Epigenetic regulators controlling osteogenic lineage commitment and bone formation.

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.

Protein arginine methyltransferase 1, a major regulator of biological processes.

Protein arginine methyltransferase 1 (PRMT1) is a major type I arginine methyltransferase that catalyzes the formation of monomethyl and asymmetric dimethylarginine in protein substrates. It was first identified to asymmetrically methylate histone H4 at the third arginine residue forming the H4R3me2a active histone mark. However, several protein substrates are now identified as being methylated by PRMT1. As a result of its association with diverse classes of substrates, PRMT1 regulates several biological processes like chromatin dynamics, transcription, RNA processing, and signal transduction. The review provides an overview of PRMT1 structure, biochemical features, specificity, regulation, and role in cellular functions. We discuss the genomic distribution of PRMT1 and its association with tRNA genes. Further, we explore the different substrates of PRMT1 involved in splicing. In the end, we discuss the proteins that interact with PRMT1 and their downstream effects in diseased states.

MeCP2 is the protector of epigenome integrity, membrane-less nuclear architecture, and stability of chromatin assembly.

Tweetable abstract MeCP2 is an epigenetic factor with global impact in epigenome integrity, membrane-less nuclear architecture, and chromatin stability. Our Editorial covers recent advances on these important topics.

Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a), a mark of super-enhancers.

Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a) is an active histone mark catalyzed by protein arginine methyltransferase 1 (PRMT1), a major arginine methyltransferase in vertebrates catalyzing asymmetric dimethylation of arginine. H4R3me2a stimulates the activity of lysine acetyltransferases such as CBP/p300, which catalyze the acetylation of H3K27, a mark of active enhancers, super-enhancers, and promoters. There are a few studies on the genomic location of H4R3me2a. In chicken polychromatic erythrocytes, H4R3me2a is found in introns and intergenic regions and binds to the globin locus control region (a super-enhancer) and globin regulatory regions. In this report, we analyzed chromatin immunoprecipitation sequencing data for the genomic location of H4R3me2a in the breast cancer cell line MCF7. As in avian cells, MCF7 H4R3me2a is present in intronic and intergenic regions. Nucleosomes with H4R3me2a and H3K27ac next to nucleosome-free regions are found at super-enhancers, enhancers, and promoter regions of expressed genes. Genes with critical roles in breast cancer cells have broad domains of nucleosomes with H4R3me2a, H3K27ac, and H3K4me3. Our results are consistent with PRMT1-mediated H4R3me2a playing a key role in the chromatin organization of regulatory regions of vertebrate genomes.

Protein arginine methyltransferases PRMT1, PRMT4/CARM1 and PRMT5 have distinct functions in control of osteoblast differentiation.

Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization (e.g., Alpl, Ibsp, Phospho1) consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators (e.g., Ezh1, Ezh2, Brd2 and Brd4). Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.

Broad histone H4 monomethylation marks expressed genes involved in translation.

H4K20me1 (histone H4 monomethylated at lysine 20) generally has a broad distribution along genes and has been reported to be associated with expressed and repressed genes. In contrast, H3K4me3 (histone H3 trimethylated at lysine 4) is positioned as a narrow peak at the 5' end of most expressed genes in vertebrate cells. A small population of genes involved in cell identity has H3K4me3 distributed throughout the gene body. In this report, we show that H4K20me1 is associated with expressed genes in estrogen receptor-positive breast cancer MCF7 cells and erythroleukemic K562 cells. Further, we identified the genes with the broadest H4K20me1 domains in these two cell types. The broad H4K20me1 domain marked gene bodies of expressed genes, but not the promoter or enhancer regions. The most significant GO term (biological processes) of these genes was cytoplasmic translation. There was little overlap between the genes marked with the broad H4K20me1 domain and those marked with H3K4me3. H4K20me1 and H3K79me2 distributions along expressed gene bodies were similar, suggesting a relationship between the enzymes catalyzing these histone modifications.

Chicken Erythrocyte: Epigenomic Regulation of Gene Activity.

The chicken genome is one-third the size of the human genome and has a similarity of sixty percent when it comes to gene content. Harboring similar genome sequences, chickens' gene arrangement is closer to the human genomic organization than it is to rodents. Chickens have been used as model organisms to study evolution, epigenome, and diseases. The chicken nucleated erythrocyte's physiological function is to carry oxygen to the tissues and remove carbon dioxide. The erythrocyte also supports the innate immune response in protecting the chicken from pathogens. Among the highly studied aspects in the field of epigenetics are modifications of DNA, histones, and their variants. In understanding the organization of transcriptionally active chromatin, studies on the chicken nucleated erythrocyte have been important. Through the application of a variety of epigenomic approaches, we and others have determined the chromatin structure of expressed/poised genes involved in the physiological functions of the erythrocyte. As the chicken erythrocyte has a nucleus and is readily isolated from the animal, the chicken erythrocyte epigenome has been studied as a biomarker of an animal's long-term exposure to stress. In this review, epigenomic features that allow erythroid gene expression in a highly repressive chromatin background are presented.

Mitogen- and stress-activated protein kinase (MSK1/2) regulated gene expression in normal and disease states.

The mitogen- and stress-activated protein kinases (MSK) are epigenetic modifiers that regulate gene expression in normal and disease cell states. MSK1 and 2 are involved in a chain of signal transduction events bringing signals from the external environment of a cell to specific sites in the genome. MSK1/2 phosphorylate histone H3 at multiple sites, resulting in chromatin remodeling at regulatory elements of target genes and the induction of gene expression. Several transcription factors (RELA of NF-κB and CREB) are also phosphorylated by MSK1/2 and contribute to induction of gene expression. In response to signal transduction pathways, MSK1/2 can stimulate genes involved in cell proliferation, inflammation, innate immunity, neuronal function, and neoplastic transformation. Abrogation of the MSK-involved signaling pathway is among the mechanisms by which pathogenic bacteria subdue the host's innate immunity. Depending on the signal transduction pathways in play and the MSK-targeted genes, MSK may promote or hinder metastasis. Thus, depending on the type of cancer and genes involved, MSK overexpression may be a good or poor prognostic factor. In this review, we focus on mechanisms by which MSK1/2 regulate gene expression, and recent studies on their roles in normal and diseased cells.

The Chromatin Structure at the MECP2 Gene and In Silico Prediction of Potential Coding and Non-Coding MECP2 Splice Variants.

Methyl CpG binding protein 2 (MeCP2) is an epigenetic reader that binds to methylated CpG dinucleotides and regulates gene transcription. Mecp2/MECP2 gene has 4 exons, encoding for protein isoforms MeCP2E1 and MeCP2E2. MeCP2 plays key roles in neurodevelopment, therefore, its gain- and loss-of-function mutations lead to neurodevelopmental disorders including Rett Syndrome. Here, we describe the structure, functional domains, and evidence support for potential additional alternatively spliced MECP2 transcripts and protein isoforms. We conclude that NCBI MeCP2 isoforms 3 and 4 contain certain MeCP2 functional domains. Our in silico analysis led to identification of histone modification and accessibility profiles at the MECP2 gene and its cis-regulatory elements. We conclude that the human MECP2 gene associated histone post-translational modifications exhibit high similarity between males and females. Between brain regions, histone modifications were found to be less conserved and enriched within larger genomic segments named as "S1-S11". We also identified highly conserved DNA accessibility regions in different tissues and brain regions, named as "A1-A9" and "B1-B9". DNA methylation profile was similar between mid-frontal gyrus of donors 35 days-25 years of age. Based on ATAC-seq data, the identified hypomethylated regions "H1-H8" intersected with most regions of the accessible chromatin (A regions).

Integrative analysis reveals novel associations between DNA methylation and the serum metabolome of adolescents with type 2 diabetes: A cross-sectional study.

Objective: Rates of type 2 diabetes (T2D) among adolescents are on the rise. Epigenetic changes could be associated with the metabolic alterations in adolescents with T2D. Methods: We performed a cross sectional integrated analysis of DNA methylation data from peripheral blood mononuclear cells with serum metabolomic data from First Nation adolescents with T2D and controls participating in the Improving Renal Complications in Adolescents with type 2 diabetes through Research (iCARE) cohort study, to explore the molecular changes in adolescents with T2D. Results: Our analysis showed that 43 serum metabolites and 36 differentially methylated regions (DMR) were associated with T2D. Several DMRs were located near the transcriptional start site of genes with established roles in metabolic disease and associated with altered serum metabolites (e.g. glucose, leucine, and gamma-glutamylisoleucine). These included the free fatty acid receptor-1 (FFAR1), upstream transcription factor-2 (USF2), and tumor necrosis factor-related protein-9 (C1QTNF9), among others. Conclusions: We identified DMRs and metabolites that merit further investigation to determine their significance in controlling gene expression and metabolism which could define T2D risk in adolescents.

Bioinformatic Analyses of Broad H3K79me2 Domains in Different Leukemia Cell Line Data Sets.

A subset of expressed genes is associated with a broad H3K4me3 (histone H3 trimethylated at lysine 4) domain that extends throughout the gene body. Genes marked in this way in normal cells are involved in cell-identity and tumor-suppressor activities, whereas in cancer cells, genes driving the cancer phenotype (oncogenes) have this feature. Other histone modifications associated with expressed genes that display a broad domain have been less studied. Here, we identified genes with the broadest H3K79me2 (histone H3 dimethylated at lysine 79) domain in human leukemic cell lines representing different forms of leukemia. Taking a bioinformatic approach, we provide evidence that genes with the broadest H3K79me2 domain have known roles in leukemia (e.g., JMJD1C). In the mixed-lineage leukemia cell line MOLM-13, the HOXA9 gene is in a 100 kb broad H3K79me2 domain with other HOXA protein-coding and oncogenic long non-coding RNA genes. The genes in this domain contribute to leukemia. This broad H3K79me2 domain has an unstable chromatin structure, as was evident by enhanced chromatin accessibility throughout. Together, we provide evidence that identification of genes with the broadest H3K79me2 domain will aid in generating a panel of genes in the diagnosis and therapeutic treatment of leukemia in the future.

The key role of differential broad H3K4me3 and H3K4ac domains in breast cancer.

Epigenetic processes are radically altered in cancer cells. The altered epigenetic events may include histone post-translational modifications (PTMs), DNA modifications, and/or alterations in the levels and modifications of chromatin modifying enzymes and chromatin remodelers. With changes in gene programming are changes in the genomic distribution of histone PTMs. Genes that are poised or transcriptionally active have histone H3 trimethylated lysine 4 (H3K4me3) located at the transcription start site and at the 5' end of the gene. However, a small population of genes that are involved in cell identity or cancer cell properties have a broad H3K4me3 domain that may stretch for several kilobases through the coding region of the gene. Each cancer cell type appears to mark a select set of cancer-related genes in this manner. In this study, we determined which genes were differentially marked with the broad H3K4me3 domain in normal-like (MCF10A), luminal-type breast cancer (MCF7), and triple-negative breast cancer (MDA-MB-231) cells. We also determined whether histone H3 acetylated lysine 4 (H3K4ac), also a mark of active promoters, had a broad domain configuration. We applied two peak callers (MACS2, PeakRanger) to analyze H3K4me3 and H3K4ac chromatin immunoprecipitation sequencing (ChIP-Seq) data. We identified genes with a broad H3K4me3 and/or H3K4ac domain specific to each cell line and show that the genes have critical roles in the breast cancer subtypes. Furthermore, we show that H3K4ac marks enhancers. The identified genes with the broad H3K4me3/H3K4ac domain have been targeted in clinical and pre-clinical studies including therapeutic treatments of breast cancer.

Differential expression of HNF1A and HNF1A-AS1 in colon cancer cells.

The human hepatocyte nuclear factor 1 homeobox A (HNF1A) gene loci express the protein-coding HNF1A transcript and a long non-coding RNA in the anti-sense (HNF1A-AS1) direction. HNF1A-AS1 is expressed in numerous types of cancers and poor clinical outcomes such as higher mortality rates, greater metastatic capacity, and poor prognosis of the disease are the results of this expression. In this study, we determined the epigenetic features of the HNF1A gene loci, and expression and cellular localization of HNF1A-AS1 RNA, HNF1A RNA, and HNF1A protein in colorectal cancer (HT-29, HTC116, RKO, and SW480) and normal colon epithelial (CCD841) cells. The HT-29 HNF1A gene had active histone marks (H3K4me3, H3K27ac) and DNase 1 accessible sites at the promoter regions of the HNF1A and HNF1A-AS1 genes. These epigenetic marks were not observed in the other colorectal cancer cells or in the normal colon epithelial cells. Consistent with the active gene epigenetic signature of the HNF1A gene in HT-29 cells, HNF1A protein, and HNF1A/HNF1A-AS1 transcripts were detected in HT-29 cells but poorly, if at all observed, in the other cell types. In HT-29 cells, HNF1A-AS1 localized to the nucleus and was found to bind to the enhancer of zeste homolog 2 (EZH2, a member of PRC2 complex) and potentially form RNA-DNA triplexes with DNase 1 accessible sites in the HT-29 genome. These activities of HNF1A-AS1 may contribute to the oncogenic properties of this long non-coding RNA.

Mitogen-induced transcriptional programming in human fibroblasts.

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

The dynamic broad epigenetic (H3K4me3, H3K27ac) domain as a mark of essential genes.

Transcriptionally active chromatin is marked by tri-methylation of histone H3 at lysine 4 (H3K4me3) located after first exons and around transcription start sites. This epigenetic mark is typically restricted to narrow regions at the 5`end of the gene body, though a small subset of genes have a broad H3K4me3 domain which extensively covers the coding region. Although most studies focus on the H3K4me3 mark, the broad H3K4me3 domain is associated with a plethora of histone modifications (e.g., H3 acetylated at K27) and is therein termed broad epigenetic domain. Genes marked with the broad epigenetic domain are involved in cell identity and essential cell functions and have clinical potential as biomarkers for patient stratification. Reducing expression of genes with the broad epigenetic domain may increase the metastatic potential of cancer cells. Enhancers and super-enhancers interact with the broad epigenetic domain marked genes forming a hub of interactions involving nucleosome-depleted regions. Together, the regulatory elements coalesce with transcription factors, chromatin modifying/remodeling enzymes, coactivators, and the Mediator and/or Integrator complex into a transcription factory which may be analogous to a liquid-liquid phase-separated condensate. The broad epigenetic domain has a dynamic chromatin structure which supports frequent transcription bursts. In this review, we present the current knowledge of broad epigenetic domains.

Transcriptionally Active Chromatin-Lessons Learned from the Chicken Erythrocyte Chromatin Fractionation.

The chicken erythrocyte model system has been valuable to the study of chromatin structure and function, specifically for genes involved in oxygen transport and the innate immune response. Several seminal features of transcriptionally active chromatin were discovered in this system. Davie and colleagues capitalized on the unique features of the chicken erythrocyte to separate and isolate transcriptionally active chromatin and silenced chromatin, using a powerful native fractionation procedure. Histone modifications, histone variants, atypical nucleosomes (U-shaped nucleosomes) and other chromatin structural features (open chromatin) were identified in these studies. More recently, the transcriptionally active chromosomal domains in the chicken erythrocyte genome were mapped by combining this chromatin fractionation method with next-generation DNA and RNA sequencing. The landscape of histone modifications relative to chromatin structural features in the chicken erythrocyte genome was reported in detail, including the first ever mapping of histone H4 asymmetrically dimethylated at Arg 3 (H4R3me2a) and histone H3 symmetrically dimethylated at Arg 2 (H3R2me2s), which are products of protein arginine methyltransferases (PRMTs) 1 and 5, respectively. PRMT1 is important in the establishment and maintenance of chicken erythrocyte transcriptionally active chromatin.

Epigenetic regulation of ACE2, the receptor of the SARS-CoV-2 virus1.

The angiotensin-converting enzyme 2 (ACE2) is the receptor for the three coronaviruses HCoV-NL63, SARS-CoV, and SARS-CoV-2. ACE2 is involved in the regulation of the renin-angiotensin system and blood pressure. ACE2 is also involved in the regulation of several signaling pathways, including integrin signaling. ACE2 expression is regulated transcriptionally and post-transcriptionally. The expression of the gene is regulated by two promoters, with usage varying among tissues. ACE2 expression is greatest in the small intestine, kidney, and heart and detectable in a variety of tissues and cell types. Herein we review the chemical and mechanical signal transduction pathways regulating the expression of the ACE2 gene and the epigenetic/chromatin features of the expressed gene.

The chicken model organism for epigenomic research.

The chicken model organism has advanced the areas of developmental biology, virology, immunology, oncology, epigenetic regulation of gene expression, conservation biology, and genomics of domestication. Further, the chicken model organism has aided in our understanding of human disease. Through the recent advances in high-throughput sequencing and bioinformatic tools, researchers have successfully identified sequences in the chicken genome that have human orthologs, improving mammalian genome annotation. In this review, we highlight the importance of chicken as an animal model in basic and pre-clinical research. We will present the importance of chicken in poultry epigenetics and in genomic studies that trace back to their ancestor, the last link between human and chicken in the tree of life. There are still many genes of unknown function in the chicken genome yet to be characterized. By taking advantage of recent sequencing technologies, it is possible to gain further insight into the chicken epigenome.

The treatment of SARS-CoV2 with antivirals and mitigation of the cytokine storm syndrome: the role of gene expression.

In the absence of a vaccine, the treatment of SARS-CoV2 has focused on eliminating the virus with antivirals or mitigating the cytokine storm syndrome (CSS) that leads to the most common cause of death: respiratory failure. Herein we discuss the mechanisms of antiviral treatments for SARS-CoV2 and treatment strategies for the CSS. Antivirals that have shown in vitro activity against SARS-CoV2, or the closely related SARS-CoV1 and MERS-CoV, are compared on the enzymatic level and by potency in cells. For treatment of the CSS, we discuss medications that reduce the effects or expression of cytokines involved in the CSS with an emphasis on those that reduce IL-6 because of its central role in the development of the CSS. We show that some of the medications covered influence the activity or expression of enzymes involved in epigenetic processes and specifically those that add or remove modifications to histones or DNA. Where available, the latest clinical data showing the efficacy of the medications is presented. With respect to their mechanisms, we explain why some medications are successful, why others have failed, and why some untested medications may yet prove useful.