Role Of Histones Research Articles

Epigenetic Alterations in Post-Traumatic Stress Disorder: Comprehensive Review of Molecular Markers

Background: Post-traumatic stress disorder (PTSD) can occur after a traumatic event. PTSD is characterized by nightmares, flashbacks and avoidance of stressors. It currently affects 2–8% of the population, with military personnel particularly susceptible. Studies show that environmental stressors can induce various epigenetic changes that shape the PTSD phenotype. Despite the significant impact of epigenetic factors on PTSD symptoms and susceptibility, they have not been widely discussed in the literature. This review focuses on describing epigenetic mechanisms in PTSD, especially DNA methylation, chromatin regulation, and noncoding RNA. Summary: The article includes relevant studies published from 2013 to 2023, excluding non-English-language studies or studies with insufficient data. This review investigated gene methylation changes in association with PTSD, including those related to the hypothalamic-pituitary-adrenal axis, brain-derived neurotrophic factor, neurotransmitters, and immune system functioning, as well as the role of histones and regulatory noncoding RNAs. Key Messages: Epigenetic alterations play a crucial role in shaping PTSD susceptibility, symptomatology, and long-term outcomes, highlighting their potential as important markers and therapeutic targets. Understanding these alterations can aid in developing clinical strategies to better predict, prevent, and treat PTSD. However, further large-scale longitudinal studies are needed to establish the temporal relationship between epigenetic changes and the onset of PTSD, as well as to classify other potential epigenetic mechanisms.

Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth.

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

Blood Clot Dynamics and Fibrinolysis Impairment in Cancer: The Role of Plasma Histones and DNA.

Blood viscoelasticity and plasma protein levels can play an important role in the diagnosis and prognosis of cancer. However, the role of histones and DNA in modulating blood clot properties remains to be investigated. This study investigates the differences in blood viscoelasticity and plasma protein levels among cancer patients, individuals with other diseases, and healthy individuals. Blood samples were collected from 101 participants, including 45 cancer patients, 22 healthy individuals, and 34 individuals with other diseases. Rheological properties of clots formed in vitro by reconstituted elements of fibrinogen or plasma were analyzed with an Anton Paar Rheometer, USA. Plasma protein levels of D-dimer, TPA, EPCR, fibrinogen, and histone H3 were measured through ELISA. Blood clots were formed with or without DNA and histones (H3) by adding thrombin and calcium to plasma samples, and were evaluated for viscoelasticity, permeability, and degradation. Cancer patients show higher blood viscoelasticity and plasma D-dimer levels compared to healthy individuals and individuals with other diseases. Our in vitro analysis showed that the addition of histone to the plasma results in a significant decrease in viscoelasticity and mean fiber thickness of the clot formed thereafter. In parallel studies, using plasma from patients, DNA and histones were detected in fibrin clots and were associated with less degradation by t-PA. Moreover, our results show that the presence of DNA and histones not only increases clots' permeability, but also makes them more prone to degradation. Plasma histones and DNA affect the structure of the clot formed and induce defective fibrinolysis. Moreover, the increased viscoelastic properties of plasma from cancer patients can be used as potential biomarkers in cancer prognosis.

Histone H3 activates caspase-1 and promotes proliferation and metastasis in hepatocellular carcinoma

Background: As a component of nucleosomes, histone H3 plays an important role in chromosome structure and gene expression. Current studies have mostly focused on the role of histones in epigenetics, but in addition to this, the role of histones themselves in tumor development and microenvironment have been less explored. Methods: Western blot and immunofluorescence were carried out to detect the content and localization of histone H3 in hepatocellular carcinoma. The changes of histone H3 were observed in hypoxia treatment cells, the specific action mechanism of histone H3 was studied by CoIP and other methods. Cell Counting Kit-8 assay, plate cloning assay and transwell assay were used to exam the effect of histone H3 on cell proliferation and metastasis, which were verified by subcutaneous tumors in mice and lung metastasis by tail vein injection in mice. Results: We found that histone H3 was overexpressed in hepatocellular carcinoma tumor tissues compared to adjacent non-tumor tissues, and there was concomitant translocation of histone H3 from the nucleus to the cytoplasm. We found that hypoxia could contribute to this phenomenon of histone H3 translocation from the nucleus to the cytoplasm in hepatocellular carcinoma cells and increased binding levels to TLR9. At the same time, hypoxia induced downstream activation of TLR9 and caspase-1, as well as cleavage and release of the pro-inflammatory cytokines IL-1β and IL-18. We further demonstrated that histone H3 could also promote proliferation and metastasis of hepatocellular carcinoma through TLR9 activation of NLRP3 inflammasome. In addition, overexpression of histone H3 was also confirmed to promote hepatocellular carcinoma proliferation and metastasis in mouse models of hepatocellular carcinoma growth assay and lung metastasis. Conclusions: In hypoxic hepatocellular carcinoma cells, histone H3 can translocate to the cytoplasm and activate caspase-1 via TLR9, thereby producing pro-inflammatory cytokines that promote tumor proliferation and metastasis.

Circulating histones contribute to monocyte and MDW alterations as common mediators in classical and COVID-19 sepsis

ObjectiveHistone proteins are physiologically involved in DNA packaging and gene regulation but are extracellularly released by neutrophil/monocyte extracellular traps and mediate thrombo-inflammatory pathways, associated to the severity of many human pathologies, including bacterial/fungal sepsis and COVID-19. Prominent and promising laboratory features in classic and viral sepsis emphasize monocyte distribution width (MDW), due to its ability to distinguish and stratify patients at higher risk of critical conditions or death. No data are available on the roles of histones as MDW modifiers.DesignComparison of MDW index was undertaken by routine hematology analyzer on whole blood samples from patients with COVID-19 and Sepsis. The impact of histones on the MDW characteristics was assessed by the in vitro time-dependent treatment of healthy control whole blood with histones and histones plus lipopolysaccharide to simulate viral and classical sepsis, respectively.Measurements and main resultsWe demonstrated the breadth of early, persistent, and significant increase of MDW index in whole blood from healthy subject treated in vitro with histones, highlighting changes similar to those found in vivo in classic and viral sepsis patients. These findings are mechanistically associated with the histone-induced modifications of cell volume, cytoplasmic granularity and vacuolization, and nuclear structure alterations of the circulating monocyte population.ConclusionsHistones may contribute to the pronounced and persistent monocyte alterations observed in both acute classical and viral sepsis. Assessment of the biological impact of circulating histone released during COVID-19 and sepsis on these blood cells should be considered as key factor modulating both thrombosis and inflammatory processes, as well as the importance of neutralization of their cytotoxic and procoagulant activities by several commercially available drugs (e.g., heparins and heparinoids).

In Memoriam: Stuart B. Moss, PhD, April 1, 1950-November 13, 2021.

Andrology lost one of its staunchest supporters with the recent death of Stuart B. Moss, PhD, in Washington, DC, on Saturday, November 13, 2021. Stuart received his undergraduate degree with honors from Union College in 1972 and then attended graduate school at the University of Rochester, where he received his PhD working in the laboratory of Jerome Kaye. From 1980 to 1985, he was a Research Fellow in Physiology at Harvard Medical School with Anthony Bellvé and, from 1985 to 1989, was Staff Scientist in the laboratory of Mark Groudine at the Fred Hutchinson Cancer Center in Seattle. In 1989, he moved to Philadelphia to join the faculty of the Temple University School of Medicine and was recruited in 1992 to join the Center of Research on Reproduction and Women's Health of the University of Pennsylvania School of Medicine. Fifteen years later, Stuart left to take administrative positions in the US National Institutes of Health (NIH), first as a Scientific Review Officer with the Center for Scientific Review and, 2 years later, as a program officer with the Fertility and Infertility Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). He also worked since 2014 in the NICHD Office of the Director on legislative matters. Stuart's research examined several important aspects of spermatogenic cell differentiation. His work on sperm nuclear structure instilled a long-term interest in the roles of histones in mouse and avian spermatogenesis. He also incorporated techniques of molecular biology to examine gene regulation during spermatogenesis, dissecting the cellular and molecular processes involved in building other sperm structures such as the flagellum. These studies led to the discovery and elucidation of several new proteins involved in anchoring and regulating signaling processes important for sperm motility. His work was some of the first to demonstrate that haploid-expressed proteins encoded by the X-chromosome are components of both the X and Y chromosome-bearing spermatids. In 2002, while on sabbatical as a Visiting Professor in the laboratory of Masaru Okabe (2017 ASA Distinguished Andrologist) in Osaka University, Japan, Stuart expanded his repertoire of techniques to include gene editing and functional fertilization studies using spermatozoa from mice containing experimentally induced mutations. As a laboratory scientist dependent on grant funding, Stuart learned first-hand how important it is to have grant proposals fairly evaluated and to have the feedback from reviews clearly communicated to the applicants. He took this understanding to his positions at NIH, first in the reviewing process and later as a Program Officer responsible for the portfolio of grants dealing with andrology. In this role, he assisted many early-stage investigators navigate the granting process and helped to guide more senior scientists entering the field for the first time. On a personal level, Stuart had a quiet, nonassuming outward demeanor. He came from a small, tight-knit family that included his parents and a younger sister. Although he had no children of his own, he was very close to his niece. He often took her or children of his friends to amusement parks or sporting events. To many, he was an honorary “uncle”. In the field of andrology, we often speak of our colleagues as “family.” At some level, conferences such as the Annual Meeting of the American Society of Andrology are “family reunions”. Stuart was an active participant in the ASA meetings where he annually presented an update of the funding climate at NIH. He also took part in symposia and luncheons for trainees. In conversations with colleagues at these meetings, the questions “Have you met Stuart yet” and “Have you spoken to Stuart about your research idea” were frequently uttered; only his first name needed to be said, we all knew who was meant. The announcements of Stuart's death released an outpouring of emotions of sorrow for his passing and gratitude for everything he had done for his scientific family and the field of andrology. Our hearts go out to all who knew him and, especially, to his companion of 20 years, Pat Pileggi; his mother, Dorothy Moss; his sister, Wendy Moss; his niece, Jordana Haviv; and his grand-nephew Julian Joshua Torres.

The Role of Histones and Heparin in Sepsis: A Review.

Septic shock with multiple organ failure is a devastating situation in clinical settings. Through the past decades, much progress has been made in the management of sepsis and its underlying pathogenesis, but a highly effective therapeutic has not been developed. Recently, macromolecules such as histones have been targeted in the treatment of sepsis. Histones primarily function as chromosomal organizers to pack DNA and regulate its transcription through epigenetic mechanisms. However, a growing body of research has shown that histone family members can also exert cellular toxicity once they relocate from the nucleus into the extracellular space. Heparin, a commonly used anti-coagulant, has been shown to possess life-saving capabilities for septic patients, but the potential interplay between heparin and extracellular histones has not been investigated. In this review, we summarize the pathogenic roles of extracellular histones and the therapeutic roles of heparin in the development and management of sepsis and septic shock.

More than meets the eye: the histones revealed as enzymes

Histones have been known as proteins that package the DNA of eukaryotes and regulate gene expression. We discovered that histones also function as enzymes that convert copper ions into a usable form for cells. The enzymatic function changes how we think about the roles of histones in health and disease. It may have also been a critical driver for emergence of eukaryotes in the first place. - submission by Oscar A. Campos, Siavash K. Kurdistani

Molecular Regulation of the Polycomb Repressive-Deubiquitinase.

Post-translational modification of histone proteins plays a major role in histone–DNA packaging and ultimately gene expression. Attachment of ubiquitin to the C-terminal tail of histone H2A (H2AK119Ub in mammals) is particularly relevant to the repression of gene transcription, and is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here, we outline recent advances in the understanding of PR-DUB regulation, which have come through structural studies of the Drosophila melanogaster PR-DUB, biochemical investigation of the human PR-DUB, and functional studies of proteins that associate with the PR-DUB. In humans, mutations in components of the PR-DUB frequently give rise to malignant mesothelioma, melanomas, and renal cell carcinoma, and increase disease risk from carcinogens. Diverse mechanisms may underlie disruption of the PR-DUB across this spectrum of disease. Comparing and contrasting the PR-DUB in mammals and Drosophila reiterates the importance of H2AK119Ub through evolution, provides clues as to how the PR-DUB is dysregulated in disease, and may enable new treatment approaches in cancers where the PR-DUB is disrupted.

Extracellular histones in lung dysfunction: a new biomarker and therapeutic target?

Extracellular histones released from injured or dying cells following trauma and other severe insults can act as potent damage-associated molecular patterns. In fact, elevated levels of histones are present in human circulation in hyperinflammatory states such as acute respiratory distress syndrome and sepsis. The molecular mechanisms owing to histone-induced pathologies are at the very beginning of elucidating. However, neutralization of histones with antibodies, histone-binding or histone-degrading proteins, and heparan sulfates have shown promising therapeutic effects in pre-clinical acute respiratory distress syndrome and sepsis models. Various cell types undergoing necrosis and apoptosis or activated neutrophils forming neutrophil extracellular traps have been implicated in excessive release of histones which further augments tissue injury and may culminate in multiple organ failure. At the molecular level, an uncontrolled inflammatory cascade has been considered as the major event; however, histone-activated coagulation and thrombosis represent additional pathologic events reflecting coagulopathy. Furthermore, epigenetic regulation and chemical modifications of circulating histones appear to be critically important in their biological functions as evidenced by increased cytotoxicity associated with citrullinated histone. Herein, we will briefly review the current knowledge on the role of histones in acute respiratory distress syndrome and sepsis, and discuss the future potential of anti-histone therapy for treatment of these life-threatening disorders.

AtFKBP53: a chimeric histone chaperone with functional nucleoplasmin and PPIase domains.

FKBP53 is one of the seven multi-domain FK506-binding proteins present in Arabidopsis thaliana, and it is known to get targeted to the nucleus. It has a conserved PPIase domain at the C-terminus and a highly charged N-terminal stretch, which has been reported to bind to histone H3 and perform the function of a histone chaperone. To better understand the molecular details of this PPIase with histone chaperoning activity, we have solved the crystal structures of its terminal domains and functionally characterized them. The C-terminal domain showed strong PPIase activity, no role in histone chaperoning and revealed a monomeric five-beta palm-like fold that wrapped over a helix, typical of an FK506-binding domain. The N-terminal domain had a pentameric nucleoplasmin-fold; making this the first report of a plant nucleoplasmin structure. Further characterization revealed the N-terminal nucleoplasmin domain to interact with H2A/H2B and H3/H4 histone oligomers, individually, as well as simultaneously, suggesting two different binding sites for H2A/H2B and H3/H4. The pentameric domain assists nucleosome assembly and forms a discrete complex with pre-formed nucleosomes; wherein two pentamers bind to a nucleosome.

Role and mechanism of extracellular histone in lung ischemia/reperfusion injury

Histone is a kind of basic structural protein of eukaryotic chromatin. When cells is damaged or necrotic, the histone proteins were released into the extracellular space and become extracellular histone proteins. Recent studies have found that extracellular group proteins play an important role in ischemic/reperfusion injury(I/RI) in the lungs. This review summarized the studies of recent years about the role of histone in I/RI in the lung and provided references for further studying the role and mechanism of histone in I/RI. Elaborating the structure and biological functions of histone and the source of the extracellular histone to investigate the possible role of the extracellular histone and the underlying mechanism of histone regulation in the I/RI in many aspects: such as toxicity, nod-like receptor protein 3 (NLRP3) pathway, injured polysaccharide-coated pathway, associated inflammatory signaling pathways, and platelet-dependent pathways, etc. Further studies of histone would provide new therapeutic target for the clinical treatment of ischemia/reperfusion injury. Key words: Extracellular histone; Ischemia-reperfusion injury; Lung

Development of Robust Yeast Strains for Lignocellulosic Biorefineries Based on Genome-Wide Studies.

Lignocellulosic biomass has been widely studied as the renewable feedstock for the production of biofuels and biochemicals. Budding yeast Saccharomyces cerevisiae is commonly used as a cell factory for bioconversion of lignocellulosic biomass. However, economic bioproduction using fermentable sugars released from lignocellulosic feedstocks is still challenging. Due to impaired cell viability and fermentation performance by various inhibitors that are present in the cellulosic hydrolysates, robust yeast strains resistant to various stress environments are highly desired. Here, we summarize recent progress on yeast strain development for the production of biofuels and biochemical using lignocellulosic biomass. Genome-wide studies which have contributed to the elucidation of mechanisms of yeast stress tolerance are reviewed. Key gene targets recently identified based on multiomics analysis such as transcriptomic, proteomic, and metabolomics studies are summarized. Physiological genomic studies based on zinc sulfate supplementation are highlighted, and novel zinc-responsive genes involved in yeast stress tolerance are focused. The dependence of host genetic background of yeast stress tolerance and roles of histones and their modifications are emphasized. The development of robust yeast strains based on multiomics analysis benefits economic bioconversion of lignocellulosic biomass.

CRISPR-Mediated Loss of Immunoglobulin Heavy Chain in Multiple Myeloma Cell Line Results in Metabolic Pathway Alterations

Aberrant over production of monoclonal immunoglobulin is a classic feature of the plasma cell (PC) malignancy multiple myeloma (MM). At diagnosis and more frequently upon relapse, some MM patients present with increased production of monoclonal free light chain (FLC) without a corresponding increase in intact immunoglobulin. This phenomenon is commonly referred to as light chain escape (LCE). As previously described (Walters et al. 2018, Experimental Hematology, Vol 57 p42-49), we have established a monoclonal IgA kappa MM cell line, designated as MC-B11/14, from the bone marrow (BM) aspirate of a 46 year old man diagnosed with MM. This cell line is non-hyperdiploid and possesses an 11;14 chromosomal translocation. Given our long standing interest in immunoglobulin function and secretion, we used CRISPR technology to knock out IgA heavy chain production in an effort to create a model of LCE. Successful knockout (KO) of IgA expression was demonstrated by immunofluorescence, flow cytometry, and western blot. As expected, secretion of intact IgA was undetectable in the MC-B11/14IgA- cells. Notably, no significant difference in morphology, phenotypic markers, or growth patterns was observed between the MC-B11/14WT and MC-B11/14IgA- cells. Examination of drug sensitivity between the MC-B11/14WT and MC-B11/14IgA- cells revealed that the response to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, the MC-B11/14IgA- cells were found to be more resistant to treatment with bortezomib. Given that increased bortezomib resistance was the only significant difference detected between the MC-B11/14WT and MC-B11/14IgA- cell lines in our initial study, we decided to take a metabolomics approach to examine whether loss of IgA heavy chain expression may alter cellular metabolism. Metabolomics or the profiling of small molecules is a powerful tool to characterize the metabolome of cells/biological systems and has proven to be useful in identifying alterations that occur in malignancy. In this regard, we employed pathway-specific targeted LC-MS/MS protocols in order to examine over 330 metabolites in 35 metabolic pathways in MC-B11/14WT and MC-B11/14IgA- methanolmetabolite extracts using an Agilent 1290 UPLC-6490 QQQ-MS liquid chromatography mass-spectrometer. Of the 330 metabolites examined, 28 were found to be significantly more abundant in the MC-B11/14IgA- cells compared to MC-B11/14WT cells while 79 were found to be significantly less abundant. In general, the majority of significant metabolic differences between the two cell lines were found to involve metabolites from various amino acid synthesis pathways. More specifically, the MC-B11/14WT cell line was found to possess significantly more citraconic acid, anthranilic acid, and homovanillic acid. By contrast, further analysis revealed that the MC-B11/14IgA- cell line was found to possess an 11.5 fold increase of the metabolite 2-hydroxyglutarate (2-HG). 2-HG has previously been shown to act as a competitive antagonist of α-ketoglutarate (α-KG), which results in the inhibition of α-KG-dependent dioxygenases including the JmjC domain-containing histone demethylases (KDMs) and the ten-eleven translocation (TET) family of DNA hydroxylases. Notably, these proteins are known to play a role in histone and DNA demethylation, respectively. Taken together, these data suggest that MM cells that have undergone LCE may also possess an accumulation of 2-HG which could lead to altered patterns of histone and DNA methylation. Given that MM patients who relapse with LCE typically have a poorer prognosis, further investigation of metabolomics in light-chain only expressing cell lines and patients who have relapsed with LCE is warranted. DisclosuresNo relevant conflicts of interest to declare.

Interfering histone deacetylase 4 inhibits the proliferation of vascular smooth muscle cells via regulating MEG3/miR-125a-5p/IRF1

ABSTRACTIn this study, we investigated the role ofhistone deacetylase 4 (HDAC4) and MEG3/miR-125a-5p/interferonregulatoryfactor 1 (IRF1) on vascular smooth muscle cell (VSMCs)proliferation. Platelet derived growth factor (PDGF)-BB was used toinduce the proliferation and migration of VSMCs. The expressionsof MEG3, miR-125a-5p, HDAC4 and IRF1in VSMCs were detectedby qRT-PCR and western blot, respectively. ChIP assay was usedto determine the relationship between MEG3 and HDAC4. Doubleluciferase reporter assay was used to test the regulation betweenmiR-125-5p and IRF1. Results showed that PDGF-BB decreasedthe expression of MEG3 and IRF1, while increased the expressionof miR-125a-5p and HDAC4. In addition, HDAC4 knockdowninhibited the proliferation and migration of VSMCs via upregulatingMEG3 and downregulating miR-125a-5p. MiR-125a-5p inhibitorcould repress the proliferation and migration of VSMCs andalleviate intimal hyperplasia (IH) by directly upregulating IRF1expression. These results suggested that HDAC4 interferenceinhibited PDGF-BB-induced VSMCs proliferation via regulatingMEG3/miR-125a-5p/IRF1 axis, and then alleviated IH.

Spermatid-specific linker histone HILS1 is a poor condenser of DNA and chromatin and preferentially associates with LINE-1 elements

BackgroundLinker histones establish and maintain higher-order chromatin structure. Eleven linker histone subtypes have been reported in mammals. HILS1 is a spermatid-specific linker histone, and its expression overlaps with the histone–protamine exchange process during mammalian spermiogenesis. However, the role of HILS1 in spermatid chromatin remodeling is largely unknown.ResultsIn this study, we demonstrate using circular dichroism spectroscopy that HILS1 is a poor condenser of DNA and chromatin compared to somatic linker histone H1d. Genome-wide occupancy study in elongating/condensing spermatids revealed the preferential binding of HILS1 to the LINE-1 (L1) elements within the intergenic and intronic regions of rat spermatid genome. We observed specific enrichment of the histone PTMs like H3K9me3, H4K20me3 and H4 acetylation marks (H4K5ac and H4K12ac) in the HILS1-bound chromatin complex, whereas H3K4me3 and H3K27me3 marks were absent.ConclusionsHILS1 possesses significantly lower α-helicity compared to other linker histones such as H1t and H1d. Interestingly, in contrast to the somatic histone variant H1d, HILS1 is a poor condenser of chromatin which demonstrate the idea that this particular linker histone variant may have distinct role in histone to protamine replacement. Based on HILS1 ChIP-seq analysis of elongating/condensing spermatids, we speculate that HILS1 may provide a platform for the structural transitions and forms the higher-order chromatin structures encompassing LINE-1 elements during spermiogenesis.

The roles of histone methyl‐transferase kmt2d in vasculogenesis

INTRODUCTIONCongenital heart defects (CHD) arise from abnormal development of the heart and major blood vessels. An estimated 40,000 infants are affected by CHD each year in the United States. Despite its high incidence, our understanding of the complex genetic anomalies leading these defects is limited. Recent studies linked mutations in the ubiquitous histone methyl‐transferase KMT2D with congenital heart defects and Kabuki Syndrome, a condition with a spectrum of CHD and other congenital disorders. While KMT2D has well‐described roles in myocardium development during cardiogenesis, it is unknown whether KMT2D has regulatory functions during vasculogenesis, which could contribute to the spectrum of heart and other defects in Kabuki Syndrome. KMTD2 has recently been shown to interact with Notch signaling in Drosophila. While Notch pathway has well‐established roles in vasculogenesis and angiogenesis, its regulation by KMT2D during cardiovascular development has not been explored. Zebrafish provide a strong model for the analysis of vasculogenesis defects, since it can survive early development in absence of blood circulation.OBJECTIVEThe primary objective of this study is to describe the roles of Kmt2d and its regulatory link with Notch signaling during zebrafish vasculogenesis.METHODSUsing the CRISPR/Cas9 technology, we generated Zebrafish kmt2d null mutants that recapitulate the phenotypic features of human Kabuki Syndrome. In order to study endocardial/endothelial development, the stable mutant kmt2dzy59 was crossed with the transgenic line tg(kdrl:GFP). kmt2dzy59;tg(kdrl:GFP) cardiovascular phenotype was characterized with super resolution confocal microscopy and fluorescent‐activated cell sorting (FACS).Kmt2d and Notch pathway interaction was assessed by G0 knockdown analysis in the Notch reporter line tg(TP1:EGFP). Results were confirmed and expanded by RNAseq of single embryos from genetic crosses at 24hpf. Single embryo transcriptome analysis allows us to capture transcriptomes in clutches of embryos before the appearance of phenotypes, and to post‐facto genotype sibling embryos by transcriptome analysis with Kallisto and SNP mapping.RESULTSIn this study we generated Zebrafish kmt2d null mutants that recapitulate the phenotypic features of Kabuki Syndrome. We characterized for the first time a cardiovascular developmental defect in kmt2dzy59 embryos, including hypoplastic ventricle, aortic arch disruption, and altered cell cycle profiles. kmt2d CRISPR/Cas9 injection in a Notch reporter line showed expanded Notch signaling in endocardial/endothelial cells. RNAseq analysis of kmt2dzy59 embryos showed a substantial upregulation of Notch signaling components.CONCLUSIONOur results demonstrate that Kmt2d regulates zebrafish vasculogenesis. We also provide evidence of regulatory interactions between Kmt2d and Notch signaling which modify endothelial/endocardial cell behavior.Support or Funding InformationThis work in HJY laboratory was supported by NHLBI CvDC U01HL0981This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histone demethylases UTX and JMJD3 are required for NKT cell development in mice

BackgroundNatural killer (NK)T cells and conventional T cells share phenotypic characteristic however they differ in transcription factor requirements and functional properties. The role of histone modifying enzymes in conventional T cell development has been extensively studied, little is known about the function of enzymes regulating histone methylation in NKT cells.ResultsWe show that conditional deletion of histone demethylases UTX and JMJD3 by CD4-Cre leads to near complete loss of liver NKT cells, while conventional T cells are less affected. Loss of NKT cells is cell intrinsic and not due to an insufficient selection environment. The absence of NKT cells in UTX/JMJD3-deficient mice protects mice from concanavalin A‐induced liver injury, a model of NKT‐mediated hepatitis. GO‐analysis of RNA-seq data indicates that cell cycle genes are downregulated in UTX/JMJD3-deleted NKT progenitors, and suggest that failed expansion may account for some of the cellular deficiency. The phenotype appears to be demethylase‐dependent, because UTY, a homolog of UTX that lacks catalytic function, is not sufficient to restore their development and removal of H3K27me3 by deletion of EZH2 partially rescues the defect.ConclusionsNKT cell development and gene expression is sensitive to proper regulation of H3K27 methylation. The H3K27me3 demethylase enzymes, in particular UTX, promote NKT cell development, and are required for effective NKT function.

KDM6B modulates MAPK pathway mediating multiple myeloma cell growth and survival.

Recent studies have delineated cancer type-specific roles of histone 3 lysine 27 (H3K27) demethylase KDM6B/JMJD3 depending on its H3K27 demethylase activity. Here we show that KDM6B is expressed in multiple myeloma (MM); and that shRNA-mediated knockdown and CRISPR-mediated knockout of KDM6B abrogate MM cell growth and survival. TNFα or bone marrow stromal cell culture supernatants induce KDM6B, which is blocked by IKKβ inhibitor MLN120B, suggesting KDM6B is regulated by NF-κB signaling in MM cells. RNA-sequencing and subsequent ChIP-qPCR analyses reveal that KDM6B is recruited to the loci of genes encoding components of MAPK signaling pathway including ELK1 and FOS, and upregulates these genes expression without affecting H3K27 methylation level. Overexpression of catalytically-inactive KDM6B activates expression of MAPK pathway-related genes, confirming its function independent of demethylase activity. We further demonstrate that downstream targets of KDM6B, ELK1 and FOS, confer MM cell growth. Our study therefore delineates KDM6B function that links NF-κB and MAPK signaling pathway mediating MM cell growth and survival, and validates KDM6B as a novel therapeutic target in MM.

Retrovirus Integration: Some Assembly Required?

Integration is a key feature of the retroviral life cycle. This process involves packaging of the viral genome into chromatin, which is often assumed to occur as a post-integration step. In this issue of Cell Host & Microbe, Wang and colleagues (Wang etal., 2016) show that chromatinization occurs before integration, raising new questions about the role of histones in retroviral integration and transcription.