Changes In CpG Methylation Research Articles

Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17α-Ethynylestradiol.

Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;43:1547-1556. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

CpG methylation changes in human mesenchymal and neural stem cells in response to in vitro niche modifications

Stem cell therapies hold promise in addressing the burden of neurodegenerative diseases with human embryonic neural stem cells (hNSC-H9s) and bone marrow-derived human mesenchymal stem cells (hMSCs) as viable candidates. The induction of hMSC neurospheres (hMSC-IN) generate a more lineage-restricted common neural progenitor-like cell population, potentially tunable by heparan sulfate proteoglycans (HSPGs). We examined CpG (5 mC) site methylation patterns using Illumina Infinium 850 K EPIC arrays in hNSC-H9, hMSCs and hMSC-IN cultures with HSPG agonist heparin at early and late phases of growth. We identified key regulatory CpG sites in syndecans (SDC2; SDC4) that potentially regulate gene expression in monolayers. Unique hMSC-IN hypomethylation in glypicans (GPC3; GPC4) underscore their significance in neural lineages with Sulfatase 1 and 2 (SULF1 &2) CpG methylation changes potentially driving the neurogenic shift. hMSC-INs methylation levels at SULF1 CpG sites and SULF2:cg25401628 were more closely aligned with hNSC-H9 cells than with hMSCs. We further suggest SOX2 regulation governed by lncSOX2-Overall Transcript (lncSOX2-OT) methylation changes with preferential activation of ENO2 over other neuronal markers within hMSC-INs. Our findings illuminate epigenetic dynamics governing neural lineage commitment of hMSC-INs offering insights for targeted mechanisms for regenerative medicine and therapeutic strategies.

Exponential dynamics of DNA methylation with age

The association of DNA methylation with age has been extensively studied. Previous work has investigated the trajectories of methylation with age, and developed predictive biomarkers of age. However, we still have a limited understanding of the functional form of methylation-age dynamics. To address this we present a theoretical framework to model the dynamics of DNA methylation at single sites. We show that this model leads to convergence to a steady-state methylation level at an exponential rate. By fitting the model to a dataset that measures changes in DNA methylation in the brain from birth to old age, we show that the timescales of this exponential convergence are heterogeneous across sites. To model this heterogeneity we generated a simulation of CpG Methylation changes with time and investigated the functional form of the dynamics of methylation with age under the empirical distribution of timescales estimated from the dataset. The resulting dynamics of the average methylation of the system were characterized and were found to closely follow an exponential trajectory. We conclude that DNA methylation can be modeled as a system that starts out of equilibrium at birth and approaches equilibrium with age in an exponential fashion. These insights illustrate the importance of accounting for nonlinear dynamics when utilizing age associated DNA methylation changes for constructing biomarkers of aging. Thus DNA methylation, along with the exponentially increasing risk of mortality with age, further establishes the exponential nature of aging.

CpG methylation changes in Alu repetitive sequences in normal aging due to diastolic hypertension in human dermal fibroblasts from the facial area.

Aging fibroblasts, an important factor contributing to skin aging, are affected by numerous mechanisms, including alterations in DNA methylation and age-related diseases. The current study aimed to investigate the role of Alu methylation in aging fibroblasts and hypertension. The Alu methylation levels in dermal fibroblasts obtained from patients of different ages and blood pressure status were analyzed using the combined bisulfite restriction analysis technique. An inverse correlation was observed between Alu methylation in dermal fibroblasts and patient age. Dermal fibroblasts from the high-normal diastolic blood pressure group had higher Alu methylation levels compared with those from the normal group. The findings of the present study suggest that Alu methylation alterations can be observed with chronological aging and hypertension, and are a potential aging marker or therapeutic target.

Effects of heated tobacco product aerosol extracts on DNA methylation and gene transcription in lung epithelial cells

AimsSmoking causes DNA methylation (DNAm) alterations that lead to lung cancer development. Although the use of heated tobacco products (HTPs) has recently increased, their impact on health remains unclear. This study aimed to evaluate the effects of HTPs on DNAm and gene transcription in human lung epithelial cells in vitro. Main methodsHuman lung adenocarcinoma (A549) cells with type II alveolar epithelial characteristics were treated with aerosol extracts of two HTPs or a smoke extract of combustible reference cigarette (RC). Global 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels were quantified using dot blot analysis. Furthermore, reduced representation of bisulfite sequencing, DNA microarray, and quantitative PCR analyses were performed to determine CpG methylation and gene transcription changes induced by HTP and RC. Key findingsGlobal 5-mC and 5-hmC levels were decreased by the RC extract but not the HTP extracts. However, an HTP extract altered the CpG methylation pattern, and Gene Ontology enrichment analysis of the differentially methylated regions of the RC and HTP groups showed a similar pattern. The HTP extract affected gene expression, albeit to a lesser extent than the RC extract. In particular, the HTP extract markedly affected the mRNA expression and promoter methylation of cytochrome P450 family 1 subfamily A member 1 (CYP1A1), which is associated with carcinogenic risk. SignificanceThe study results suggest that HTPs as well as conventional combustible cigarettes can alter CpG methylation and gene transcription in lung epithelial cells.

Integrative analysis of DNA methylomes reveals novel cell-free biomarkers in lung adenocarcinoma.

Lung cancer is a leading cause of cancer-related deaths worldwide, with a low 5-year survival rate due in part to a lack of clinically useful biomarkers. Recent studies have identified DNA methylation changes as potential cancer biomarkers. The present study identified cancer-specific CpG methylation changes by comparing genome-wide methylation data of cfDNA from lung adenocarcinomas (LUAD) patients and healthy donors in the discovery cohort. A total of 725 cell-free CpGs associated with LUAD risk were identified. Then XGBoost algorithm was performed to identify seven CpGs associated with LUAD risk. In the training phase, the 7-CpGs methylation panel was established to classify two different prognostic subgroups and showed a significant association with overall survival (OS) in LUAD patients. We found that the methylation of cg02261780 was negatively correlated with the expression of its representing gene GNA11. The methylation and expression of GNA11 were significantly associated with LAUD prognosis. Based on bisulfite PCR, the methylation levels of five CpGs (cg02261780, cg09595050, cg20193802, cg15309457, and cg05726109) were further validated in tumor tissues and matched non-malignant tissues from 20 LUAD patients. Finally, validation of the seven CpGs with RRBS data of cfDNA methylation was conducted and further proved the reliability of the 7-CpGs methylation panel. In conclusion, our study identified seven novel methylation markers from cfDNA methylation data which may contribute to better prognosis for LUAD patients.

Abstract 6009: An end-to-end workflow for improved methylation detection

Abstract Introduction: DNA methylation at CpG nucleotide sites in eukaryotes is a key epigenetic mark that can help regulate gene expression. Specific changes in CpG methylation occur in many human cancers, making them a promising biomarker for early cancer detection. However, existing assays can be costly, lack specificity to regions of interest and often provide only semi-quantitative estimates of the methylation fraction. Here, we present a targeted methylome panel to decrease costs associated with next generation sequencing (NGS), methylation controls to calibrate quantitative assays and UMIs for accurate deduplication in low-diversity samples. Experimental Procedures: Genomic DNA (gDNA) was prepared for sequencing using the Twist Methylation Detection System consisting of enzymatic methylation conversion and hybrid capture using the Twist Human Methylome Panel. Twist’s synthetic CpG methylation level controls were spiked-in to gDNA and taken through the Twist Methylation Detection System to demonstrate their utility in calibrating methylation assays. Additionally, libraries were generated using cell free DNA (cfDNA) and either conventional or UMI-containing adapters to investigate the impact on quantitative detection and total unique coverage. Results: Using the Twist Human Methylome Panel at 150x raw coverage achieves uniform coverage with low off-bait Picard metrics. 6.59 million CpG sites were detected using a minimum depth of 10X. Target capture with the Human Methylome Panel allows for informative CpG calling of up to 82 samples on a single Illumina Novaseq S4 flowcell, compared to 3 samples per flowcell with a tradition whole-genome bisulfite sequencing (WGBS) assay. The Twist CpG methylation specific controls are constructed of 48 unique contrived sequences that contain a total of 8 different levels of methylation, ranging from 100% to 0%. Including these controls allows for quantitation of methylation levels in the experimental samples and qualification of the enzymatic conversion process. Conclusions: Our study leverages the Twist methylation detection portfolio to interrogate genome-wide methylation patterns for various applications. In combination, these tools can be used to design cost effective end-to-end assays. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Lydia Bonar, Kristin Butcher, Michael Bocek, Holly Corbitt, Bryan Hoglund, Cibelle Nassif, Patrick Cherry, Derek Murphy, Jean Challacombe, Esteban Toro. An end-to-end workflow for improved methylation detection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6009.

Genome-wide DNA methylation profile of peripheral blood lymphocytes from subjects with nonsteroidal anti-inflammatory drug-induced respiratory diseases.

Significant changes in CpG methylation have been identified in nasal polyps, which are the main targets of nonsteroidal anti-inflammatory drug-exacerbated respiratory disease (NERD); however, these polyps are composed of various cellular components. In the present study, whole-genome CpG methylation in peripheral blood lymphocytes (PBLs) was analyzed to define the epigenetic changes in lymphocytes, which are the primary immune cells involved in NERD. Genomic DNA from peripheral blood mononuclear cells from 27 NERD and 24 aspirin-tolerant asthma (ATA) was subjected to bisulfate conversion and a methylation array. Quantitative CpG methylation, the β-values as a quantitative measure of DNA methylation, in lymphocytes were calculated after adjustments for cellular composition. Fifty-six hypermethylated and three hypomethylated differentially methylated CpGs (DMCs) in PBLs in the NERD compared with ATA. The top 10 CpG loci predicted the methylation risk score, with a positive predictive value of 91.3%, a negative predictive value of 81.5% and an accuracy of 84.3%. As demonstrated in the nasal polyps, 30 DMCs were predicted to bind to the following 10 transcription factors, ranked in descending order: AP-2alphaA, TFII-1, STAT4, FOXP3, GR, c-Est-1, E2F-1, XBP1, ENKTF-1 and NF-1. Gene ontology analysis identified 13 categories such as regulation of T-helper 17 cell differentiation, including SMAD7 and NFKBIZ. PBLs in NERD contained no DMCs in genes associated with the prostaglandin and leukotriene pathways, which were found in ATA. PBLs in NERD form a unique pattern of DNA CpG methylation, and the combined analysis may provide predictive values for NERD.

An antisense Alu transposon insertion/deletion polymorphism of ALDH1A1 may functionally associate with Parkinson’s disease

BackgroundAldehyde dehydrogenase 1 (encoded by ALDH1A1) has been shown to protect against Parkinson’s disease (PD) by reducing toxic metabolites of dopamine. We herein revealed an antisense Alu element insertion/deletion polymorphism in intron 4 of ALDH1A1, and hypothesized that it might play a role in PD. MethodsA Han Chinese cohort comprising 488 PD patients and 515 controls was recruited to validate the Alu insertion/deletion polymorphism following a previous study of tag-single nucleotide polymorphisms, where rs7043217 was shown to be significantly associated with PD. Functional analyses of the Alu element insertion were performed.ResultsThe Alu element of ALDH1A1 was identified to be a variant of Yb8 subfamily and termed as Yb8c4. The antisense Yb8c4 insertion/deletion polymorphism (named asYb8c4ins and asYb8c4del, respectively) appeared to be in a complete linkage disequilibrium with rs7043217 and was validated to be significantly associated with PD susceptibility with asYb8c4ins serving as a risk allele (P = 0.030, OR = 1.224, 95% CI = 1.020–1.470). Multiple functional analyses including ALDH1A1 mRNA expression in blood cells of carriers, and reporters of EGFP and luciferase showed that the asYb8c4ins had a suppressive activity on gene transcription. Mechanistic explorations suggested that the asYb8c4ins induced no changes in CpG methylation and mRNA splicing of ALDH1A1 and appeared no binding of transcription factors.ConclusionsOur results consolidate an involvement of ALDH1 in PD pathogenesis. The asYb8c4 polymorphism may be a functional output of its linkage disequilibrium-linked single nucleotide polymorphisms.

Prolonged diet-induced obesity modifies DNA methylation and gene expression in the hippocampus

Obesity and age independently have negative effects on the brain, increasing the risk of neurodegenerative events. How these events synergize to increase disease susceptibility remains poorly understood. To better understand the epigenetic implications of poor diet with increased age, we sought to determine CpG methylation changes in the dorsal hippocampus attributable to age and/or diet-induced obesity. Male C57BL/6J mice were fed either normal chow or an obesogenic diet until six or 14 months of age. Hippocampus was analyzed by Reduced Representation Bisulfite Sequencing. In total, 107 differentially methylated regions (DMRs) were identified across all four conditions; older obese mice accounted for 63% of those DMRs. Gene ontology analysis showed two affected pathways, nervous system development and regulation of multicellular organismal process, in older obese mice only. From the RRBS results, five genes were selected for qPCR quantification: Bridging Integrator 1 (BIN1), Histone Deacetylase 5 (HDAC5), Folliculin Interacting Protein 2 (FNIP2), Proline, Glutamate and Leucine Rich Protein 1 (PELP1), and Protein Tyrosine Phosphatase Non-Receptor Type 1 (PTPN1). Generally, DMR changes aligned with mRNA expression changes in BIN1, HDAC5, FNIP2, PELP1, and PTPN1. Notably BIN1 decreased and HDAC5 increased due to obesity and age. In summary, obesity interacts with aging to significantly affect the hippocampal methylome, changing the expression of genes implicated in neurodegeneration and metabolism.

Alu repetitive sequence CpG methylation changes in burn scars

Alu elements are retrotransposons related to epigenetic modifications. To date, the role of epigenetics in hypertrophic scars from burn remains unknown. Here, our aim was to examine the pathophysiology of hypertrophic scars from an epigenetic perspective. For that, we performed a cross-sectional analytical study using tissue and blood samples from burned and healthy patients (n = 23 each) to detect Alu methylation levels and patterns. The results of the combined bisulfite restriction analysis technique were categorized into four groups based on the methylation status at the CpG dinucleotides from the 5′ to the 3′ ends of the Alu sequence: hypermethylated (mCmC), hypomethylated (uCuC), and partially methylated (uCmC and mCuC). Alu methylation levels were significantly lower in hypertrophic scar tissues than in normal skin (29.37 ± 2.49% vs. 35.56 ± 3.18%, p = 0.0002). In contrast, the levels were significantly higher in white blood cells from blood samples of burned patients than in those of control blood samples (26.92 ± 4.04% vs. 24.58 ± 3.34%, p = 0.0278). Alu total methylation (mC) and the uCmC pattern were significantly lower, whereas uCuC was significantly higher, in hypertrophic scar tissues than in normal skin (p < 0.0001). Receiver operating characteristic analysis indicated that the uCmC and uCuC patterns are useful as hypertrophic scar DNA methylation markers after burn, with 91.30% sensitivity and 96.23% specificity and 100% sensitivity and 94.23% specificity, respectively. Our findings suggest that epigenetic modifications play a major role in hypertrophic scar pathogenesis, and may be the starting point for developing a novel technique for burn scar treatment.

Weaning Induces Stress-Dependent DNA Methylation and Transcriptional Changes in Piglet PBMCs.

Changes to the epigenome, including those to DNA methylation, have been proposed as mechanisms by which stress can induce long-term physiological changes in livestock species. Pig weaning is associated with dietary and social stress, both of which elicit an immune response and changes to the hypothalamic–pituitary–adrenal (HPA) axis. While differential methylation following stress has been assessed in model organisms, it remains poorly understood how the pig methylome is altered by stressors in production settings. We quantified changes in CpG methylation and transcript abundance in piglet peripheral blood mononuclear cells (PBMCs) following weaning and also assessed differential patterns in pigs exhibiting high and low stress response as measured by cortisol concentration and lesion scores. Blood was collected from nine gilt piglets 24 h before and after weaning, and whole-genome bisulfite sequencing (WGBS) and RNA-sequencing were performed on six and nine animals, respectively, at both time points. We identified 2,674 differentially methylated regions (DMRs) that were enriched within promoters of genes associated with lymphocyte stimulation and transcriptional regulation. Stress groups displayed unique differential methylation and expression patterns associated with activation and suppression of T cell immunity in low and high stress animals, respectively. Differential methylation was strongly associated with differential expression; specifically, upregulated genes were enriched among hypomethylated genes. We observed post-weaning hypermethylation of the glucocorticoid receptor (NR3C1) promoter and a significant decrease in NR3C1 expression (n = 9, p = 6.1 × 10–3). Our results indicate that weaning-associated stress elicits genome-wide methylation changes associated with differential gene expression, reduced T cell activation, and an altered HPA axis response.

DNA Methylation Dynamics in Atlantic Salmon (Salmo salar) Challenged With High Temperature and Moderate Hypoxia

The marine environment is predicted to become warmer and more hypoxic, and these conditions may become a challenge for marine fish species. Phenotypically plastic responses facilitating acclimatization to changing environments can be mediated by DNA methylation through the modulation of gene expression. To investigate whether temperature and hypoxia exposure induce DNA methylation changes, we challenged post-smolt Atlantic salmon (Salmo salar) to increasing temperatures (12 → 20°C, 1°C week–1) under normoxia or moderate hypoxia (∼70% air saturation) and compared responses in the liver after 3 days or 4 weeks at 20°C. DNA methylation was studied in six genes related to temperature stress (cirbp, serpinh1), oxidative stress (prdx6, ucp2), apoptosis (jund), and metabolism (pdk3). Here, we report that exposure to high temperature, alone or combined with hypoxia, affected the methylation of CpG sites within different genomic regulatory elements around the transcription start of these temperature/hypoxia biomarker genes. Yet, we uncovered distinct CpG methylation profiles for each treatment group, indicating that each environmental condition may induce different epigenetic signatures. These CpG methylation responses were strongly dependent on the duration of stress exposure, and we found reversible, but also persistent, CpG methylation changes after 4 weeks of exposure to 20°C. Further, several of these changes in CpG methylation correlated with transcriptional changes, and thus, can be considered as regulatory epigenetic marks (epimarkers). Our study provides insights into the dynamic associations between CpG methylation and transcript expression in Atlantic salmon, and suggests that this epigenetic mechanism may mediate physiological acclimation to short-term and long-term environmental changes.

Telomerase Reverse Transcriptase (TERT) Regulation in Thyroid Cancer: A Review.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of the enzyme telomerase and is essential for telomerase activity. Upregulation of TERT expression and resulting telomerase activity occurs in the large majority of malignancies, including thyroid cancer. This upregulation results in continued cellular proliferation and avoidance of cellular senescence and cell death. In this review we will briefly introduce TERT and telomerase activity as it pertains to thyroid cancer and, highlight the effects of TERT on cancer cells. We will also explore in detail the different TERT regulatory strategies and how TERT is reactivated in thyroid cancer cells, specifically. These regulatory mechanisms include both activating single base pair TERT promoter mutations and epigenetic changes at the promoter, including changes in CpG methylation and histone modifications that affect chromatin structure. Further, regulation includes the allele-specific regulation of the TERT promoter in thyroid cancer cells harboring the TERT promoter mutation. These entail allele-specific transcriptional activator binding, DNA methylation, histone modifications, and mono-allelic expression of TERT. Lastly, TERT copy number alterations and alternative splicing are also implicated. Both amplifications of the TERT locus and increased full-length transcripts and decreased inactive and dominant negative isoforms result in active telomerase. Finally, the clinical significance of TERT in thyroid cancer is also reviewed.

Transcriptomic and epigenomics atlas of myotubes reveals insight into the circadian control of metabolism and development

Aim: Innate circadian rhythms are critical for optimal tissue-specific functions, including skeletal muscle, a major insulin-sensitive tissue responsible for glucose homeostasis. We determined whether transcriptional oscillations are associated with CpG methylation changes in skeletal muscle. Materials & methods: We performed rhythmicity analysis on the transcriptome and CpG methylome of circadian synchronized myotubes. Results: We identified several transcripts and CpG-sites displaying oscillatory behavior, which were enriched with Gene Ontology terms related to metabolism and development. Oscillating CpG methylation was associated with rhythmic expression of 31 transcripts. Conclusion: Although circadian oscillations may be regulated by rhythmic DNA methylation, strong rhythmic associations between transcriptome and CpG methylation were not identified. This resource constitutes a transcriptomic/epigenomic atlas of skeletal muscle and regulation of circadian rhythms.

Triterpenoid corosolic acid modulates global CpG methylation and transcriptome of tumor promotor TPA induced mouse epidermal JB6 P+ cells

Epigenetic regulation is one of the driving forces in the process of carcinogenesis. Corosolic acid (CA); triterpenoid abundantly found in Lagerstroemia speciosa L. is known to modulate various cellular process including cellular oxidative stress and signaling kinases in various diseases, including skin cancer. Genetic mutations in early stages of skin cancer are well-documented, the epigenetic alterations remain elusive. In the present study, we identified the transcriptomic gene expression changes with RNAseq and genome-wide DNA CpG methylation changes with DNA methylseq to profile the early stage transcriptomic and epigenomic changes using tumor promoter TPA-mediated mouse epidermal epithelial JB6 P+ cells. JB6 P+ cells were treated with TPA and Corosolic acid by 7.5uM optimized by MTS assay. Differentiated expressed genes (DEGs) and Differentially methylated genes (DMRs) were analyzed by R software. Ingenuity Pathway Analysis (IPA) was employed to understand the differential regulation of specific pathways. Novel TPA induced differentially overexpressed genes like tumor promoter Prl2c2, small prolin rich protein (Sprr2h) was reported which was downregulated by corosolic acid treatment. Several cancer related pathways were identified by Ingenuity Pathways Analysis (IPA) including p53, Erk, TGF beta signaling pathways. Moreover, differentially methylated regions (DMRs) in genes like Dusp22 (Dual specificity protein phosphatase 22), Rassf (tumor suppressor gene family, Ras association domain family) in JB6 P+ cells were uncovered which are altered by TPA and are reversed by CA treatment. Interestingly, genes like CDK1 (Cyclin-dependent kinases 1) and RASSF2 (Ras association domain family member 2) observed to be differentially methylated and expressed which was further modulated by corosolic acid treatment, validated by qPCR. Given study indicated gene expression changes to DNA CpG methylation epigenomic changes modulated various molecular pathways in TPA-induced JB6 cells and revealed that CA can potentially reverse these changes which deciphering novel molecular targets for future prevention of early stages of skin cancer studies in human.

Prenatal Glucocorticoid Exposure Results in Changes in Gene Transcription and DNA Methylation in the Female Juvenile Guinea Pig Hippocampus Across Three Generations

Synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery, to mature the fetal lung and decrease neonatal morbidity. sGC also profoundly affect the fetal brain. The hippocampus expresses high levels of glucocorticoid (GR) and mineralocorticoid receptor (MR), and its development is affected by elevated fetal glucocorticoid levels. Antenatal sGC results in neuroendocrine and behavioral changes that persist in three generations of female guinea pig offspring of the paternal lineage. We hypothesized that antenatal sGC results in transgenerational changes in gene expression that correlate with changes in DNA methylation. We used RNASeq and capture probe bisulfite sequencing to investigate the transcriptomic and epigenomic effects of antenatal sGC exposure in the hippocampus of three generations of juvenile female offspring from the paternal lineage. Antenatal sGC exposure (F0 pregnancy) resulted in generation-specific changes in hippocampal gene transcription and DNA methylation. Significant changes in individual CpG methylation occurred in RNApol II binding regions of small non-coding RNA (snRNA) genes, which implicates alternative splicing as a mechanism involved in transgenerational transmission of the effects of antenatal sGC. This study provides novel perspectives on the mechanisms involved in transgenerational transmission and highlights the importance of human studies to determine the longer-term effects of antenatal sGC on hippocampal-related function.

PTEN deletion drives aberrations of DNA methylome and transcriptome in different stages of prostate cancer.

Phosphatase and tensin homolog located on chromosome 10 (PTEN) is a tumor suppressor gene and one of the most frequently mutated/deleted genes in human prostate cancer (PCa). However, how PTEN deletion would impact the epigenome and transcriptome alterations remain unknown. This hypothesis was tested in a prostate-specific PTEN-/- (KO) mouse prostatic adenocarcinoma model through DNA methyl-Seq and RNA-Seq analyses. Examination of cancer genomic datasets revealed that PTEN is expressed at lower levels in PTEN-deleted tumor samples than in normal solid tissue samples. Methylome and transcriptome profiling identified several inflammatory responses and immune response signaling pathways, including NF-kB signaling, IL-6 signaling, LPS/IL-1-mediated inhibition of RXR Function, PI3K in B lymphocytes, iCOS-iCOSL in T helper cells, and the role of NFAT in regulating the immune response, were affected by PTEN deletion. Importantly, a small subset of genes that showed DNA hypermethylation or hypomethylation was correlated with decreased or increased gene expression including CXCL1. quantitative polymerase chain reaction analyses of representative genes validated the RNA-Seq results. Histopathological examinations showed that the severity of prostatic intraepithelial neoplasia and inflammation development gradually increased as PTEN null mice aged. Collectively, these findings suggest that loss of PTEN drives global changes in DNA CpG methylation and transcriptomic gene expression and highly associated with several inflammatory and immune molecular pathways during PCa development. These biomarkers could be valuable molecular targets for cancer drug discovery and development against PCa.

Whole-genome bisulfite sequencing in systemic sclerosis provides novel targets to understand disease pathogenesis

BackgroundSystemic sclerosis (SSc) is a rare autoimmune connective tissue disease whose pathogenesis remains incompletely understood. Increasing evidence suggests that both genetic susceptibilities and changes in DNA methylation influence pivotal biological pathways and thereby contribute to the disease. The role of DNA methylation in SSc has not been fully elucidated, because existing investigations of DNA methylation predominantly focused on nucleotide CpGs within restricted genic regions, and were performed on samples containing mixed cell types.MethodsWe performed whole-genome bisulfite sequencing on purified CD4+ T lymphocytes from nine SSc patients and nine controls in a pilot study, and then profiled genome-wide cytosine methylation as well as genetic variations. We adopted robust statistical methods to identify differentially methylated genomic regions (DMRs). We then examined pathway enrichment associated with genes located in these DMRs. We also tested whether changes in CpG methylation were associated with adjacent genetic variation.ResultsWe profiled DNA methylation at more than three million CpG dinucleotides genome-wide. We identified 599 DMRs associated with 340 genes, among which 54 genes exhibited further associations with adjacent genetic variation. We also found these genes were associated with pathways and functions that are known to be abnormal in SSc, including Wnt/β-catenin signaling pathway, skin lesion formation and progression, and angiogenesis.ConclusionThe CD4+ T cell DNA cytosine methylation landscape in SSc involves crucial genes in disease pathogenesis. Some of the methylation patterns are also associated with genetic variation. These findings provide essential foundations for future studies of epigenetic regulation and genome-epigenome interaction in SSc.

Artificial Intelligence and the detection of pediatric concussion using epigenomic analysis

Concussion, also referred to as mild traumatic brain injury (mTBI) is the most common type of traumatic brain injury. Currently concussion is an area ofintensescientific interest to better understand the biological mechanisms and for biomarker development. We evaluated whole genome-wide blood DNA cytosine (‘CpG’) methylation in 17 pediatric concussion isolated cases and 18 unaffected controls using Illumina Infinium MethylationEPIC assay. Pathway analysis was performed using Ingenuity Pathway Analysis to help elucidate the epigenetic and molecular mechanisms of the disorder. Area under the receiver operating characteristics (AUC) curves and FDR p-values were calculated for mTBI detection based on CpG methylation levels. Multiple Artificial Intelligence (AI) platforms including Deep Learning (DL), the newest form of AI, were used to predict concussion based on i) CpG methylation markers alone, and ii) combined epigenetic, clinical and demographic predictors. We found 449 CpG sites (473 genes), those were statistically significantly methylated in mTBI compared to controls. There were four CpGs with excellent individual accuracy (AUC ≥ 0.90–1.00) while 119 displayed good accuracy (AUC ≥ 0.80–0.89) for the prediction of mTBI. The CpG methylation changes ≥10% were observed in many CpG loci after concussion suggesting biological significance. Pathway analysis identified several biologically important neurological pathways that were perturbed including those associated with: impaired brain function, cognition, memory, neurotransmission, intellectual disability and behavioral change and associated disorders. The combination of epigenomic and clinical predictors were highly accurate for the detection of concusion using AI techniques. Using DL/AI, a combination of epigenomic and clinical markers had sensitivity and specificity ≧95% for prediction of mTBI. In this novel study, we identified significant methylation changes in multiple genes in response to mTBI. Gene pathways that were epigenetically dysregulated included several known to be involved in neurological function, thus giving biological plausibility to our findings.