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.

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.

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 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.

Atypical chromatin structure of immune-related genes expressed in chicken erythrocytes.

The major biological role of red blood cells is to carry oxygen to the tissues in the body. However, another role of the erythroid cell is to participate in the immune response. Mature erythrocytes from chickens express Toll-like receptors and several cytokines in response to stimulation of the immune system. We previously reported the application of a biochemical fractionation protocol to isolate highly enriched transcribed DNA from polychromatic erythrocytes from chickens. In conjunction with next-generation DNA, RNA sequencing, chromatin immunoprecipitation-DNA sequencing, and formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing, we identified the active chromosomal compartments and determined their structural signatures in relation to expression levels. Here, we present the detailed chromatin characteristics of erythroid genes participating in the innate immune response. Our studies revealed an atypical chromatin structure for several genes coding for Toll-like receptors, interleukins, and interferon regulatory factors. The body of these genes had nucleosome-free regions intermingled with nucleosomes modified with H3K4me3 and H3K27ac, suggesting a dynamic unstable chromatin structure. We further show that human genes involved in cell identity have gene bodies with the same chromatin-instability features as the chicken polychromatic erythrocyte genes participating in the innate immune response.

SARS-CoV-2 multifaceted interaction with human host. Part I: What we have learnt and done so far, and the still unknown realities.

SARS-CoV-2, the causing agent of the ongoing COVID-19 pandemic, is a beta-coronavirus which has 80% genetic homology with SARS-CoV, but displays increased virulence and transmissibility. Initially, SARS-CoV-2 was considered a respiratory virus generally causing a mild disease, only severe and fatal in the elderly and individuals with underlying conditions. Severe illnesses and fatalities were attributed to a cytokine storm, an excessive response from the host immune system. However, with the number of infections over 10 millions and still soaring, the insidious and stealthy nature of the virus has emerged, as it causes a vast array of diverse unexpected symptoms among infected individuals, including the young and healthy. It has become evident that besides infecting the respiratory tract, SARS-CoV-2 can affect many organs, possibly through the infection of the endothelium. This review presents an overview of our learning curve with the novel virus emergence, transmission, pathology, biological properties and host-interactions. It also briefly describes remedial measures taken until an effective vaccine is available, that is non-pharmaceutical interventions to reduce the viral spread and the repurposing of existing drugs, approved or in development for other conditions to eliminate the virus or mitigate the cytokine storm.

SARS-CoV-2 multifaceted interaction with the human host. Part II: Innate immunity response, immunopathology, and epigenetics.

The SARS-CoV-2 makes its way into the cell via the ACE2 receptor and the proteolytic action of TMPRSS2. In response to the SARS-CoV-2 infection, the innate immune response is the first line of defense, triggering multiple signaling pathways to produce interferons, pro-inflammatory cytokines and chemokines, and initiating the adaptive immune response against the virus. Unsurprisingly, the virus has developed strategies to evade detection, which can result in delayed, excessive activation of the innate immune system. The response elicited by the host depends on multiple factors, including health status, age, and sex. An overactive innate immune response can lead to a cytokine storm, inflammation, and vascular disruption, leading to the vast array of symptoms exhibited by COVID-19 patients. What is known about the expression and epigenetic regulation of the ACE2 gene and the various players in the host response are explored in this review.

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.

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.

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.

Genomic landscape of transcriptionally active histone arginine methylation marks, H3R2me2s and H4R3me2a, relative to nucleosome depleted regions.

Protein arginine methyltransferase 1 (PRMT1) and the product of this enzyme (histone H4 asymmetrically dimethylated at Arg 3; H4R3me2a) are important in the establishment and maintenance of chicken and murine erythrocyte transcriptionally active chromatin. Silencing the expression of PRMT1 results in loss of acetylated histones H3 and H4 and methylated H3K4 and prevents erythropoiesis. Here, we show that H4R3me2a and the PRMT5-catalyzed histone H3 symmetrically dimethylated at Arg 2 (H3R2me2s) locate largely to introns of expressed genes and intergenic regions, with both marks co-localizing in the chicken polychromatic erythrocyte genome. H4R3me2a and H3R2me2s were associated with histone marks of active promoters and enhancers, as well as with the body of genes that have an atypical chromatin structure, with nucleosome depleted regions. H4R3me2a co-localized with acetylated H3K27. Previous studies have shown that PRMT1 was bound to CBP/p300, suggesting a role of PRMT1-mediated H4R3me2a in CBP/p300 recruitment and H3K27 acetylation. Moreover, PRMT1 might be a key enzyme affected when S-adenosyl methionine levels are reduced in metabolic disorders.

Chromatin organization of transcribed genes in chicken polychromatic erythrocytes.

Transcriptional regulation is impacted by the organization of the genome into chromatin compartments and domains. We previously reported the application of a biochemical fractionation protocol to isolate highly enriched transcribed DNA from chicken polychromatic erythrocytes. In conjunction with next-generation DNA and RNA sequencing as well as chromatin immunoprecipitation-DNA sequencing, we identified all the active chromosomal compartments and determined their structural signatures in relation to expression levels. Highly expressed genes were found in broad dynamically highly acetylated, salt-soluble chromatin compartments, while poorly or moderately expressed genes exhibited a narrow stretch of salt-soluble chromatin limited to their 5' or body region. Here, we present the detailed characteristics, including the location of nucleosome-free regions and CpG islands, of several transcriptionally active chromatin compartments. These chromatin patterns illustrate how the salt solubility profile of a genomic region aids in the annotation of genes expressed in erythroid cells and contributes to the identification of functional features such as regulatory regions.