci to secondary positions, often enriched for Rap1, Abf1 or Reb1 binding sites, whereas H3E50 does not. The redistribution of Sir3 in the genome can be reproduced by an equilibrium model based on primary and secondary binding sites with different affinities for Sir3. The redistributed Sir3 cause transcriptional repression at most of the new loci, including of genes where null mutants were previously shown to extend chronological lifespan. The transcriptomic profiles of H4K16 and H4H18 mutant strains are very similar, and compatible with a DNA replication stress response. This is distinct from the transcriptomic profile of H3E50, which matches strong induction of oxidative phosphorylation. We propose that the different groups of residues are involved in binding to heterochromatin proteins, in destabilizing the association of the nucleosome DNA, disrupting binding of the H3-H4 dimer in the nucleosome, or disrupting the structural stability of the octamer, each category impacting on chronological lifespan by a different mechanism., Competing Interests: The authors have declared that no competing interests exist.

A quick, reliable, and reproducible biological assay to distinguish individuals with possible life-threatening risk following radiological or nuclear incidents remains a quest in biodosimetry. In this paper, we examined the use of a γ-H2AX assay as an early dose estimation for rapid triage based on both flow cytometry and image analyses. In the experiment, whole blood from 11 donors was irradiated ex vivo inside a water phantom by gamma rays from Co-60 at 0.51 Gy/min. After the lysis of red blood cells, the white blood cells were collected for immunofluorescence labeling of γ-H2AX, CD45, and nuclear stained for signal collection and visualization. Analysis by flow cytometry showed that the relative γ-H2AX intensities of lymphocytes and granulocytes increased linearly with absorbed doses from 0 to 6 Gy with a large variation among individuals observed above 2 Gy. The relative γ-H2AX intensities of lymphocytes assessed by two different laboratories were highly correlated (ICC = 0.979). Using confocal microscopic images, γ-H2AX foci were observed to be discretely distributed inside the nuclei and to increase proportionally with doses from 0 to 2 Gy, whereas large plagues of merged foci appeared at 4 and 6 Gy, resulting in the saturation of foci counts above 4 Gy. The number of total foci per cell as well as the number of foci per plane were significantly different at 0 vs 1 and 2 vs 4 Gy doses (p < 0.01). Blind tests at 0.5 Gy and 1 Gy doses showed that dose estimation by flow cytometry had a mean absolute difference of less than 0.5 Gy from the actual value. In conclusion, while flow cytometry can provide a dose estimation with an uncertainty of 0.5 Gy at doses ≤ 1 Gy, foci counting can identify merged foci that are prominent at doses ≥ 4 Gy., Competing Interests: The authors have declared that no competing interests exist.

Epigenetic mechanisms regulate osteogenic lineage differentiation of mesenchymal stromal cells. Histone methylation is controlled by multiple lysine demethylases and is an important step in controlling local chromatin structure and gene expression. Here, we show that the lysine-specific histone demethylase Kdm1A/Lsd1 is abundantly expressed in osteoblasts and that its suppression impairs osteoblast differentiation and bone nodule formation in vitro. Although Lsd1 knockdown did not affect global H3K4 methylation levels, genome-wide ChIP-Seq analysis revealed high levels of Lsd1 at gene promoters and its binding was associated with di- and tri-methylation of histone 3 at lysine 4 (H3K4me2 and H3K4me3). Lsd1 binding sites in osteoblastic cells were enriched for the Runx2 consensus motif suggesting a functional link between the two proteins. Importantly, inhibition of Lsd1 activity decreased osteoblast activity in vivo. In support, mesenchymal-targeted knockdown of Lsd1 led to decreased osteoblast activity and disrupted primary spongiosa ossification and reorganization in vivo. Together, our studies demonstrate that Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 and its activity is required for proper bone formation., Competing Interests: The authors have declared that no competing interests exist.

Cardiac hypertrophy is a complex process induced by the activation of multiple signaling pathways. We previously reported that anacardic acid (AA), a histone acetyltransferase (HAT) inhibitor, attenuates phenylephrine (PE)-induced cardiac hypertrophy by downregulating histone H3 acetylation at lysine 9 (H3K9ac). Unfortunately, the related upstream signaling events remained unknown. The mitogen-activated protein kinase (MAPK) pathway is an important regulator of cardiac hypertrophy. In this study, we explored the role of JNK/MAPK signaling pathway in cardiac hypertrophy induced by PE. The mice cardiomyocyte hypertrophy model was successfully established by treating cells with PE in vitro. This study showed that p-JNK directly interacts with HATs (P300 and P300/CBP-associated factor, PCAF) and alters H3K9ac. In addition, both the JNK inhibitor SP600125 and the HAT inhibitor AA attenuated p-JNK overexpression and H3K9ac hyperacetylation by inhibiting P300 and PCAF during PE-induced cardiomyocyte hypertrophy. Moreover, we demonstrated that both SP600125 and AA attenuate the overexpression of cardiac hypertrophy-related genes (MEF2A, ANP, BNP, and β-MHC), preventing cardiomyocyte hypertrophy and dysfunction. These results revealed a novel mechanism through which AA might protect mice from PE-induced cardiomyocyte hypertrophy. In particular, AA inhibits the effects of JNK signaling on HATs-mediated histone acetylation, and could therefore be used to prevent and treat pathological cardiac hypertrophy., Competing Interests: The authors have declared that no competing interests exist.

Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay., Competing Interests: The authors report that there were material support and technical advice from VERITAS corporation for this study that could have influenced its outcome and that one of the authors, Y.T. is its employee. This does not alter our adherence to PLOS ONE policies on sharing data and materials." We believe our revised manuscript would suffice the reviewer’s concerns. It would be greatly appreciated if the editorial office and reviewers could consider this revision. All the authors have read the manuscript and have approved this submission. All study participants provided informed consent, and the study design was approved by an ethics review board.

Lycoris species have various chromosome numbers and karyotypes, but all have a constant total number of chromosome major arms. In addition to three fundamental types, including metacentric (M-), telocentric (T-), and acrocentric (A-) chromosomes, chromosomes in various morphology and size were also observed in natural populations. Both fusion and fission translocation have been considered as main mechanisms leading to the diverse karyotypes among Lycoris species, which suggests the centromere organization playing a role in such arrangements. We detected several chromosomal structure changes in Lycoris including centric fusion, inversion, gene amplification, and segment deletion by using fluorescence in situ hybridization (FISH) probing with rDNAs. An antibody against centromere specific histone H3 (CENH3) of L. aurea (2n = 14, 8M+6T) was raised and used to obtain CENH3-associated DNA sequences of L. aurea by chromatin immunoprecipitation (ChIP) cloning method. Immunostaining with anti-CENH3 antibody could label the centromeres of M-, T-, and A-type chromosomes. Immunostaining also revealed two centromeres on one T-type chromosome and a centromere on individual mini-chromosome. Among 10,000 ChIP clones, 500 clones which showed abundant in L. aurea genome by dot-blotting analysis were FISH mapped on chromosomes to examine their cytological distribution. Five of these 500 clones could generate intense FISH signals at centromeric region on M-type but not T-type chromosomes. FISH signals of these five clones rarely appeared on A-type chromosomes. The five ChIP clones showed similarity in DNA sequences and could generate similar but not identical distribution patterns of FISH signals on individual chromosomes. Furthermore, the distinct distribution patterns of FISH signals on each chromosome generated by these five ChIP clones allow to identify individual chromosome, which is considered difficult by conventional staining approaches. Our results suggest a different organization of centromeres of the three chromosome types in Lycoris species., Competing Interests: The author, Ching-Chi Tsai is currently employed by CH Biotech R & D Co., LTD (CHB); but, before she moved to CHB, she worked as a post-doctor and joined this study which was supported by the funding from the Institute of Plant and Microbial Biology, Academia Sinica, Taiwan, ROC. That commercial company, CH Biotech R & D Co., LTD, did not play any role in this study. Therefore, the author’s affiliation is “Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan”, and “CH Biotech R & D Co., LTD “is her current address. The authors have declared that no competing interests exist.

Lysine 2-hydroxyisobutyrylation (Khib) is a recently discovered post-translational modification (PTM) showing diverse biological functions and effects in living organisms. However, the study of Khib in plant species is still relatively limited. Wheat (Triticum aestivum L.) is a global important cereal plant. In this study, the systematic Khib analysis was performed in wheat leave tissues. A total of 3004 Khib sites in 1104 proteins were repeatedly identified. Structure characterization of these Khib peptides revealed 12 conserved sequence motifs. Function classification and enrichment analysis indicated these Khib proteins showed a wide function and pathway distribution, of which ribosome activity, protein biosynthesis and photosynthesis were the preferred biological processes. Subcellular location predication indicated chloroplast was the dominant subcellular compartment where Khib was distributed. There may be some crosstalks among Khib, lysine acetylation and lysine succinylation modification because some proteins and sites were modified by all these three acylations. The present study demonstrated the critical role of Khib in wheat biological and physiology, which has expanded the scope of Khib in plant species. Our study is an available resource and reference of Khib function demonstration and structure characterization in cereal plant, as well as in plant kingdom., Competing Interests: The authors have declared that no competing interests exist.

The acetylation of H4 lysine 16 (H4 K16Ac) in Saccharomyces cerevisiae counteracts the binding of the heterochromatin complex SIR to chromatin and inhibits gene silencing. Contrary to other histone acetylation marks, the H4 K16Ac level is high on genes with low transcription, whereas highly transcribed genes show low H4 K16Ac. Approximately 60% of cellular H4 K16Ac in S. cerevisiae is provided by the SAS-I complex, which consists of the MYST-family acetyltransferase Sas2, Sas4 and Sas5. The absence of SAS-I causes inappropriate spreading of the SIR complex and gene silencing in subtelomeric regions. Here, we investigated the genome-wide dynamics of SAS-I dependent H4 K16Ac during DNA replication. Replication is highly disruptive to chromatin and histone marks, since histones are removed to allow progression of the replication fork, and chromatin is reformed with old and new histones after fork passage. We found that H4 K16Ac appears in chromatin immediately upon replication. Importantly, this increase depends on the presence of functional SAS-I complex. Moreover, the appearance of H4 K16Ac is delayed in genes that are strongly transcribed. This indicates that transcription counteracts SAS-I-mediated H4 K16 acetylation, thus "sculpting" histone modification marks at the time of replication. We furthermore investigated which acetyltransferase acts redundantly with SAS-I to acetylate H4 K16Ac. esa1Δ sds3Δ cells, which were also sas2Δ sir3Δ in order to maintain viability, contained no detectable H4 K16Ac, showing that Esa1 and Sas2 are redundant for cellular H4 K16 acetylation. Furthermore, esa1Δ sds3Δ sas2Δ sir3Δ showed a more pronounced growth defect compared to the already defective esa1Δ sds3Δ sir3Δ. This indicates that SAS-I has cellular functions beyond preventing the spreading of heterochromatin., Competing Interests: The authors have declared that no competing interests exist.

Peptidylarginine deiminase 4 (PAD4) catalyzes posttranslational modification of many target proteins through converting protein arginine or mono-methylarginine to citrulline. Neutrophil extracellular trap (NET) formation is the most dramatic manifestation of PAD4-mediated hypercitrullination reaction in neutrophils, which is characterized by the release of nuclear chromatin to form a chromatin network in the extracellular space. Histones H4, one of the major protein components of chromatin, is released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury and can also be released during the process of NET formation, along with its citrullinated form. The present study showed that histone H4 can induce NET formation in a calcium and PAD4 dependent manner. Histone H4 caused permeabilization of the neutrophil membrane and sustained rise in intracellular calcium that is necessary for activation of PAD4. In comparison, citrullinated histone H4 induced less calcium influx compared with its native form, leading to reduced NET formation. These studies suggest that citrullinated histone H4 could serve as a brake in the pathology of NETs, slowing down the vicious circle between histone H4 and NETs., Competing Interests: The authors have declared that no competing interests exist.

Research into the epigenome is of growing importance as a loss of epigenetic control has been implicated in the development of neurodegenerative diseases. Previous studies have implicated aberrant DNA and histone methylation in multiple sclerosis (MS) disease pathogenesis. We have previously reported that the methyl donor betaine is depleted in MS and is linked to changes in histone H3 trimethylation (H3K4me3) in neurons. We have also shown that betaine increases histone methyltransferase activity by activating chromatin bound betaine homocysteine S-methyltransferase (BHMT). Here, we investigated the role of the BHMT-betaine methylation pathway in oligodendrocytes. Immunocytochemistry in the human MO3.13 cell line, primary rat oligodendrocytes, and tissue from MS postmortem brain confirmed the presence of the BHMT enzyme in the nucleus in oligodendrocytes. BHMT expression is increased 2-fold following oxidative insult, and qRT-PCR demonstrated that betaine can promote an increase in expression of oligodendrocyte maturation genes SOX10 and NKX-2.2 under oxidative conditions. Chromatin fractionation provided evidence of a direct interaction of BHMT on chromatin and co-IP analysis indicates an interaction between BHMT and DNMT3a. Our data show that both histone and DNA methyltransferase activity are increased following betaine administration. Betaine effects were shown to be dependent on BHMT expression following siRNA knockdown of BHMT. This is the first report of BHMT expression in oligodendrocytes and suggests that betaine acts through BHMT to modulate histone and DNA methyltransferase activity on chromatin. These data suggest that methyl donor availability can impact epigenetic changes and maturation in oligodendrocytes., Competing Interests: The authors have declared that no competing interests exist.

The interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. However, it remains poorly understood how the nuclear architecture of neurons adapts in response to behaviorally relevant stimuli that trigger fast alterations in gene expression patterns. Imaging of fluorescently tagged nucleosomes revealed that pharmacological manipulation of neuronal activity in vitro and auditory cued fear conditioning in vivo induce nucleus-scale restructuring of chromatin within minutes. Furthermore, the acquisition of auditory fear memory is impaired after infusion of a drug into auditory cortex which blocks chromatin reorganization in vitro. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional adaptations at fast time scales. Introducing a transgenic mouse line for photolabeling of histones, we extend the realm of systems available for imaging of chromatin dynamics to living animals., Competing Interests: The authors have declared that no competing interests exist.

Understanding the factors that underlie the epigenetic regulation of genes is crucial to understand the gene regulatory machinery as a whole. Several experimental and computational studies examined the relationship between different factors involved. Here we investigate the relationship between transcription factors (TFs) and histone modifications (HMs), based on ChIP-seq data in cell lines. As it was shown that gene regulation by TFs differs depending on the CpG class of a promoter, we study the impact of the CpG content in promoters on the associations between TFs and HMs. We suggest an approach based on sparse linear regression models to infer associations between TFs and HMs with respect to CpG content. A study of the partial correlation of HMs for the two classes of high and low CpG content reveals possible CpG dependence and potential candidates for confounding factors in our models. We show that the models are accurate, inferred associations reflect known biological relationships, and we give new insight into associations with respect to CpG content. Moreover, analysis of a ChIP-seq dataset in HepG2 cells of the HM H3K122ac, an HM about little is known, reveals novel TF associations and supports a previously established link to active transcription., Competing Interests: The authors have declared that no competing interests exist.

Long noncoding RNAs (lncRNAs) are RNA fragments that generally do not code for a protein but are involved in epigenetic gene regulation. In this study, lncRNAs of Brassica rapa were classified into long intergenic noncoding RNAs, natural antisense RNAs, and intronic noncoding RNAs and their expression analyzed in relation to genome-wide 24-nt small interfering RNAs (siRNAs), DNA methylation, and histone H3 lysine 27 trimethylation marks (H3K27me3). More than 65% of the lncRNAs analyzed consisted of one exon, and more than 55% overlapped with inverted repeat regions (IRRs). Overlap of lncRNAs with IRRs or genomic regions encoding for 24-nt siRNAs resulted in increased DNA methylation levels when both were present. LncRNA did not overlap greatly with H3K27me3 marks, but the expression level of intronic noncoding RNAs that did coincide with H3K27me3 marks was higher than without H3K27me3 marks. The Brassica genus comprises important vegetables and oil seed crops grown across the world. B. rapa is a diploid (AA genome) thought to be one of the ancestral species of both B. juncea (AABB genome) and B. napus (AACC) through genome merging (allotetrapolyploidization). Complex genome restructuring and epigenetic alterations are thought to be involved in these allotetrapolyploidization events. Comparison of lncRNAs between B. rapa and B. nigra, B. oleracea, B. juncea, and B. napus showed the highest conservation with B. oleracea. This study presents a comprehensive analysis of the epigenome structure of B. rapa at multi-epigenetic levels (siRNAs, DNA methylation, H3K27me3, and lncRNAs) and identified a suite of candidate lncRNAs that may be epigenetically regulated in the Brassica genus., Competing Interests: The authors have declared that no competing interests exist.

Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif (AAUAAUAA) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1., Competing Interests: The authors have declared that no competing interests exist.

Determining the function of proteins remains a key task of modern biology. Classical genetic approaches to knocking out protein function in plants still face limitations, such as the time-consuming nature of generating homozygous transgenic lines or the risk of non-viable loss-of-function phenotypes. We aimed to overcome these limitations by acting downstream of the protein level. Chimeric E3 ligases degrade proteins of interest in mammalian cell lines, Drosophila melanogaster embryos, and transgenic tobacco. We successfully recruited the 26S proteasome pathway to directly degrade a protein of interest located in plant nuclei. This success was achieved via replacement of the interaction domain of the E3 ligase adaptor protein SPOP (Speckle-type POZ adapter protein) with a specific anti-GFP nanobody (VHHGFP4). For proof of concept, the target protein CENH3 of A. thaliana fused to EYFP was subjected to nanobody-guided proteasomal degradation in planta. Our results show the potential of the modified E3-ligase adapter protein VHHGFP4-SPOP in this respect. We were able to point out its capability for nucleus-specific protein degradation in plants., Competing Interests: The authors have declared that no competing interests exist.

Methionine (Met) is an essential precursor of S-adenosylmethionine (SAM), which is the primary methyl donor required for biological processes such as DNA and histone methylation, which alter gene expression. In dairy cows, dietary Met has been observed to exert transcriptional alterations with beneficial effects on milk biosynthesis; however, the extent of these effects via SAM remains unknown. Therefore, we evaluated the effect of Met supply on histone methylation in lysine residues K9 and K27 in the histone tail H3 via a fluorescence resonance energy transfer (FRET) system in immortalized bovine mammary alveolar epithelial cells (MACT) incubated varying concentration of Met. The histone methylation data was complemented with global DNA methylation, cellular protein synthesis, and RT-qPCR analysis of genes related to Met cycle, DNA and histone methylation, AA transporters, and protein synthesis. The histone methylation data was performed on MACT cells seeded at 30,000 cells/well in 96-well plates 24 h prior to transfection. The transfections of FRET gene reporter plasmids H3K9 and H3K27 was performed with 0.3 μL/well of Lipofectamine® 3000 and 50 ng of plasmid DNA per well. At 24 h post-transfection, cells were treated with 0, 125, 250, and 500 μM of Met, and quantification of histone methylation was performed at 0, 12, and 24 h post-treatment as well as cell viability at 24 h using CellProfiler software. An inverted microscope for live imagining (EVOS® FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37˚C and 5.0% of CO2 was used to take 4 pictures/well at 4x magnification. A more defined response on histone methylation was observed in H3K9 than H3K27 to Met supply, where maximal histone methylation in H3K9 was observed with 125 μM of Met. This greater histone methylation in H3K9 at 125 μM was accompanied by greater cellular protein concentration. The linear increase in Met supply causes a linear decrease in global DNA methylation, while linearly upregulating genes related to the Met cycle (i.e., MAT1A, PEMT, SAHH, and MTR). The histone methylation data suggest that, to some extent, methyl-donors such as Met may affect the methylation sites, H3K9 and H3K27, and consequently causing a different epigenetic alteration. In the context of the dairy cow, further refinement to this FRET assay to study histone methylation could lead to establishing novel potential mechanisms of how dietary methyl donors may control the structural conformation of the bovine genome and, by extension, gene expression., Competing Interests: The authors have declared that no competing interest exist.

Atrazine is a common agricultural herbicide previously shown to promote epigenetic transgenerational inheritance of disease to subsequent generations. The current study was designed as an epigenome-wide association study (EWAS) to identify transgenerational sperm disease associated differential DNA methylation regions (DMRs) and differential histone retention regions (DHRs). Gestating female F0 generation rats were transiently exposed to atrazine during the period of embryonic gonadal sex determination, and then subsequent F1, F2, and F3 generations obtained in the absence of any continued exposure. The transgenerational F3 generation males were assessed for disease and sperm collected for epigenetic analysis. Pathology was observed in pubertal onset and for testis disease, prostate disease, kidney disease, lean pathology, and multiple disease. For these pathologies, sufficient numbers of individual males with only a single specific disease were identified. The sperm DNA and chromatin were isolated from adult one-year animals with the specific diseases and analyzed for DMRs with methylated DNA immunoprecipitation (MeDIP) sequencing and DHRs with histone chromatin immunoprecipitation (ChIP) sequencing. Transgenerational F3 generation males with or without disease were compared to identify the disease specific epimutation biomarkers. All pathologies were found to have disease specific DMRs and DHRs which were found to predominantly be distinct for each disease. No common DMRs or DHRs were found among all the pathologies. Epimutation gene associations were identified and found to correlate to previously known disease linked genes. This is one of the first observations of potential sperm disease biomarkers for histone retention sites. Although further studies with expanded animal numbers are required, the current study provides evidence the EWAS analysis is effective for the identification of potential pathology epimutation biomarkers for disease susceptibility., Competing Interests: The authors declare no competing interests.

X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression., Competing Interests: The authors have declared that no competing interests exist.

Objective: The role of neutrophil extracellular traps (NETs) in acute heart failure is unknown. We recently showed that interleukin 8, a putative NETs stimulator, was associated with myocardial recovery in acute heart failure complicating ST-elevation myocardial infarction (STEMI). In this exploratory post-hoc study, we aimed to investigate the role of NETs components in relation to myocardial function and interleukin 8 in STEMI patients with symptomatic acute heart failure., Methods: In 61 STEMI patients developing acute heart failure within 48 hours of successful revascularization, wall motion score index (WMSI), global longitudinal strain (GLS) and left ventricular ejection fraction (LVEF) were assessed by echocardiography at baseline and on day 5. Blood drawn at baseline and days 1, 2 and 5 was used to quantify double-stranded DNA (dsDNA), myeloperoxidase-DNA complexes (MPO-DNA) and citrullinated histone 3 (CitH3). The area under the curve (AUC) of each NETs marker and interleukin 8 was approximated for the first 5 days., Results: dsDNAAUC and MPO-DNAAUC correlated significantly with change in WMSI from baseline to day 5 (rs = 0.28 for both, p≤0.05), whereas NETs AUCs did not correlate with changes in GLS and LVEF. dsDNAAUC was significantly correlated with interleukin 8AUC (r = 0.40, p = 0.003). However, mixed model regression could not identify a significant effect of the NETs components on myocardial function parameters., Conclusions: In this cohort with acute heart failure complicating STEMI, NETs components were partly correlated with myocardial function and interleukin 8 levels, yet no causal relationship between NETs components and myocardial recovery could be established., Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT00324766., Competing Interests: The authors have declared that no competing interests exist.

Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations., Competing Interests: The authors have declared that no competing interests exist.

Cruciform DNA is a causing factor of genome instability and chromosomal translocation, however, most studies about cruciform DNA in mammalian cells were based on palindromic sequences containing plasmids and reports about endogenous cruciform DNA are rare. In this study we observed the dynamics of endogenous cruciform DNA in mouse growing oocytes using immunofluorescence labeling method. We found cruciform DNA foci exist in transcription active growing oocytes but not in transcription inactive fully grown oocytes and colocalized with Parp1 but not with DNA damage marker γH2A.X. By analyzing the Genotype-Tissue Expression data, we found cruciform DNA-mediated chromosomal translocation in human spermatocytes is associated with the specific DNA transcription in testis. When inhibiting the transcription with α-amanitin in mouse oocytes, we found oocyte cruciform DNA foci decreased significantly. In summary, we observed the endogenous cruciform DNA in growing oocytes and our results showed that the cruciform DNA formation is transcription-dependent., Competing Interests: The authors have declared that no competing interests exist.

Background: Suicide is a common phenomenon affecting people of all ages. There is a strong relationship between suicidal ideation and depressive disorders. Increasing number of studies suggest that epigenetic modifications in certain brain areas are the main mechanism through which environmental and genetic factors interact with each other contributing to the development of mental disorders. To verify this hypothesis, some epigenetic marks: H3K9/14ac, HDAC2/3, H3K27me2 and Sin3a, as well as p-S421-MeCP2/MeCP2 were examined. On the other hand, BDNF protein level were studied., Materials and Methods: Western blot analysis were performed in the frontal cortex (FCx) and hippocampus (HP) of suicide victims (n = 14) and non-suicidal controls (n = 8). The differences between groups and correlations between selected proteins were evaluated using Mann-Whitney U-test and Spearman's rank correlation., Results: Statistically significant decrease in H3K9/14ac (FCx:↓~23%;HP:↓~33%) combined with increase in HDAC3 (FCx:↑~103%;HP:↑~85% in HP) protein levels in suicides compared to the controls was shown. These alterations were accompanied by an increase in H3K27me2 (FCx:↑45%;HP:↑~59%) and Sin3a (HP:↑50%) levels and decrease in p-S421-MeCP2/MeCP2 protein ratio (HP:↓~55%;FCx:↓~27%). Moreover, reduced BDNF protein level (FCx:↓~43%;HP:↓~28%) in suicides was observed. On the other hand, some significant correlations (e.g. between H3K9/14ac and HDAC2 or between BDNF and p-S421-MeCP2/MeCP2) were demonstrated., Conclusions: Our findings confirm the role of epigenetic component and BDNF protein in suicidal behavior. Lowered BDNF protein level in suicides is probably due to decrease in histone acetylation and increased level of factors related with deacetylation and methylation processes, including MeCP2 factor, which may operate bidirectionally (an activator or inhibitor of transcription)., Competing Interests: The authors declare no conflicts of interest.

It has been suggested that DNA methylation can work in concert with other epigenetic factors, leading to changes in cellular phenotypes. For example, DNA demethylation modifications producing 5-hydroxymethylcytosine (5hmC) are thought to interact with histone modifications to influence the acquisition of embryonic stem cell (ESC) potency. However, the mechanism by which this occurs is still unknown. Thus, we systematically analysed the co-occurrence of DNA and histone modifications at genic regions as well as their relationship with ESC-specific expression using a number of heterogeneous public datasets. From a set of 19 epigenetic factors, we found remarkable co-occurrence of 5hmC and H4K8ac, accompanied by H3K4me1. This enrichment was more prominent at gene body regions. The results were confirmed using data obtained from different detection methods and species. Our analysis shows that these marks work cooperatively to influence ESC-specific gene expression. We also found that this trivalent mark is relatively enriched in genes related with immunity, which is a bit specific in ESCs. We propose that a trivalent epigenetic mark, composed of 5hmC, H4K8ac and H3K4me1, regulates gene expression and modulates the nature of human ESCs as a novel epigenetic code., Competing Interests: The authors have declared that no competing interests exist.

Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells., Competing Interests: The authors have declared that no competing interests exist.

Mutations of the SWI/SNF chromatin remodeling complex occur in 20% of all human cancers, including ovarian cancer. Approximately half of ovarian clear cell carcinomas (OCCC) carry mutations in the SWI/SNF subunit ARID1A, while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) presents with inactivating mutations of the SWI/SNF ATPase SMARCA4 alongside epigenetic silencing of the ATPase SMARCA2. Loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A), which regulates the chromatin landscape and gene expression by demethylating proteins such as histone H3. Cross-cancer analysis of the TCGA database shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. SCCOHT and OCCC cell lines have shown sensitivity to the reversible LSD1 inhibitor SP-2577 (Seclidemstat), suggesting that SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Moreover, it has been shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of endogenous retroviral elements and may thereby overcome resistance to checkpoint blockade. In this study, we investigated the ability of SP-2577 to promote anti-tumor immunity and T-cell infiltration in SCCOHT and OCCC cell lines. We found that SP-2577 stimulated IFN-dependent anti-tumor immunity in SCCOHT and promoted the expression of PD-L1 in both SCCOHT and OCCC. Together, these findings suggest that the combination therapy of SP-2577 with checkpoint inhibitors may induce or augment immunogenic responses of SWI/SNF-mutated ovarian cancers and warrants further investigation., Competing Interests: Dr. Sunil Sharma declares ownership of stocks from Salarius Pharmaceuticals and he is an inventor on the patent for SP-2577 compound. Dr. Soldi declares ownership of stocks from Salarius Pharmaceuticals. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors have declared that no other competing interests exist.

Triple Negative Breast Cancer (TNBC) is a heterogeneous disease lacking known molecular drivers and effective targeted therapies. Cytotoxic chemotherapy remains the mainstay of treatment for TNBCs, which have significantly poorer survival rates compared to other breast cancer subtypes. In addition to changes within the coding genome, aberrant enhancer activity is a well-established contributor to tumorigenesis. Here we use H3K27Ac chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map the active cis-regulatory landscape in TNBC. We identify distinct disease subtypes associated with specific enhancer activity, and over 2,500 unique superenhancers acquired by tumor cells but absent from normal breast tissue. To identify potential actionable disease drivers, we probed the dependency on genes that associate with tumor-specific enhancers by CRISPR screening. In this way we identify a number of tumor-specific dependencies, including a previously uncharacterized dependency on the TGFβ pseudo-receptor BAMBI., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Ryan Raisner, Peter M Haverty, & Karen E Gascoigne are employees of Genentech and shareholders of Roche. This does not alter our adherence to PLOS ONE policies on sharing data and materials.