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.

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.

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.

Histone H3K4 trimethylation: dynamic interplay with pre-mRNA splicing.

Histone H3 lysine 4 trimethylation (H3K4me3) is often stated as a mark of transcriptionally active promoters. However, closer study of the positioning of H3K4me3 shows the mark locating primarily after the first exon at the 5' splice site and overlapping with a CpG island in mammalian cells. There are several enzyme complexes that are involved in the placement of the H3K4me3 mark, including multiple protein complexes containing SETD1A, SETD1B, and MLL1 enzymes (writers). CXXC1, which is associated with SETD1A and SETD1B, target these enzymes to unmethylated CpG islands. Lysine demethylases (KDM5 family members, erasers) demethylate H3K4me3. The H3K4me3 mark is recognized by several proteins (readers), including lysine acetyltransferase complexes, chromatin remodelers, and RNA bound proteins involved in pre-mRNA splicing. Interestingly, attenuation of H3K4me3 impacts pre-mRNA splicing, and inhibition of pre-mRNA splicing attenuates H3K4me3.

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.

Increased genomic instability and altered chromosomal protein phosphorylation timing in HRAS-transformed mouse fibroblasts.

The RAS-mitogen-activated protein kinase signaling pathway is often deregulated in cancer cells. In metastatic HRAS-transformed mouse fibroblasts (Ciras-3), the RAS-MAPK pathway is constitutively activated. We show here that Ciras-3 cells exhibit a higher incidence of chromosomal instability than 10T1/2 cells, including higher levels of clonal and nonclonal chromosomal aberrations. Stimulation of serum starved 10T1/2 and Ciras-3 cells with phorbol esters (TPA) results in the phosphorylation of histone H3 at serine 10 and serine 28. Regardless of the increased genomic instability in Ciras-3 cells, TPA-induced H3 phosphorylated at serine 10 and H3 phosphorylated at serine 28 partitioned into distinct nuclear subdomains as they did in the parental cells. However, the timing of the response of the H3 phosphorylation event to TPA induction was delayed in Ciras-3 cells. Further Ciras-3 cells, which have a more open chromatin structure, had increased steady state levels of phosphorylated H3 and HMGN1 relative to parental 10T1/2 cells. TPA-induced H3 phosphorylated at serine 10 and 28 were colocalized with the transcriptionally initiated form of RNA polymerase II in 10T1/2 and Ciras-3 cells. Chromatin immunoprecipitation assays demonstrated that TPA-induced H3 phosphorylation at serine 28 was associated with the immediate early JUN promoter, providing direct evidence that this histone post-translational modification is associated with transcriptionally active genes. Together our results demonstrate the increased genomic instability and alterations in the epigenetic program in HRAS-transformed cells.

Epigenetic control.

Epigenetics refers to mitotically and/or meiotically heritable variations in gene expression that are not caused by changes in DNA sequence. Epigenetic mechanisms regulate all biological processes from conception to death, including genome reprogramming during early embryogenesis and gametogenesis, cell differentiation and maintenance of a committed lineage. Key epigenetic players are DNA methylation and histone post-translational modifications, which interplay with each other, with regulatory proteins and with non-coding RNAs, to remodel chromatin into domains such as euchromatin, constitutive or facultative heterochromatin and to achieve nuclear compartmentalization. Besides epigenetic mechanisms such as imprinting, chromosome X inactivation or mitotic bookmarking which establish heritable states, other rapid and transient mechanisms, such as histone H3 phosphorylation, allow cells to respond and adapt to environmental stimuli. However, these epigenetic marks can also have long-term effects, for example in learning and memory formation or in cancer. Erroneous epigenetic marks are responsible for a whole gamut of diseases including diseases evident at birth or infancy or diseases becoming symptomatic later in life. Moreover, although epigenetic marks are deposited early in development, adaptations occurring through life can lead to diseases and cancer. With epigenetic marks being reversible, research has started to focus on epigenetic therapy which has had encouraging success. As we witness an explosion of knowledge in the field of epigenetics, we are forced to revisit our dogma. For example, recent studies challenge the idea that DNA methylation is irreversible. Further, research on Rett syndrome has revealed an unforeseen role for methyl-CpG-binding protein 2 (MeCP2) in neurons.

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.

Mitotic partitioning of transcription factors.

Mitosis is a highly orchestrated process involving numerous protein kinases and phosphatases. At the onset of mitosis, the chromatin condensation into metaphase chromosomes is correlated with global phosphorylation of histone H3. The bulk of transcription is silenced while many of the transcription-associated proteins, including transcription and chromatin remodeling factors, are excluded from chromatin, typically as a consequence of their phosphorylation. Components of the transcription machinery and regulatory proteins are recycled and equally partitioned between newly divided cells by mechanisms that may involve microtubules, microfilaments or intermediate filaments. However, as demonstrated in the case of Runx2, a subset of transcription factors involved in lineage-specific control, likely remain associated with their target genes to direct the deposition or removal of epigenetic marks. The displacement and re-entry into daughter cells of transcription and chromatin remodeling factors are temporally defined and regulated. Reformation of daughter nuclei is a critical time to re-establish the proper gene expression pattern. The mechanisms involved in the marking and re-establishment of gene expression has been elucidated for few genes. The elucidation of how the memory of a programmed expression profile is transmitted to daughter cells represents a challenge.

Mitogen-induced distinct epialleles are phosphorylated at either H3S10 or H3S28, depending on H3K27 acetylation.

Stimulation of the MAPK pathway results in mitogen- and stress-activated protein kinase 1/2 (MSK1/2)-catalyzed phosphorylation of histone H3 at serine 10 or 28 and expression of immediate-early (IE) genes. In 10T1/2 mouse fibroblasts, phosphorylation of H3S10 and H3S28 occurs on different H3 molecules and in different nuclear regions. Similarly, we show that mitogen-induced H3S10 and H3S28 phosphorylation occurs in separate pools in human primary fibroblasts. High-resolution imaging studies on both cell types reveal that H3S10 and H3S28 phosphorylation events can be induced in a single cell but on different alleles, giving rise to H3S10ph and H3S28ph epialleles. Coimmunoprecipitation and inhibition studies demonstrate that CBP/p300-mediated H3K27 acetylation is required for MSK1/2 to phosphorylate S28. Although the K9ac and S10ph marks coexist on H3, S10 phosphorylation is not dependent on K9 acetylation by PCAF. We propose that random targeting of H3S10 or H3S28 results from the stochastic acetylation of H3 by CBP/p300 or PCAF, a process comparable to transcriptional bursting causing temporary allelic imbalance. In 10T1/2 cells expressing Jun, at least two of three alleles per cell were induced, a sign of high expression level. The redundant roles of H3S10ph and H3S28ph might enable rapid and efficient IE gene induction.

The chicken erythrocyte epigenome.

BACKGROUND: Transcriptional regulation is impacted by multiple layers of genome organization. A general feature of transcriptionally active chromatin is sensitivity to DNase I and association with acetylated histones. However, very few of these active DNase I-sensitive domains, such as the chicken erythrocyte ß-globin domain, have been identified and characterized. In chicken polychromatic erythrocytes, dynamically acetylated histones associated with DNase I-sensitive, transcriptionally active chromatin prevent histone H1/H5-induced insolubility at physiological ionic strength. RESULTS: Here, we identified and mapped out all the transcriptionally active chromosomal domains in the chicken polychromatic erythrocyte genome by combining a powerful chromatin fractionation method with next-generation DNA and RNA sequencing. Two classes of transcribed chromatin organizations were identified on the basis of the extent of solubility at physiological ionic strength. Highly transcribed genes were present in multigenic salt-soluble chromatin domains ranging in length from 30 to over 150 kb. We identified over 100 highly expressed genes that were organized in broad dynamically highly acetylated, salt-soluble chromatin domains. Highly expressed genes were associated with H3K4me3 and H3K27ac and produced discernible antisense transcripts. The moderately- and low-expressing genes had highly acetylated, salt-soluble chromatin regions confined to the 5' end of the gene. CONCLUSIONS: Our data provide a genome-wide profile of chromatin signatures in relation to expression levels in chicken polychromatic erythrocytes.

Targeting class I histone deacetylases in cancer therapy.

INTRODUCTION: Class I histone deacetylases (HDACs) are often overexpressed in cancer, and their inhibition typically leads cancer cells, but not normal cells, to apoptosis. Hence, the field of cancer therapy has experienced a continued surge in the development of HDAC inhibitors. AREAS COVERED: Class I comprises of HDAC1, 2, 3 and 8. HDAC1, 2 and 3 are active as subunits of multiprotein complexes while an HDAC8 complex has not been identified. Besides being a major contributor to poor prognosis in childhood neuroblastoma, little is known of HDAC8 functions and substrates. The targeting and activities of HDAC1 - 3 are modulated by post-translational modifications and association with numerous proteins. The composition of the various HDAC complexes is cell type dependent and fluctuates with intra- and intercellular stimuli. These HDAC complexes play roles at multiple levels in gene expression and genome stability. The application of isoform-specific HDAC inhibitors has met with varying success in clinical trials. EXPERT OPINION: To elucidate the mechanism and cellular impact of HDAC inhibitors, we need to identify the spectrum of class I HDAC complexes and their functions. In the cases of HDAC1 - 3, selectivity of HDAC inhibitors should be directed against relevant complexes. HDAC8 active site unique features facilitate the design of selective inhibitors.

Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors.

The zinc-dependent mammalian histone deacetylase (HDAC) family comprises 11 enzymes, which have specific and critical functions in development and tissue homeostasis. Mounting evidence points to a link between misregulated HDAC activity and many oncologic and nononcologic diseases. Thus the development of HDAC inhibitors for therapeutic treatment garners a lot of interest from academic researchers and biotechnology entrepreneurs. Numerous studies of HDAC inhibitor specificities and molecular mechanisms of action are ongoing. In one of these studies, mass spectrometry was used to characterize the affinities and selectivities of HDAC inhibitors toward native HDAC multiprotein complexes in cell extracts. Such a novel approach reproduces in vivo molecular interactions more accurately than standard studies using purified proteins or protein domains as targets and could be very useful in the isolation of inhibitors with superior clinical efficacy and decreased toxicity compared to the ones presently tested or approved. HDAC inhibitor induced-transcriptional reprogramming, believed to contribute largely to their therapeutic benefits, is achieved through various and complex mechanisms not fully understood, including histone deacetylation, transcription factor or regulator (including HDAC1) deacetylation followed by chromatin remodeling and positive or negative outcome regarding transcription initiation. Although only a very low percentage of protein-coding genes are affected by the action of HDAC inhibitors, about 40% of noncoding microRNAs are upregulated or downregulated. Moreover, a whole new world of long noncoding RNAs is emerging, revealing a new class of potential targets for HDAC inhibition. HDAC inhibitors might also regulate transcription elongation and have been shown to impinge on alternative splicing.

Selective association of peroxiredoxin 1 with genomic DNA and COX-2 upstream promoter elements in estrogen receptor negative breast cancer cells.

In a search for proteins differentially cross-linked to DNA by cisplatin or formaldehyde in normal breast epithelial and breast cancer cell lines, we identified peroxiredoxin 1 (PRDX1) as a protein preferentially cross-linked to DNA in estrogen receptor negative (ER-) MDA-MB-231 but not in estrogen receptor positive (ER+) MCF7 breast cancer cells. Indirect immunofluorescence microscopic analyses showed that PRDX1 was located in the cytoplasm and nucleus of normal and breast cancer cells, with nuclear PRDX1 associated with promyelocytic leukemia protein bodies. We demonstrated that PRDX1 association with the transcription factor nuclear factor-kappaB (NF-kappaB) in MDA-MB-231 but not in MCF7 cells contributed to PRDX1-selective recruitment to MDA-MB-231 genomic DNA. Furthermore, PRDX1 was associated with the cyclooxygenase (COX)-2 upstream promoter region at sites occupied by NF-kappaB in ER- but not in ER+ breast cancer cells. PRDX1 knockdown attenuated COX-2 expression by reducing NF-kappaB occupancy at its upstream promoter element in MDA-MB-231 but not in MCF7 cells. A phosphorylated form of PRDX1 was only present in ER- breast cancer cells. Because PRDX1 phosphorylation is known to inhibit its peroxidase activity and to promote PRDX1 oligomerization, we propose that PRDX1 acts as a chaperone to enhance the transactivation potential of NF-kappaB in ER- breast cancer cells.