Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function

Background: DNA hypomethylation at the F2RL3 locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. Methods: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3,205), we explored the relationship between smoking, DNA hypomethylation at F2RL3 and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract (CSE). Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. Results: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 stimulation. In cells, CSE exposure was associated with a 4.9 to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7 (95% CI: 1.2, 2.4, p=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. Conclusions: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk, but from any feature potentially influencing F2RL3 regulation in a similar manner.

Establishment of developmental gene silencing by ordered polycomb complex recruitment in early zebrafish embryos

Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal the order of chromatin states in establishing developmental gene poising in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with 'Placeholder' nucleosomes (bearing H2A.Z, H3K4me1, and H3K27ac), and DNA hypomethylation. Silencing is initiated by the recruitment of Polycomb Repressive Complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 - establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos.

Promotor Hypomethylation Mediated Upregulation of miR-23b-3p Targets PTEN to Promote Bronchial Epithelial-Mesenchymal Transition in Chronic Asthma

Chronic asthma is characterized by airway inflammation and irreversible airway remodeling. Epithelial-mesenchymal transition (EMT) is a typical pathological change of airway remodeling. Our previous research demonstrated miR-23b inhibited airway smooth muscle proliferation while the function of miR-23b-3p has not been reported yet. Besides, miRNA is regulated by many factors, including DNA methylation. The function of miR-23b-3p and whether it is regulated by DNA methylation are worth exploring. Balb/c mice were given OVA sensitization to develop the asthmatic model. Expression of miR-23b-3p and EMT markers were measured by RT-qPCR, WB and immunohistochemistry (IHC). DNA methylation was detected by methylation-specific PCR (MSP) and the MassARRAY System. Asthmatic mice and TGF-β1-stimulated bronchial epithelial cells (BEAS-2B) showed EMT with increased miR-23b-3p. Overexpression of miR-23b-3p promoted EMT and migration, while inhibition of miR-23b-3p reversed these transitions. DNA methyltransferases were decreased in asthmatic mice. MSP and MassARRAY System detected the promotor of miR-23b showed DNA hypomethylation. DNA methyltransferase inhibitor 5’-AZA-CdZ increased the expression of miR-23b-3p. Meanwhile, PTEN was identified as a target gene of miR-23b-3p. Our results indicated that promotor hypomethylation mediated upregulation of miR-23b-3p targets PTEN to promote EMT in chronic asthma. miR-23b-3p and DNA methylation might be potential therapeutic targets for irreversible airway remodeling.

Detection of oncometabolite nicotine imine in the nail of oral cancer patients and predicted as an inhibitor of DNMT1

Background: Nicotine-metabolized product nicotine imine is suggested to play a role in metabolic changes in oral cancer. There is a significant gap in the detection of oncometabolite nicotine imine in biological fluids and nails of oral cancer patients. Oncometabolites are designated as metabolites those are usually elevated in cancer cells over normal cells. Interestingly, a direct or indirect link is missing that establishes a role of nicotine imine in pro-cancer cellular events including global DNA hypomethylation, a potential metabolic-epigenetic axis in oral cancer. Methods: A novel vertical tube gel electrophoresis (VTGE) system assisted purification and liquid chromatography-high resolution mass spectrometry (LC-HRMS) based identification of nicotine imine in the nails of oral cancer patients. Further, nicotine imine was evaluated for its molecular interactions with various methyltransferases including DNA methyltransferase 1 (DNMT1) by molecular docking and molecular dynamics (MD) simulations. Results: Data suggested the presence of nicotine imine in the nails of oral cancer patients. Molecular docking and MD simulations revealed a specific binding affinity by nicotine imine with DNMT1. Binding by nicotine imine is within the CXCC regulatory domain of DNMT1 including key residues as ARG690, PRO574, VAL658, PRO692 and ALA695. Similar binding residues are displayed by DNMT1 inhibitor 5'-Aza-2'-deoxycytidine. Conclusion : Nicotine imine is suggested as a predictive biomarker for oral cancer patients in nails and this finding is a first report. Molecular docking and dynamics simulation propose the role of nicotine imine as an inhibitor of DNMT1. This work supports the involvement of synergistic pro-tumor metabolic-epigenomic axis by nicotine imine that may contribute towards potential mutagenesis of normal squamous epithelium.

Epigenetics of Mitochondria-Associated Genes in Striated Muscle

Striated muscle has especially large energy demands. We identified 97 genes preferentially expressed in skeletal muscle and heart, but not in aorta, and found significant enrichment for mitochondrial associations among them. We compared the epigenomic and transcriptomic profiles of the 27 genes associated with striated muscle and mitochondria. Many showed strong correlations between their tissue-specific transcription levels, and their tissue-specific promoter, enhancer, or open chromatin as well as their DNA hypomethylation. Their striated muscle-specific enhancer chromatin was inside, upstream, or downstream of the gene, throughout much of the gene as a super-enhancer (CKMT2, SLC25A4, and ACO2), or even overlapping a neighboring gene (COX6A2, COX7A1, and COQ10A). Surprisingly, the 3′ end of the 1.38 Mb PRKN (PARK2) gene (involved in mitophagy and linked to juvenile Parkinson’s disease) displayed skeletal muscle/myoblast-specific enhancer chromatin, a myoblast-specific antisense RNA, as well as brain-specific enhancer chromatin. We also found novel tissue-specific RNAs in brain and embryonic stem cells within PPARGC1A (PGC-1α), which encodes a master transcriptional coregulator for mitochondrial formation and metabolism. The tissue specificity of this gene’s four alternative promoters, including a muscle-associated promoter, correlated with nearby enhancer chromatin and open chromatin. Our in-depth epigenetic examination of these genes revealed previously undescribed tissue-specific enhancer chromatin, intragenic promoters, regions of DNA hypomethylation, and intragenic noncoding RNAs that give new insights into transcription control for this medically important set of genes.

Specific hypomethylation programs underpin B cell activation in early multiple sclerosis

Epigenetic changes have been consistently detected in different cell types in multiple sclerosis (MS). However, their contribution to MS pathogenesis remains poorly understood partly because of sample heterogeneity and limited coverage of array-based methods. To fill this gap, we conducted a comprehensive analysis of genome-wide DNA methylation patterns in four peripheral immune cell populations isolated from 29 MS patients at clinical disease onset and 24 healthy controls. We show that B cells from new-onset untreated MS cases display more significant methylation changes than other disease-implicated immune cell types, consisting of a global DNA hypomethylation signature. Importantly, 4,933 MS-associated differentially methylated regions in B cells were identified, and this epigenetic signature underlies specific genetic programs involved in B cell differentiation and activation. Integration of the methylome to changes in gene expression and susceptibility-associated regions further indicates that hypomethylated regions are significantly associated with the up-regulation of cell activation transcriptional programs. Altogether, these findings implicate aberrant B cell function in MS etiology.

Early Pregnancy Exposure to Ambient Air Pollution among Late-Onset Preeclamptic Cases Is Associated with Placental DNA Hypomethylation of Specific Genes and Slower Placental Maturation

Exposure to ambient air pollution during pregnancy has been associated with an increased risk of preeclampsia (PE). Some suggested mechanisms behind this association are changes in placental DNA methylation and gene expression. The objective of this study was to identify how early pregnancy exposure to ambient nitrogen oxides (NOx) among PE cases and normotensive controls influence DNA methylation (EPIC array) and gene expression (RNA-seq). The study included placentas from 111 women (29 PE cases/82 controls) in Scania, Sweden. First-trimester NOx exposure was assessed at the participants’ residence using a dispersion model and categorized via median split into high or low NOx. Placental gestational epigenetic age was derived from the DNA methylation data. We identified six differentially methylated positions (DMPs, q < 0.05) comparing controls with low NOx vs. cases with high NOx and 14 DMPs comparing cases and controls with high NOx. Placentas with female fetuses showed more DMPs (N = 309) than male-derived placentas (N = 1). Placentas from PE cases with high NOx demonstrated gestational age deceleration compared to controls with low NOx (p = 0.034). No differentially expressed genes (DEGs, q < 0.05) were found. In conclusion, early pregnancy exposure to NOx affected placental DNA methylation in PE, resulting in placental immaturity and showing sexual dimorphism.

Multiomics profiling of the expression and prognosis of MCMs in endometrial carcinoma

Minichromosome maintenance (MCM) family members are a group of genes involved in regulating DNA replication and cell division and have been identified as oncogenes in various cancer types. Several experimental studies have suggested that MCMs are dysregulated in endometrial carcinoma (EC). However, the expression pattern, clinical value and functions of different MCMs have yet to be analyzed systematically and comprehensively. We analyzed expression, survival rate, DNA alteration, PPT network, GGI network, functional enrichment cancer hallmarks and drug sensitivity of MCMs in patients with EC based on diverse datasets, including Oncomine, GEPIA, Kaplan-Meier Plotter, HPA, Sangerbox and GSCALite databases. The results indicated that most MCM members were increased in EC and showed a prognostic value in survival analysis, which were considerately well in terms of PFS and OS prognostic prediction. Importantly, functional enrichment, PPI network and GGI network suggested that MCMs interact with proteins related to DNA replication and cell division, which may be the mechanism of MCM promote EC progression. Further data mining illustrated that MCMs have broad DNA hypomethylation levels and high levels of copy number aberrations in tumor tissue samples, which may be the mechanism causing the high expression level of MCMs. Moreover, MCM2 can activate or suppress diverse cancer-related pathways and is implicated in EC drug sensitivity. Taking together, our findings illustrate the expression pattern, clinical value and function of MCMs in EC and implies that MCMs are potential targets for precision therapy and new biomarkers for the prognosis of patients with EC.

SWI/SNF chromatin remodeling complex is required for initiation of sex-dependent differentiation in mouse germline

Sexual reproduction involves the creation of sex-dependent gametes, oocytes and sperm. In mammals, sexually dimorphic differentiation commences in the primordial germ cells (PGCs) in embryonic gonads; PGCs in ovaries and testes differentiate into meiotic primary oocytes and mitotically quiescent prospermatogonia, respectively. Here, we show that the transition from PGCs to sex-specific germ cells was abrogated in conditional knockout mice carrying a null mutation of Smarcb1 (also known as Snf5) gene, which encodes a core subunit of the SWI/SNF chromatin remodeling complex. In female mutant mice, failure to upregulate meiosis-related genes resulted in impaired meiotic entry and progression, including defects in synapsis formation and DNA double strand break repair. Mutant male mice exhibited delayed mitotic arrest and DNA hypomethylation in retrotransposons and imprinted genes, resulting from aberrant expression of genes related to growth and de novo DNA methylation. Collectively, our results demonstrate that the SWI/SNF complex is required for transcriptional reprogramming in the initiation of sex-dependent differentiation of germ cells.

Identification of glucocorticoid-related molecular signature by whole blood methylome analysis

Objective: Cushing’s syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing’s syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing’s syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers. Design: We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing’s syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. Methods: Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850000 CpG sites). Both unsupervised (Principal Component Analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. Results: Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing’s syndrome correction. In Cushing’s syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. Conclusions: Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assess glucocorticoid action beyond hormone assays.