Epigenetic control of SOX9 gene by the histone acetyltransferase P300 in human Sertoli cells.

Background: The transcription factor SOX9 is a key regulator of male sexual development and Sertoli cell differentiation. Altered SOX9 expression has been implicated in the pathogenesis of disorders of sexual development (DSD) in mammals. However, limited information exists regarding the epigenetic mechanisms governing its transcriptional control during sexual development. Methods: This study employed real-time PCR (qPCR), immunofluorescence (IIF), and chromatin immunoprecipitation (ChIP) assays to investigate the epigenetic mechanisms associated with SOX9 gene transcriptional control in human and mouse Sertoli cell lines. To identify the specific epigenetic enzymes involved in SOX9 epigenetic control, functional assays using siRNAs for P300, GCN5, and WDR5 were performed. Results: The transcriptional activation of SOX9 was associated with selective deposition of active histone modifications, such as H3K4me3 and H3K27ac, at its enhancer and promoter regions. Importantly, the histone acetyltransferase P300 was found to be significantly enriched at the SOX9 enhancers, co-localizing with the H3K27ac and the SOX9 transcription factor. Silencing of P300 led to decreased SOX9 expression and reduced H3K27ac levels at the eSR-A and e-ALDI enhancers, demonstrating the crucial role of P300-mediated histone acetylation in SOX9 transcriptional activation. Interestingly, another histone lysine acetyltransferases like GNC5 and methyltransferases as the Trithorax/COMPASS-like may also have a relevant role in male sexual differentiation. Conclusions: Histone acetylation by P300 at SOX9 enhancers, is a key mechanism governing the transcriptional control of this essential regulator of male sexual development. These findings provide important insights into the epigenetic basis of sexual differentiation and the potential pathogenesis of DSDs.

Association of DNA Methylation with Infant Birth Weight in Women with Gestational Diabetes.

Offspring exposed to gestational diabetes mellitus (GDM) exhibit greater adiposity at birth. This early-life phenotype may increase offspring risk of developing obesity, metabolic syndrome, type 2 diabetes, and cardiovascular disease later in life. Infants born to women with GDM have a dysregulation of several hormones, cytokines, and growth factors related to fetal fat mass growth. One of the molecular mechanisms of GDM influencing these factors is epigenetic alterations, such as DNA methylation (DNAm). This review will examine the role of DNAm as a potential biomarker for monitoring fetal growth during pregnancy in women with GDM. This information is relevant since it may provide useful new biomarkers for the diagnosis, prognosis, and treatment of fetal growth and its later-life health consequences.

Revisiting Epigenetics Fundamentals and Its Biomedical Implications.

In light of the post-genomic era, epigenetics brings about an opportunity to better understand how the molecular machinery works and is led by a complex dynamic set of mechanisms, often intricate and complementary in many aspects. In particular, epigenetics links developmental biology and genetics, as well as many other areas of knowledge. The present work highlights substantial scopes and relevant discoveries related to the development of the term from its first notions. To our understanding, the concept of epigenetics needs to be revisited, as it is one of the most relevant and multifaceted terms in human knowledge. To redirect future novel experimental or theoretical efforts, it is crucial to compile all significant issues that could impact human and ecological benefit in the most precise and accurate manner. In this paper, the reader can find one of the widest compilations of the landmarks and epistemic considerations of the knowledge of epigenetics across the history of biology from the earliest epigenetic formulation to genetic determinism until the present. In the present work, we link the current body of knowledge and earlier pre-genomic concepts in order to propose a new definition of epigenetics that is faithful to its regulatory nature.

De Novo Design of Inhibitors of DNA Methyltransferase 1: A Critical Comparison of Ligand- and Structure-Based Approaches.

Designing and developing inhibitors against the epigenetic target DNA methyltransferase (DNMT) is an attractive strategy in epigenetic drug discovery. DNMT1 is one of the epigenetic enzymes with significant clinical relevance. Structure-based de novo design is a drug discovery strategy that was used in combination with similarity searching to identify a novel DNMT inhibitor with a novel chemical scaffold and warrants further exploration. This study aimed to continue exploring the potential of de novo design to build epigenetic-focused libraries targeted toward DNMT1. Herein, we report the results of an in-depth and critical comparison of ligand- and structure-based de novo design of screening libraries focused on DNMT1. The newly designed chemical libraries focused on DNMT1 are freely available on GitHub.

Sex differences in sensitivity to fentanyl effects in mice: Behavioral and molecular findings during late adolescence.

PURPOSE: Sex differences play a crucial role in understanding vulnerability to opioid addiction, yet there have been limited preclinical investigations of this effect during the transition from adolescence to adulthood. The present study compared the behaviors of male and female rodents in response to fentanyl treatment and targeted molecular correlates in the striatum and medial prefrontal cortex. MATERIALS AND METHODS: Thirty adolescent C57BL/6J mice underwent a 1-week fentanyl treatment with an escalating dose. In addition to evaluating locomotor activity and anxiety-related parameters, we also assessed naloxone-induced fentanyl acute withdrawal jumps. We employed real-time quantitative PCR (qPCR) to assess overall gene expression of dopaminergic receptors (Drd1, Drd2, Drd4 and Drd5) and the µ-opioid receptor Oprm1. The levels of epigenetic base modifications including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were assessed on CpG islands of relevant genes. RESULTS: Females had higher locomotor activity than males after chronic fentanyl treatment, and they exhibited higher fentanyl withdrawal jumping behavior induced by naloxone. Females also presented lower Drd4 gene expression and DNA methylation (5mC + 5hmC) in the striatum. We found that locomotor activity and fentanyl withdrawal jumps were negatively correlated with Drd4 methylation and gene expression in the striatum, respectively. CONCLUSIONS: The findings suggested that female mice displayed heightened sensitivity to the effects of fentanyl treatment during the transition from adolescence to adulthood. This effect may be associated with molecular alterations related to the Drd4 gene.

Epigenetic regulation by hypoxia, N-acetylcysteine and hydrogen sulphide of the fetal vasculature in growth restricted offspring: A study in humans and chicken embryos.

Fetal growth restriction (FGR) is a common outcome in human suboptimal gestation and is related to prenatal origins of cardiovascular dysfunction in offspring. Despite this, therapy of human translational potential has not been identified. Using human umbilical and placental vessels and the chicken embryo model, we combined cellular, molecular, and functional studies to determine whether N-acetylcysteine (NAC) and hydrogen sulphide (H2S) protect cardiovascular function in growth-restricted unborn offspring. In human umbilical and placental arteries from control or FGR pregnancy and in vessels from near-term chicken embryos incubated under normoxic or hypoxic conditions, we determined the expression of the H2S gene CTH (i.e. cystathionine γ-lyase) (via quantitative PCR), the production of H2S (enzymatic activity), the DNA methylation profile (pyrosequencing) and vasodilator reactivity (wire myography) in the presence and absence of NAC treatment. The data show that FGR and hypoxia increased CTH expression in the embryonic/fetal vasculature in both species. NAC treatment increased aortic CTH expression and H2S production and enhanced third-order femoral artery dilator responses to the H2S donor sodium hydrosulphide in chicken embryos. NAC treatment also restored impaired endothelial relaxation in human third-to-fourth order chorionic arteries from FGR pregnancies and in third-order femoral arteries from hypoxic chicken embryos. This NAC-induced protection against endothelial dysfunction in hypoxic chicken embryos was mediated via nitric oxide independent mechanisms. Both developmental hypoxia and NAC promoted vascular changes in CTH DNA and NOS3 methylation patterns in chicken embryos. Combined, therefore, the data support that the effects of NAC and H2S offer a powerful mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy. KEY POINTS: Gestation complicated by chronic fetal hypoxia and fetal growth restriction (FGR) increases a prenatal origin of cardiovascular disease in offspring, increasing interest in antenatal therapy to prevent against a fetal origin of cardiovascular dysfunction. We investigated the effects between N-acetylcysteine (NAC) and hydrogen sulphide (H2S) in the vasculature in FGR human pregnancy and in chronically hypoxic chicken embryos. Combining cellular, molecular, epigenetic and functional studies, we show that the vascular expression and synthesis of H2S is enhanced in hypoxic and FGR unborn offspring in both species and this acts to protect their vasculature. Therefore, the NAC/H2S pathway offers a powerful therapeutic mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy.

Quantification of DNA Methylation by ELISA in Epigenetic Studies in Plant Tissue Culture: A Theoretical-Practical Guide.

This chapter describes a step-by-step protocol for rapid serological quantification of global DNA methylation by enzyme-linked immunosorbent assay (ELISA) in plant tissue culture specimens. As a case study model, we used the coconut palm (Cocos nucifera), from which plumules were subjected to somatic embryogenesis followed by embryogenic calli multiplication. DNA methylation is one of the most common epigenetic markers in the regulation of gene expression. DNA methylation is generally associated with non-expressed genes, that is, gene silencing under certain conditions, and the degree of DNA methylation can be used as a marker of various physiological processes, both in plants and in animal cells. Methylation consists of adding a methyl radical to carbon 5 of the DNA cytosine base. Herein, the global DNA methylation was quantified by ELISA with antibodies against methylated cytosines using a commercial kit (Zymo-Research™). The method allowed the detection of methylation in total DNA extracts from coconut palm embryogenic calli (arising from somatic embryogenesis) cultivated in liquid or solid media by using antibodies against methylated cytosines and enzymatic development with a colorimetric substrate. Control samples of commercially provided Escherichia coli bacterial DNA with previously known methylation percentages were included in the ELISA test to construct an experimental methylation standard curve. The logarithmic regression of this E. coli standard curve allowed methylation quantification in coconut palm samples. The present ELISA methodology, applied to coconut palm tissue culture specimens, is promising for use in other plant species and botanical families. This chapter is presented in a suitable format for use as a step-by-step laboratory procedure manual, with theoretical introduction information, which makes it easy to apply the protocol in samples of any biological nature to evaluate DNA global methylation associated with any physiological process.

Combined exercise training decreases blood pressure in OLDER women with NOS3 polymorphism providing changes in differentially methylated regions (DMRs).

The mechanisms by which the ageing process is associated to an unhealthy lifestyle and how they play an essential role in the aetiology of systemic arterial hypertension have not yet been completely elucidated. Our objective is to investigate the influence of NOS3 polymorphisms [-786T > C and (Glu298Asp)] on systolic blood pressure (SBP) and diastolic blood pressure (DBP) response, differentially methylated regions (DMRs), and physical fitness of adult and older women after a 14-week combined training intervention. The combined training was carried out for 14 weeks, performed 3 times a week, totalling 180 minutes weekly. The genotyping experiment used Illumina Infinium Global Screening Array version 2.0 (GSA V2.0) and Illumina's EPIC Infinium Methylation BeadChip. The participants were separated into SNP rs2070744 in TT (59.7 ± 6.2 years) and TC + CC (60.0 ± 5.2 years), and SNP rs17999 in GluGlu (58.8 ± 5.7 years) and GluAsp + AspAsp (61.6 ± 4.9 years). We observed an effect of time for variables BP, physical capacities, and cholesterol. DMRs related to SBP and DBP were identified for the rs2070744 and rs17999 groups pre- and decreased numbers of DMRs post-training. When we analysed the effect of exercise training in pre- and post-comparisons, the GluGlu SNP (rs17999) showed 10 DMRs, and after enrichment, we identified several biological biases. The combined training improved the SBP and DBP values of the participants regardless of the SNPs. In addition, exercise training affected DNA methylation differently between the groups of NOS3 polymorphisms.

Probiotics as a strategy for addressing helminth infections in low-income countries: Working smarter rather than richer.

Helminth infections, which affect approximately 1.5 billion individuals worldwide (mainly children), are common in low- and middle-income tropical countries and can lead to various diseases. One crucial factor affecting the occurrence of these diseases is the reduced diversity of the gut microbiome due to antibiotic use. This reduced diversity compromises immune health in hosts and alters host gene expression through epigenetic mechanisms. Helminth infections may produce complex biochemical signatures that could serve as therapeutic targets. Such therapies include next-generation probiotics, live biotherapeutic products, and biochemical drug approaches. Probiotics can bind ferric hydroxide, reducing the iron that is available to opportunistic microorganisms. They also produce short-chain fatty acids associated with immune response modulation, oral tolerance facilitation, and inflammation reduction. In this review, we examine the potential link between these effects and epigenetic changes in immune response-related genes by analyzing methyltransferase-related genes within probiotic strains discussed in the literature. The identified genes were only correlated with methylation in bacterial genes. Various metabolic interactions among hosts, helminth parasites, and intestinal microbiomes can impact the immune system, potentially aiding or hindering worm expulsion through chemical signaling. Implementing a comprehensive strategy using probiotics may reduce the impact of drug-resistant helminth strains.

Lysine methyltransferase 2 plays a key role in the encystation process in the parasite Giardia lamblia.

Histone post-translational modifications are extensively studied for their role in regulating gene transcription and cellular environmental adaptation. Research into these modifications has recently begun in the protozoan parasite Giardia lamblia, focusing on histone-modifying enzymes and specific post-translational changes. In the transformation from the trophozoite to the cyst form in the life cycle of this parasite, significant morphological and genetic alterations occur, culminating in the synthesis of cyst wall proteins responsible for forming the protective cyst wall. It has been previously demonstrated that histone deacetylation is required during encystation and that the enzyme lysine methyltransferase 1 is involved in the upregulation of encystation. Our study aims to extend the analysis to lysine methyltransferase 2 (GlKMT2) function. For this, two constructs were generated: one that downregulate the expression of GLKMT2 via antisense (glkmt2-as transgenic cells) and the other overexpressing GlKMT2 (glkmt2-ha transgenic cells). We found that the glktm2-as transgenic cells showed an arrest in progress at the late encystation stage. Consequently, the number of cysts produced was lower than that of the control cells. On the other hand, we found that the overexpression of GlKMT2 acts as a negative mutant of the enzyme. In this way, these glktm2-ha transgenic cells showed the same behavior during growth and encystation as glkmt2-as transgenic cells. This interplay between different enzymes acting during encystation reveals the complex process behind the differentiation of the parasite. Understanding how these enzymes play their role during the encystation of the parasite would allow the design of inhibitors to control the parasite.

Emerging roles of plant transcriptional gene silencing under heat.

Plants continuously endure unpredictable environmental fluctuations that upset their physiology, with stressful conditions negatively impacting yield and survival. As a contemporary threat of rapid progression, global warming has become one of the most menacing ecological challenges. Thus, understanding how plants integrate and respond to elevated temperatures is crucial for ensuring future crop productivity and furthering our knowledge of historical environmental acclimation and adaptation. While the canonical heat-shock response and thermomorphogenesis have been extensively studied, evidence increasingly highlights the critical role of regulatory epigenetic mechanisms. Among these, the involvement under heat of heterochromatic suppression mediated by transcriptional gene silencing (TGS) remains the least understood. TGS refers to a multilayered metabolic machinery largely responsible for the epigenetic silencing of invasive parasitic nucleic acids and the maintenance of parental imprints. Its molecular effectors include DNA methylation, histone variants and their post-translational modifications, and chromatin packing and remodeling. This work focuses on both established and emerging insights into the contribution of TGS to the physiology of plants under stressful high temperatures. We summarized potential roles of constitutive and facultative heterochromatin as well as the most impactful regulatory genes, highlighting events where the loss of epigenetic suppression has not yet been associated with corresponding changes in epigenetic marks.

Investigating the correlation between genotype and phenotype in Prader-Willi syndrome: a study of 45 cases from Brazil.

BACKGROUND: Prader-Willi syndrome (PWS) is a genetic disorder characterized by abnormalities in the 15q11-q13 region. Understanding the correlation between genotype and phenotype in PWS is crucial for improved genetic counseling and prognosis. In this study, we aimed to investigate the correlation between genotype and phenotype in 45 PWS patients who previously underwent methylation-sensitive high-resolution melting (MS-HRM) for diagnosis. RESULTS: We employed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and Sanger sequencing, along with collecting phenotypic data from the patients for comparison. Among the 45 patients, 29 (64%) exhibited a deletion of 15q11-q13, while the remaining 16 (36%) had uniparental disomy. No statistically significant differences were found in the main signs and symptoms of PWS. However, three clinical features showed significant differences between the groups. Deletion patients had a higher prevalence of myopia than those with uniparental disomy, as well as obstructive sleep apnea and an unusual skill with puzzles. CONCLUSIONS: The diagnostic tests (MS-HRM, MS-MLPA, and Sanger sequencing) yielded positive results, supporting their applicability in PWS diagnosis. The study's findings indicate a general similarity in the genotype-phenotype correlation across genetic subtypes of PWS.

Genetic Alterations of SMYD4 in Solid Tumors Using Integrative Multi-Platform Analysis.

SMYD4 is a member of the SMYD family that has lysine methyltransferase function. Little is known about the roles of SMYD4 in cancer. The aim of this study is to investigate genetic alterations in the SMYD4 gene across the most prevalent solid tumors and determine its potential as a biomarker. We performed an integrative multi-platform analysis of the most common mutations, copy number alterations (CNAs), and mRNA expression levels of the SMYD family genes using cohorts available at the Cancer Genome Atlas (TCGA), cBioPortal, and the Catalogue of Somatic Mutations in Cancer (COSMIC). SMYD genes displayed a lower frequency of mutations across the studied tumors, with none of the SMYD4 mutations detected demonstrating sufficient discriminatory power to serve as a biomarker. In terms of CNAs, SMYD4 consistently exhibited heterozygous loss and downregulation across all tumors evaluated. Moreover, SMYD4 showed low expression in tumor samples compared to normal samples, except for stomach adenocarcinoma. SMYD4 demonstrated a frequent negative correlation with other members of the SMYD family and a positive correlation between CNAs and mRNA expression. Additionally, patients with low SMYD4 expression in STAD and LUAD tumors exhibited significantly poorer overall survival. SMYD4 demonstrated its role as a tumor suppressor in the majority of tumors evaluated. The consistent downregulation of SMYD4, coupled with its association with cancer progression, underscores its potential usefulness as a biomarker.

Epigenetic reprogramming of H3K4me3 in adipose-derived stem cells by HFS diet consumption leads to a disturbed transcriptomic profile in adipocytes.

Adipose tissue metabolism is actively involved in the regulation of energy balance. Adipose-derived stem cells (ASCs) play a critical role in maintaining adipose tissue function through their differentiation into mature adipocytes (Ad). This study aimed to investigate the impact of an obesogenic environment on the epigenetic landscape of ASCs and its impact on adipocyte differentiation and its metabolic consequences. Our results showed that ASCs from rats on a high-fat sucrose (HFS) diet displayed reduced adipogenic capacity, increased fat accumulation, and formed larger adipocytes than the control (C) group. Mitochondrial analysis revealed heightened activity in undifferentiated ASC-HFS but decreased respiratory and glycolytic capacity in mature adipocytes. The HFS diet significantly altered the H3K4me3 profile in ASCs on genes related to adipogenesis, mitochondrial function, inflammation, and immunomodulation. After differentiation, adipocytes retained H3K4me3 alterations, confirming the upregulation of genes associated with inflammatory and immunomodulatory pathways. RNA-seq confirmed the upregulation of genes associated with inflammatory and immunomodulatory pathways in adipocytes. Overall, the HFS diet induced significant epigenetic and transcriptomic changes in ASCs, impairing differentiation and causing dysfunctional adipocyte formation.NEW & NOTEWORTHY Obesity is associated with the development of chronic diseases like metabolic syndrome and type 2 diabetes, and adipose tissue plays a crucial role. In a rat model, our study reveals how an obesogenic environment primes adipocyte precursor cells, leading to epigenetic changes that affect inflammation, adipogenesis, and mitochondrial activity after differentiation. We highlight the importance of histone modifications, especially the trimethylation of histone H3 to lysine 4 (H3K4me3), showing its influence on adipocyte expression profiles.

Factors Associated with Neonatal Arterial Hypertension: Case and Control Study.

Background. Neonatal high blood pressure has been diagnosed more frequently in recent years, and its impact extends to adulthood. However, the knowledge gaps on associated factors, diagnosis, and treatment are challenging for medical personnel. The incidence of this condition varies depending on neonatal conditions. Patients in the Newborn Unit are at increased risk of developing high blood pressure. The persistence of this condition beyond the neonatal stage increases the risk of cardiovascular disease and chronic kidney disease in childhood and adulthood. Methodology. A case-control study was carried out. It included hospitalized patients with neonatal hypertension as cases. Three controls were randomly selected for each case and matched by gestational age. The variables were analyzed based on their nature. Multivariate analysis was performed using a multivariate conditional regression model to identify variables associated with the outcome. Finally, the model was adjusted for possible confounders. Results. 37 cases were obtained and matched with 111 controls. In the univariate analysis, heart disease (OR 2.86; 95% CI 1.22-6.71), kidney disease (OR 7.24; 95% CI 1.92-28.28), bronchopulmonary dysplasia (OR 6.62; 95% CI 1.42-50.82) and major surgical procedures (OR 3.71; 95% CI 1.64-8.39) had an association with neonatal arterial hypertension. Only the latter maintained this finding in the multivariate analysis (adjusted OR 2.88; 95% CI 1.14-7.30). A significant association of two or more comorbidities with neonatal arterial hypertension was also found (OR 3.81; 95% CI 1.53-9.49). Conclusions. The study analyzed the factors related to high blood pressure in hospitalized neonates, finding relevant associations in the said population. The importance of meticulous neonatal care and monitoring of risk factors such as birth weight and major surgeries is highlighted.

Histone methylation: at the crossroad between circadian rhythms in transcription and metabolism.

Circadian rhythms, essential 24-hour cycles guiding biological functions, synchronize organisms with daily environmental changes. These rhythms, which are evolutionarily conserved, govern key processes like feeding, sleep, metabolism, body temperature, and endocrine secretion. The central clock, located in the suprachiasmatic nucleus (SCN), orchestrates a hierarchical network, synchronizing subsidiary peripheral clocks. At the cellular level, circadian expression involves transcription factors and epigenetic remodelers, with environmental signals contributing flexibility. Circadian disruption links to diverse diseases, emphasizing the urgency to comprehend the underlying mechanisms. This review explores the communication between the environment and chromatin, focusing on histone post-translational modifications. Special attention is given to the significance of histone methylation in circadian rhythms and metabolic control, highlighting its potential role as a crucial link between metabolism and circadian rhythms. Understanding these molecular intricacies holds promise for preventing and treating complex diseases associated with circadian disruption.

Etiología del autismo / Etiology of autism

Resumen El Trastorno del Espectro Autista es una patología de base neurobiológica con alto porcentaje de hereda bilidad y amplia lista de posibles etiologías, que pre senta cambios muy heterogéneos en la arquitectura, conectividad y sinaptogénesis neuronal, con manifes taciones clínicas características, cuyo origen apunta a causas ambientales, inmunológicas, genéticas y otras, sin haberse confirmado biomarcadores específicos. El diagnóstico se sigue basando en características típicas que incluyen conductas repetitivas y comunicación e interacción social deterioradas. Se revisan sus factores de riesgo genéticos y no genéticos para avanzar en el conocimiento sobre los procesos patológicos que pueden relacionarse a su origen.

Abstract The Autism Spectrum Disorder is a neurobiological based disorder with a high percentage of heritability and a wide list of possible etiologies that presents very heterogeneous changes in neuronal architecture, con nectivity and synaptogenesis with characteristic clinical manifestations whose origin points to environmental, immunological, genetic and other causes, without hav ing been confirmed specific biomarkers. Diagnosis con tinues to be based on typical features including repeti tive behaviors and impaired communication and social interaction. Their genetic and non-genetic risk factors are reviewed to advance knowledge about the pathologi cal processes that may be related to their origin.

Polyphenolic Compounds: Orchestrating Intestinal Microbiota Harmony during Aging.

In the aging process, physiological decline occurs, posing a substantial threat to the physical and mental well-being of the elderly and contributing to the onset of age-related diseases. While traditional perspectives considered the maintenance of life as influenced by a myriad of factors, including environmental, genetic, epigenetic, and lifestyle elements such as exercise and diet, the pivotal role of symbiotic microorganisms had been understated. Presently, it is acknowledged that the intestinal microbiota plays a profound role in overall health by signaling to both the central and peripheral nervous systems, as well as other distant organs. Disruption in this bidirectional communication between bacteria and the host results in dysbiosis, fostering the development of various diseases, including neurological disorders, cardiovascular diseases, and cancer. This review aims to delve into the intricate biological mechanisms underpinning dysbiosis associated with aging and the clinical ramifications of such dysregulation. Furthermore, we aspire to explore bioactive compounds endowed with functional properties capable of modulating and restoring balance in this aging-related dysbiotic process through epigenetics alterations.

Nrf2 pre-recruitment at Enhancer 2 is a hallmark of H2 O2 -induced epigenetic transcriptional memory in the HMOX1 gene in human umbilical artery endothelial cells.

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.