Global DNA Hypomethylation Research Articles

ARGONAUTE4 and the DNA demethylase ROS1c mediate dehydroascorbate-induced intergenerational nematode resistance in rice.

Plants can transmit information to the next generation and modulate the phenotype of their offspring through epigenetic mechanisms. In this study, we demonstrate the activation of 'intergenerational acquired resistance' (IAR) in the progeny of rice (Oryza sativa) plants exogenously treated with dehydroascorbate (DHA). The offspring of lifelong DHA-treated plants (DHA-IAR) were significantly less susceptible to the root-knot nematode Meloidogyne graminicola and partially inherited the DHA-induced transcriptional response found in the parental plants. Phytohormone analyses on the DHA-IAR plants unveiled higher basal abscisic acid (ABA) levels and a primed induction of the jasmonic acid (JA) pathway. RNA-seq analysis on the embryonic tissues of immature seeds of DHA-treated plants revealed major shifts in the expression of genes associated with epigenetic pathways. We confirmed that DHA treatment leads to a significant but transient pattern of global DNA hypomethylation in the parental plants at 12 to 24 hours after treatment. The induction of resistance in the parental plants requires the DNA demethylase REPRESSOR OF SILENCING 1C (ROS1c) and ARGONAUTE 4 (AGO), suggesting a role for DNA demethylation and subsequent re-methylation in establishment of this phenotype. Confirming the transience of global hypomethylation upon DHA treatment, no significant change in global DNA methylation levels was observed in DHA-IAR versus naïve plants. Finally, DHA could not induce IAR in the Ros1c mutant line and in the ARGONAUTE 4 (ago4ab)-RNAi line. These data indicate that a controlled collaboration between transient DNA demethylation and remethylation underlies the induced resistance and IAR phenotypes upon DHA treatment.

Hypertension and global DNA methylation: a population-based study in rural, Punjab, India

Hypertension is a significant public health concern and a modifiable risk factor for increased mortality worldwide. It is reported to be influenced by gene-environment interactions and micronutrient intake. This study aims to understand the relationship between global DNA methylation levels and hypertension, independently and in the context of micronutrient status, among rural population in Punjab, India. A total of 2300 individuals, aged 30–75 years, (54.9% females) were screened for blood pressure. Of 2300 screened individuals, 900 (age sex matched 450 cases and 450 controls of hypertension) individuals were selected to examine the relationship between hypertension, global DNA methylation (5mC), and biochemicals (Folate, Vitamin B12, and Homocysteine). Folate, vitamin B12, and homocysteine levels were estimated using chemiluminescence technique. The ELISA-based colorimetric technique was used for performing peripheral blood leucocyte (PBL) global DNA methylation (5mC). Statistical analyses were performed using SPSS version 22.0. Hypertensives were found to have significantly lower levels of global DNA methylation than normotensives (0.65 vs. 0.72 respectively; p-value = 0.01*). Individuals in the 1st quartile of 5mC were at significantly (OR: 1.671; 95% CI: 1.206–2.315; p-value = 0.01*) increased risk for hypertension in comparison to those in the 4th quartile (reference). Further hypertensives on medication with controlled blood pressure (BP) were significantly hypermethylated as compared to hypertensives on medication with uncontrolled BP (0.70 vs. 0.62 respectively; p-value = 0.04*). Folate appeared to mediate global DNA methylation among hypertensives on medication-controlled BP. Further hypertension driven hypomethylation hints towards accelerated biological aging among younger hypertensives. Hypertension may be associated with Global DNA hypomethylation in the studied rural population of Punjab, India. Folate sufficiency may prove to be an aid in improving the methylation levels among the cases of hypertension who were on medication and had controlled BP.

Maternal Malnutrition in Mice Impairs Nephrogenesis by Disrupting DNA Methylation of Regulatory Regions.

Maternal caloric restriction during pregnancy significantly impacts kidney development, influencing susceptibility to chronic kidney disease in adulthood. This study explores DNA methylation changes in nephron progenitor cells resulting from caloric restriction and their implications for kidney health. Global DNA hypomethylation is observed in nephron progenitors from caloric-restricted embryos, with specific genomic regions displaying distinct methylation patterns, including hypomethylation and hypermethylation. Differentially methylated regions exhibit enhanced chromatin accessibility, indicating biological relevance. Hypomethylated regions are enriched for genes associated with developmental processes, reflecting changes in gene expression and highlighting their functional relevance in kidney development. The study also reveals that supplementing methionine, an essential amino acid, restores disrupted DNA methylation patterns, particularly in enhancer regions, emphasizing methionine's critical role in regulating nephron progenitor cell epigenetics and ensuring proper kidney development. The intricate relationship between maternal nutrition, dynamic DNA methylation, and kidney development is highlighted, emphasizing the enduring impact of early-life nutritional challenges on kidney function. This research elucidates epigenetic mechanisms as mediators for the lasting effects of maternal caloric restriction on kidney health. The study contributes valuable insights into the origins of chronic kidney diseases during early developmental stages, offering potential interventions to mitigate adverse outcomes.

Molecular and cell phenotype programs in oral epithelial cells directed by co-exposure to arsenic and smokeless tobacco.

Chronic arsenic exposure can lead to various health issues, including cancer. Concerns have been mounting about the enhancement of arsenic toxicity through co-exposure to various prevalent lifestyle habits. Smokeless tobacco products are commonly consumed in South Asian countries, where their use frequently co-occurs with exposure to arsenic from contaminated groundwater. To decipher the in vitro molecular and cellular responses to arsenic and/or smokeless tobacco, we performed temporal multi-omics analysis of the transcriptome and DNA methylome remodelling in exposed hTERT-immortalized human normal oral keratinocytes (NOK), as well as arsenic and/or smokeless tobacco genotoxicity and mutagenicity investigations in NOK cells and in human p53 knock-in murine embryonic fibroblasts (Hupki MEF). RNAseq results from acute exposures to arsenic alone and in combination with smokeless tobacco extract revealed upregulation of genes with roles in cell cycle changes, apoptosis and inflammation responses. This was in keeping with global DNA hypomethylation affecting genes involved in the same processes in response to chronic treatment in NOK cells. At the phenotypic level, we observed a dose-dependent decrease in NOK cell viability, induction of DNA damage, cell cycle changes and increased apoptosis, with the most pronounced effects observed under arsenic and SLT co-exposure conditions. Live-cell imaging experiments indicated that the DNA damage likely resulted from induction of apoptosis, an observation validated by a lack of exome-wide mutagenesis in response to chronic exposure to arsenic and/or smokeless tobacco. In sum, our integrative omics study provides novel insights into the acute and chronic responses to arsenic and smokeless tobacco (co-)exposure, with both types of responses converging on several key mechanisms associated with cancer hallmark processes. The generated rich catalogue of molecular programs in oral cells regulated by arsenic and smokeless tobacco (co-)exposure may provide bases for future development of biomarkers for use in molecular epidemiology studies of exposed populations at risk of developing oral cancer.

Impact of smoking hookah and bidi on global DNA methylation: a community-based study from Haryana, North India

Smoking is a major contributor to the increased burden of chronic diseases worldwide. Smoking has been reported to be associated with several adverse epigenetic modifications like altered DNA methylation patterns. Despite adverse effects, smoking hookah and bidi is accepted as a cultural practice in several communities, viz., the Jats of Haryana. This study aims to explore the smoking-induced alterations in global DNA methylation in the Jat community, where smoking is a cultural practice. A total of 1075 participants, aged 30–75 years (69.8%, females), belonging to the Jat community from Palwal, Haryana, were recruited. Data on socio-demographic and lifestyle variables were collected through pre-tested interview schedules. The ELISA-based colorimetric technique was used for performing peripheral blood leucocyte global DNA methylation. The difference between median global DNA methylation (5mC%) levels of non-smokers (median 5mC% = 0.66) and smokers (median 5mC% = 0.62) was not found to be statistically significant (p value = 0.309). However, the median 5mC level among former smokers (median 5mC% = 0.41) was found to be significantly lower than smokers and non-smokers (p value < 0.01). The adjusted regression model revealed former smoker status to be significantly associated with global DNA hypomethylation. Further, no significant difference in global DNA methylation levels was found between heavy and light smokers. The study suggests that continuous exposure to tobacco smoke during the prenatal and later stages of life may be contributing to the new environmental adaptations resulting in no significant effect of smoking on epigenetic alterations. A unique DNA hypomethylation signature of former smokers may be due to smoking-related comorbidities.

DIPG-87. TARGETING GENE-REPRESSIVE EPIGENETIC REGULATION TO INDUCE INTERFERON SIGNALING IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brainstem tumor with no known effective treatment. DIPG is driven by the H3K27M mutant histone, which promotes epigenetic dysregulation, leading to loss of gene-repressive H3K27me3 marks, global DNA hypomethylation, and a distinct gene expression profile. DIPG cells retain histone methylation at histone H3 lysine 9 (H3K9me2/3), suggesting that the tumor may rely on these marks to repress unwanted expression. Our prior work has demonstrated that treatment with the DNA hypomethylating agent decitabine (DAC) elicits expression of cancer-specific antigens and immune activation, including activation of interferon signaling, but modulating gene-repressive H3K9 methylation in DIPG has not been extensively investigated. METHODS We treated four primary patient-derived DIPG neurosphere cell lines with BIX01294 and UNC0642, inhibitors of H3K9 methyltransferases G9a/GLP, and analyzed gene expression changes by RNA-sequencing and Western blot. We assessed the effect of combination treatments on these four cell lines with both DAC and either BIX01294 or UNC0642 and evaluated impact on gene expression, innate immune signaling, and interferon pathway activation. RESULTS We found that treatment with either of the G9a/GLP inhibitors resulted in the upregulation of genes involved in immune signaling. Given the similarities in the immune signaling response elicited from treatment with DAC and the G9a/GLP inhibitors, we evaluated the effect of combination treatments with both DAC and either BIX01294 or UNC0642. These combinations resulted in an increased induction of genes involved in immune signaling, including STING. CONCLUSIONS Pharmacologic inhibition of DNA methylation and histone H3K9 methylation elicits immune activation in DIPG cells. Upregulation of interferon and STING signaling with this combination treatment may be significant in enhancing the immunogenicity of DIPG cells to allow effective immunotherapy.

DIPG-58. TARGETING DISORDERED DNA METHYLATION IN DIPG TO CONSTRAIN VARIABILITY AND INDUCE IMMUNE SIGNALING

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal pediatric brain cancer characterized by the histone H3 K27M mutation. The known downstream consequences of this mutation include loss of repressive chromatin marks, global DNA hypomethylation, and altered gene expression. METHODS We sought to investigate the role of epigenetic variability as a basis of DIPG cellular heterogeneity and plasticity by using Whole Genome Bisulfite Sequencing (WGBS). Existing methods of analysis of WGBS are limited in that they average/smooth methylation data and fail to capture intrinsic variability. We performed (WGBS) on 23 primary patient samples of DIPG and applied an analysis that captures the variability of DNA methylation reads to characterize methylation stochasticity. We then sought to modulate the epigenome pharmacologically using a DNA methyltransferase inhibitor and assessed changes through RNA-Seq and immunopeptidomics. RESULTS We find that DIPG has a marked increase in methylation stochasticity (quantified as methylation entropy). Additionally, while DIPG is globally hypomethylated, it retains DNA methylation at specific regulatory regions, such as bivalent promoters, and key genes, such as FOXG1, CXCR4, and KLF4. We sought to reverse these changes using the DNA methyltransferase inhibitor, decitabine, in 4 DIPG neurosphere cell lines. Treatment with decitabine reversed the entropy seen in DIPG and induced innate immune signaling and interferon signaling in DIPG cells. Using RNA-seq, we found that decitabine treatment dramatically alters gene expression, including activation of endogenous retroviral elements, activation of interferon signaling, de-repression of hypermethylated targets, and expression of putative neoantigens, such as PRAME and DAZL. Immunopeptidomics profiling of MHC Class I bound canonical and non-canonical peptides of DIPG cell lines after decitabine treatment also revealed decitabine-induced differences. CONCLUSIONS Taken together, our study finds that the methylome of DIPG is highly disordered but is also highly responsive to pharmacologic modulation by hypomethylating agents. Furthermore, hypomethylating drugs can increase the immunogenicity of DIPG, thus offering the potential for future combination with immunotherapy.

Patulin alters alpha-adrenergic receptor signalling and induces epigenetic modifications in the kidneys of C57BL/6 mice

Patulin (PAT) is a food-borne mycotoxin produced by Penicillium and Byssochlamys species. It is widely known for its mutagenic, carcinogenic, and genotoxic effects and has been associated with kidney injury; however, the mechanism of toxicity remains unclear. To address this gap, we conducted a study to explore the changes in α-adrenergic receptor signalling pathways and epigenetic modifications induced by PAT in the kidneys of C57BL/6 mice during acute (1 day) and prolonged (10 days) exposure. The mice (20–22 g) were orally administered PAT (2.5 mg/kg; at 1 and 10 days), and post-treatment, the kidneys were harvested, homogenised and extracted for RNA, DNA, and protein. The relative gene expression of the α-adrenergic receptors (ADRA1, ADRA2A, ADRA2B) and associated signalling pathways (MAPK, MAPK14, ERK, PI3K, and AKT) was assessed by qPCR. The protein expression of ERK1/2 and MAPK was determined by western blot. The impact of PAT on DNA methylation was evaluated by quantifying global DNA methylation; qPCR was used to determine gene expression levels of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and demethylase (MBD2). PAT downregulated the expression of ADRA1, ADRA2A, ADRA2B, PI3K, and AKT and upregulated ERK1/2 and MAPK protein expression. Furthermore, PAT induced alterations in DNA methylation patterns by upregulating DNMT1 and MBD2 expressions and downregulating DNMT3A and DNMT3B expressions, resulting in global DNA hypomethylation. In conclusion, PAT disrupts α-1 and α-2 adrenergic receptor signalling pathways and induces epigenetic modifications, that can lead to kidney injury.

DNMT1-targeting remodeling global DNA hypomethylation for enhanced tumor suppression and circumvented toxicity in oral squamous cell carcinoma

BackgroundThe faithful maintenance of DNA methylation homeostasis indispensably requires DNA methyltransferase 1 (DNMT1) in cancer progression. We previously identified DNMT1 as a potential candidate target for oral squamous cell carcinoma (OSCC). However, how the DNMT1- associated global DNA methylation is exploited to regulate OSCC remains unclear.MethodsThe shRNA-specific DNMT1 knockdown was employed to target DNMT1 on oral cancer cells in vitro, as was the use of DNMT1 inhibitors. A xenografted OSCC mouse model was established to determine the effect on tumor suppression. High-throughput microarrays of DNA methylation, bulk and single-cell RNA sequencing analysis, multiplex immunohistochemistry, functional sphere formation and protein immunoblotting were utilized to explore the molecular mechanism involved. Analysis of human samples revealed associations between DNMT1 expression, global DNA methylation and collaborative molecular signaling with oral malignant transformation.ResultsWe investigated DNMT1 expression boosted steadily during oral malignant transformation in human samples, and its inhibition considerably minimized the tumorigenicity in vitro and in a xenografted OSCC model. DNMT1 overexpression was accompanied by the accumulation of cancer-specific DNA hypomethylation during oral carcinogenesis; conversely, DNMT1 knockdown caused atypically extensive genome-wide DNA hypomethylation in cancer cells and xenografted tumors. This novel DNMT1-remodeled DNA hypomethylation pattern hampered the dual activation of PI3K-AKT and CDK2-Rb and inactivated GSK3β collaboratively. When treating OSCC mice, targeting DNMT1 achieved greater anticancer efficacy than the PI3K inhibitor, and reduced the toxicity of blood glucose changes caused by the PI3K inhibitor or combination of PI3K and CDK inhibitors as well as adverse insulin feedback.ConclusionsTargeting DNMT1 remodels a novel global DNA hypomethylation pattern to facilitate anticancer efficacy and minimize potential toxic effects via balanced signaling synergia. Our study suggests DNMT1 is a crucial gatekeeper regarding OSCC destiny and treatment outcome.

Epigenetic processes involved in response to pesticide exposure in human populations: a systematic review and meta-analysis.

In recent decades, the use of pesticides in agriculture has increased dramatically. This has resulted in these substances being widely dispersed in the environment, contaminating both exposed workers and communities living near agricultural areas and via contaminated foodstuffs. In addition to acute poisoning, chronic exposure to pesticides can lead to molecular changes that are becoming better understood. Therefore, the aim of this study was to assess, through a systematic review of the literature, what epigenetic alterations are associated with pesticide exposure. We performed a systematic review and meta-analysis including case-control, cohort and cross-sectional observational epidemiological studies to verify the epigenetic changes, such as DNA methylation, histone modification and differential microRNA expression, in humans who had been exposed to any type of pesticide. Articles published between the years 2005 and 2020 were collected. Two different reviewers performed a blind selection of the studies using the Rayyan QCRI software. Post-completion, the data of selected articles were extracted and analyzed. Most of the 28 articles included evaluated global DNA methylation levels, and the most commonly reported epigenetic modification in response to pesticide exposure was global DNA hypomethylation. Meta-analysis revealed a significant negative correlation between Alu methylation levels and β-hexachlorocyclohexane, p,p'-dichlorodiphenyldichloroethane and p,p'-dichlorodiphenylethylene levels. In addition, some specific genes were reported to be hypermethylated in promoter regions, such as CDKN2AIGF2, WRAP53α and CDH1, while CDKN2B and H19 were hypomethylated due to pesticide exposure. The expression of microRNAs was also altered in response to pesticides, as miR-223, miR-518d-3p, miR-597, miR-517b and miR-133b that are associated with many human diseases. Therefore, this study provides evidence that pesticide exposure could lead to epigenetic modifications, possibly altering global and gene-specific methylation levels, epigenome-wide methylation and microRNA differential expression.

The Role of Methylation in Chronic Lymphocytic Leukemia and Its Prognostic and Therapeutic Impacts in the Disease: A Systematic Review.

Epigenetic regulation has been thoroughly investigated in recent years and has emerged as an important aspect of chronic lymphocytic leukemia (CLL) biology. Characteristic aberrant features such as methylation patterns and global DNA hypomethylation were the early findings of the research during the last decades. The investigation in this field led to the identification of a large number of genes where methylation features correlated with important clinical and laboratory parameters. Gene-specific analyses investigated methylation in the gene body enhancer regions as well as promoter regions. The findings included genes and proteins involved in key pathways that play central roles in the pathophysiology of the disease. Τhe application of these findings beyond the theoretical understanding can not only lead to the creation of prognostic and predictive models and scores but also to the design of novel therapeutic agents. The following is a review focusing on the present knowledge about single gene/gene promoter methylation or mRNA expression in CLL cases as well as records of older data that have been published in past papers.

Evidence of epigenetic landscape shifts in mucopolysaccharidosis IIIB and IVA

Lysosomal storage diseases (LSDs) are a group of monogenic diseases characterized by mutations in genes coding for proteins associated with the lysosomal function. Despite the monogenic nature, LSDs patients exhibit variable and heterogeneous clinical manifestations, prompting investigations into epigenetic factors underlying this phenotypic diversity. In this study, we focused on the potential role of epigenetic mechanisms in the pathogenesis of mucopolysaccharidosis IIIB (MPS IIIB) and mucopolysaccharidosis IVA (MPS IVA). We analyzed DNA methylation (5mC) and histone modifications (H3K14 acetylation and H3K9 trimethylation) in MPS IIIB and MPS IVA patients’ fibroblasts and healthy controls. The findings revealed that global DNA hypomethylation is present in cell lines for both diseases. At the same time, histone acetylation was increased in MPS IIIB and MPS IVA cells in a donor-dependent way, further indicating a shift towards relaxed open chromatin in these MPS. Finally, the constitutive heterochromatin marker, histone H3K9 trimethylation, only showed reduced clustering in MPS IIIB cells, suggesting limited alterations in heterochromatin organization. These findings collectively emphasize the significance of epigenetic mechanisms in modulating the phenotypic variations observed in LSDs. While global DNA hypomethylation could contribute to the MPS pathogenesis, the study also highlights individual-specific epigenetic responses that might contribute to phenotypic heterogeneity. Further research into the specific genes and pathways affected by these epigenetic changes could provide insights into potential therapeutic interventions for these MPS and other LSDs.

CDCA7-associated global aberrant DNA hypomethylation translates to localized, tissue-specific transcriptional responses.

Disruption of cell division cycle associated 7 (CDCA7) has been linked to aberrant DNA hypomethylation, but the impact of DNA methylation loss on transcription has not been investigated. Here, we show that CDCA7 is critical for maintaining global DNA methylation levels across multiple tissues in vivo. A pathogenic Cdca7 missense variant leads to the formation of large, aberrantly hypomethylated domains overlapping with the B genomic compartment but without affecting the deposition of H3K9 trimethylation (H3K9me3). CDCA7-associated aberrant DNA hypomethylation translated to localized, tissue-specific transcriptional dysregulation that affected large gene clusters. In the brain, we identify CDCA7 as a transcriptional repressor and epigenetic regulator of clustered protocadherin isoform choice. Increased protocadherin isoform expression frequency is accompanied by DNA methylation loss, gain of H3K4 trimethylation (H3K4me3), and increased binding of the transcriptional regulator CCCTC-binding factor (CTCF). Overall, our in vivo work identifies a key role for CDCA7 in safeguarding tissue-specific expression of gene clusters via the DNA methylation pathway.

Inhibition of protein kinase C increases Prdm14 level to promote self-renewal of embryonic stem cells through reducing Suv39h-induced H3K9 methylation

Inhibition of protein Kinase C (PKC) efficiently promoted the self-renewal of embryonic stem cells (ESCs). However, information about the function of PKC inhibition remains lacking. Here, RNA- sequencing showed that the addition of Go6983 significantly inhibited the expression of de novo methyltransferases (Dnmt3a and Dnmt3b) and their regulator Dnmt3l, resulting in global hypomethylation of DNA in mouse ESCs. Mechanistically, PR domain-containing 14 (Prdm14), a site-specific transcriptional activator, partially contributed to Go6983-mediated repression of Dnmt3 genes. Administration of Go6983 increased Prdm14 expression mainly through the inhibition of PKCδ. High constitutive expression of Prdm14 phenocopied the ability of Go6983 to maintain` mouse ESC stemness in the absence of self-renewal-promoting cytokines. In contrast, the knockdown of Prdm14 eliminated the response to PKC inhibition and substantially impaired the Go6983-induced resistance of mouse ESCs to differentiation. Furthermore, LC‒MS profiling and Western blotting revealed low levels of Suv39h1 and Suv39h2 in Go6983-treated mouse ESCs. Suv39h enzymes are histone methyltransferases that recognize di- and trimethylated histone H3K9 specifically and usually function as transcriptional repressors. Consistently, the inhibition of Suv39h1 by RNA interference or the addition of the selective inhibitor chaetocin increased Prdm14 expression. Moreover, ChIP assays showed that Go6983 treatment led to decreased enrichment of di- and trimethylation of H3K9 at the Prdm14 promoter but increased RNA polymerase Ⅱ binding affinity. Together, our results provide novel insights into the pivotal association between PKC inhibition-mediated self-renewal and epigenetic changes, which will help us better understand the regulatory network of stem cell pluripotency.

DNA methylome analysis reveals novel insights into active hypomethylated regulatory mechanisms of temperature-dependent flower opening in Osmanthus fragrans.

Short-term ambient low temperature (ALT) stimulation is necessary for Osmanthus fragrans to facilitate continued flower opening after floral bud development reaches maturity. DNA methylation, a vital epigenetic modification, regulates various biological processes in response to temperature fluctuations. However, its role in temperature-driven flower opening remains elusive. In this study, we identified the pivotal timeframe during which O. fragrans promptly detected temperature cues. Using whole-genome bisulfite sequencing, we explored global DNA hypomethylation during this phase, with the most significant changes occurring in CHH sequence contexts. Auxin transport inhibitor (TIBA) application revealed that ALT-induced endogenous auxin accumulation promoted peduncle elongation. In our mRNA-seq analysis, we discovered that the differentially expressed genes (DEGs) with hypo-differentially methylated regions (hypo-DMRs) were mainly enriched in auxin and temperature response, RNA processing, and carbohydrate and lipid metabolism. Transcripts of three DNA demethylase genes (OfROS1a, OfDML3, OfDME) showed upregulation. Furthermore, all DNA methylase genes, except OfCMT2b, also displayed increased expression, specifically with two of them, OfCMT3a and OfCMT1, being associated with hypo-DMRs. Promoter assays showed that OfROS1a, with promoters containing low-temperature- and auxin-responsive elements, were activated by ALT and exogenous IAA at low concentrations but inhibited at high concentrations. Overexpression of OfROS1 reduced endogenous auxin levels but enhanced the expression of genes related to auxin response and spliceosome in petunia. Furthermore, OfROS1 promoted sucrose synthesis in petunia corollas. Our data characterized the rapid response of active DNA hypomethylation to ALT and suggested a possible epiregulation of temperature-dependent flower opening in O. fragrans. This study revealed the pivotal role of DNA hypomethylation in O. fragrans during the ALT-responsive phase before flower opening, involving dynamic DNA demethylation, auxin signaling modulation, and a potential feedback loop between hypomethylation and methylation.

Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

Whole-genome sequencing reveals the molecular implications of the stepwise progression of lung adenocarcinoma

The mechanism underlying the development of tumors, particularly at early stages, still remains mostly elusive. Here, we report whole-genome long and short read sequencing analysis of 76 lung cancers, focusing on very early-stage lung adenocarcinomas such as adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma. The obtained data is further integrated with bulk and spatial transcriptomic data and epigenomic data. These analyses reveal key events in lung carcinogenesis. Minimal somatic mutations in pivotal driver mutations and essential proliferative factors are the only detectable somatic mutations in the very early-stage of AIS. These initial events are followed by copy number changes and global DNA hypomethylation. Particularly, drastic changes are initiated at the later AIS stage, i.e., in Noguchi type B tumors, wherein cancer cells are exposed to the surrounding microenvironment. This study sheds light on the pathogenesis of lung adenocarcinoma from integrated pathological and molecular viewpoints.

Relationship between hysterectomy and global DNA methylation levels in peripheral blood: an exploratory follow-up study

ObjectiveThe present study aims to understand the relationship between hysterectomy and peripheral blood leukocyte (PBL) global DNA methylation independently and in light of selected biochemicals (vitamin B12, folate, homocysteine). Material and methodsFor the present population-based follow-up study, a total of 616 women aged 30–70 years were recruited from Haryana, India. Recruited participants underwent a baseline assessment, which included the determination of menopausal and hysterectomy status and estimation of vitamin B12, folate, homocysteine, and PBL global DNA methylation (5mC) levels. The 5mC levels were analyzed using ELISA-based colorimetric technique. Participants with no history of hysterectomy were followed up after 8 years, and their current menopausal and hysterectomy statuses were determined. ResultThe median PBL 5mC level of the group with hysterectomy was found to be significantly lower than that of those without hysterectomy in the baseline assessment (PBL 5mC = 0.47 vs. 0.74, respectively; p-value = 0.016). However, there was no significant difference in baseline median PBL 5mC levels between women who eventually underwent hysterectomy and those who did not. When stratified for biochemical levels, the trend of PBL global DNA hypomethylation in the hysterectomy group, compared to the no-hysterectomy group, was statistically significant only within the normal folate group. ConclusionThis study suggests a potential association between hysterectomy (or related factors) and global DNA hypomethylation. Further research is warranted to validate the findings of the present study and to advance the existing knowledge regarding the epigenetic implications of hysterectomy.

Abstract A042: Targeting vulnerabilities arising from global DNA hypomethylation in cancer

Abstract Background: DNA methylation alterations are a universal feature of cancer. In addition to site specific gain of DNA methylation (hypermethylation), a global loss of methylation (hypomethylation) was noted in most cancer genomes. Whereas numerous studies have focused on the role of hypermethylation in disease progression, less is known about the biology of hypomethylation in cancer. Recently, we have identified tumors across all major cancer types that are characterized by severe loss of DNA methylation. Our preliminary data suggest that these DNA hypomethylated cancers (hereafter DHMCs) show distinct alterations influencing gene expression and tumor microenvironment composition. Objective: We hypothesize that since DNA methylation is critical for genome organization and gene expression. Severe global hypomethylation is likely to result in distinct epigenomic and genomic alterations. We therefore investigate common molecular changes associated with DNA hypomethylation to develop novel therapeutic approaches targeting unique vulnerabilities arising in DHMCs. Study design: To delineate the biology of DHMCs, we determined methylation changes with validated orthogonal methods in large representative cohorts of patient samples (The Cancer Genome Atlas (TCGA), University of Washington rapid autopsy cohorts), patient derived xenografts and cancer cell lines. We assessed the patterns of common molecular alterations by performing genome wide epigenome mapping experiments and by defining common driver gene events in DHMCs. We further determined tumor cell intrinsic therapeutic vulnerabilities in broad pharmacologic and CRISPR-Cas9 genomic screens. Results: We observed that DNA hypomethylation is a relatively common feature of solid tumors affecting overall 15-20% of all cancers, with highest rates observed in urothelial, melanoma, hepatocellular and lung and head and neck squamous cell carcinoma. In in vivo tumor models of DHMCs, we observe distinct shifts in epigenetic states upon DNA hypomethylation. Most notably, we found that DHMCs are characterized by unique intrinsic drug sensitivities to drug classes affecting core histone levels. Impact: This is the first study to show druggable vulnerabilities arising from DNA hypomethylation. Collectively, these studies define a novel epigenetic subtype of cancer, determine its clinical and molecular features, and pave the way for new targeted therapies. Citation Format: Pallabi Mustafi, Brian Hanratty, Ilsa Coleman, Radhika Patel, Adil Mohamed, Jay Sarthy, Gavin Ha, Michael Haffner. Targeting vulnerabilities arising from global DNA hypomethylation in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A042.

Egr2 Deletion in Autoimmune-Prone C57BL6/lpr Mice Suppresses the Expression of Methylation-Sensitive Dlk1-Dio3 Cluster MicroRNAs.

We previously demonstrated that the upregulation of microRNAs (miRNAs) at the genomic imprinted Dlk1-Dio3 locus in murine lupus is correlated with global DNA hypomethylation. We now report that the Dlk1-Dio3 genomic region in CD4+ T cells of MRL/lpr mice is hypomethylated, linking it to increased Dlk1-Dio3 miRNA expression. We evaluated the gene expression of methylating enzymes, DNA methyltransferases (DNMTs), and demethylating ten-eleven translocation proteins (TETs) to elucidate the molecular basis of DNA hypomethylation in lupus CD4+ T cells. There was a significantly elevated expression of Dnmt1 and Dnmt3b, as well as Tet1 and Tet2, in CD4+ T cells of three different lupus-prone mouse strains compared to controls. These findings suggest that the hypomethylation of murine lupus CD4+ T cells is likely attributed to a TET-mediated active demethylation pathway. Moreover, we found that deletion of early growth response 2 (Egr2), a transcription factor gene in B6/lpr mice markedly reduced maternally expressed miRNA genes but not paternally expressed protein-coding genes at the Dlk1-Dio3 locus in CD4+ T cells. EGR2 has been shown to induce DNA demethylation by recruiting TETs. Surprisingly, we found that deleting Egr2 in B6/lpr mice induced more hypomethylated differentially methylated regions at either the whole-genome level or the Dlk1-Dio3 locus in CD4+ T cells. Although the role of methylation in EGR2-mediated regulation of Dlk1-Dio3 miRNAs is not readily apparent, these are the first data to show that in lupus, Egr2 regulates Dlk1-Dio3 miRNAs, which target major signaling pathways in autoimmunity. These data provide a new perspective on the role of upregulated EGR2 in lupus pathogenesis.