5hmC Levels Research Articles

Preservation of 5-Hydroxymethylcytosine Levels in LRIG1 across Genomic DNA and Cell-Free DNA in Glioma Patients.

Cell-free DNA (cfDNA) has recently emerged as a promising minimally invasive diagnostic biomarker for various cancers. In this study, our aim was to identify cfDNA biomarkers by investigating genes that displayed significant differences between glioma patients and their corresponding controls. To accomplish this, we utilized publicly available data from the Gene Expression Omnibus, focusing on 5-hydroxymethylcytosine (5hmC) profiles in both cfDNA and genomic DNA (gDNA) from glioma patients and healthy individuals. The intersection of gene lists derived from these comparative analyses unveiled LRIG1 and ZNF703 as the two genes with elevated 5hmC levels in both the cfDNA of glioma patients and gDNA of glioma tissue compared to their respective controls. The gene expression data revealed both genes were upregulated in glioma tissue compared to normal brain tissue. Integration of 5hmC data revealed a strong positive correlation in the glioma tissue group between 5hmC and the gene expression of the LRIG1 gene. Furthermore, exploration using the AmiCa web tool indicated that LRIG1 gene expression was elevated compared to 17 other cancers included in the database, emphasizing its potential as a distinctive biomarker across multiple cancer types.

5-Hydroxymethylcytosine in Cell-Free DNA Predicts Immunotherapy Response in Lung Cancer.

Immune checkpoint inhibitors (ICIs) drastically improve therapeutic outcomes for lung cancer, but accurately predicting individual patient responses to ICIs remains a challenge. We performed the genome-wide profiling of 5-hydroxymethylcytosine (5hmC) in 85 plasma cell-free DNA (cfDNA) samples from lung cancer patients and developed a 5hmC signature that was significantly associated with progression-free survival (PFS). We built a 5hmC predictive model to quantify the 5hmC level and validated the model in the validation, test, and control sets. Low weighted predictive scores (wp-scores) were significantly associated with a longer PFS compared to high wp-scores in the validation [median 7.6 versus 1.8 months; p = 0.0012; hazard ratio (HR) 0.12; 95% confidence interval (CI), 0.03-0.54] and test (median 14.9 versus 3.3 months; p = 0.00074; HR 0.10; 95% CI, 0.02-0.50) sets. Objective response rates in patients with a low or high wp-score were 75.0% (95% CI, 42.8-94.5%) versus 0.0% (95% CI, 0.0-60.2%) in the validation set (p = 0.019) and 80.0% (95% CI, 44.4-97.5%) versus 0.0% (95% CI, 0.0-36.9%) in the test set (p = 0.0011). The wp-scores were also significantly associated with PFS in patients receiving single-agent ICI treatment (p < 0.05). In addition, the 5hmC predictive signature demonstrated superior predictive capability to tumor programmed death-ligand 1 and specificity to ICI treatment response prediction. Moreover, we identified novel 5hmC-associated genes and signaling pathways integral to ICI treatment response in lung cancer. This study provides proof-of-concept evidence that the cfDNA 5hmC signature is a robust biomarker for predicting ICI treatment response in lung cancer.

Abstract 6218: Haplotype-resolved analysis of cancer genomes and epigenomes using Oxford Nanopore sequencing

Abstract Cancer is a complex and dynamic disease driven by somatic genomic and epigenomic alterations that accumulate over time. These changes give rise to heterogeneous collections of cells or clones, each with distinct (epi)genomic profiles within a single tumour. Accurate characterisation of these changes is crucial for understanding the mechanisms driving the disease, identifying potential therapeutic targets, and personalising treatment strategies. Due to the technological constraints of short-read and array-based approaches, cancer research has historically had a strong focus on detecting small genomic changes like SNPs and small indels (SNVs) as well as the broad characterisation of large-scale copy number changes (CNVs), mostly ignoring other important variant classes and epigenetic modifications. Here we demonstrate how Oxford Nanopore native long-read sequencing enables the direct detection of not only SNVs, but also break point-resolved simple and complex structural variants (SVs and CNVs), haplotype phasing of all variant types, and identification of DNA modifications like 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) from a single tumour-normal dataset. We illustrate how our end-to-end somatic workflow streamlines analysis for the different variant classes and benchmark somatic SNV and SV calling performance using well characterised cancer cell lines and in-silico benchmarking datasets at different sequencing depths. Finally, we use real-world tumour-normal pairs to showcase a comprehensive sample to answer tumour-normal analysis using Nanopore sequencing. This includes the characterisation of complex patterns of somatic SVs in the different cancer samples, the identification of unique 5mC methylation patterns, exploring characteristic differences in 5hmC levels between tumour and normal samples as well as showing how Nanopore long reads enable haplotype and clone specific CNV calling as well as phasing of genetic and epigenetic variation. Citation Format: Philipp Rescheneder, Phill James, Sean McKenzie, Andrea Talenti, Sergey Aganezov, Dan Turner, Sissel Juul. Haplotype-resolved analysis of cancer genomes and epigenomes using Oxford Nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6218.

Abstract 7008: NEBNext® E5hmC-seqTM: Direct detection of 5-hydroxymethylcytosine at single base resolution

Abstract DNA methylation is a critical epigenetic regulator of gene expression during development and in diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), can be detected by sequencing libraries generated using existing methods: NEBNext® EM-seq™ or bisulfite sequencing. However, these methods cannot differentiate between 5mC and 5hmC. Interest in the role of 5hmC in regulating gene expression and as a cancer biomarker has increased, resulting in the need for robust methods for the identification of specific 5hmC sites. Methods currently exist to enable discrimination of 5mC and 5hmC (for example, oxBS-seq and TAB-seq), however these are based on modifications of bisulfite sequencing, and suffer from reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC, termed NEBNext® Enzymatic 5hmC-seq (E5hmC-seqTM). E5hmC-seq libraries were generated for 0.1 ng to 200 ng DNA isolated from human brain and E14 mouse embryonic stem cells. Libraries were prepared using NEBNext® Ultra IITM reagents followed by two enzymatic steps to detect 5hmC. In the first step, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. This conversion allows discrimination of 5hmC from cytosine and 5mC. Finally, libraries were PCR amplified using NEBNext® Q5U® and sequenced on the Illumina platform. The E5hmC-seq libraries have similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. Moreover, E5hmC-seq libraries were well-correlated between inputs and replicates at higher sequencing depths. Fully hydroxymethylated T4147 phage DNA was used as an internal control, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for a period of 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. E5hmC-seq libraries provide accurate measurements of 5hmC across a wide input range with expected insert sizes and minimal GC bias. Additionally, subtracting E5hmC-seq data from EM-seq data, which detects both 5mC and 5hmC, enables the precise location of individual 5mC and 5hmC sites. The ability to discriminate between 5mC and 5hmC will allow key insights to be made into the role of cytosine modifications in development and cancer. Citation Format: Garrett M. Cammarata, Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matt A. Campbell, Nan Dai, Bradley W. Langhorst, Louise Williams. NEBNext® E5hmC-seqTM: Direct detection of 5-hydroxymethylcytosine at single base resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7008.

Abstract 7007: 5hmc-sequencing identifies candidate genes in Puerto Rican Hispanic/Latino men with aggressive prostate cancer

Abstract Introduction: Puerto Rican (PR) Hispanic/Latino (H/L) men are an understudied population which has the highest prostate cancer (PCa) specific mortality when compared with other Hispanic populations. According to the recent PR Cancer Registry data, PCa is the leading cancer type in terms of incidence (38% of all cancer cases) and mortality (16% of all cancer deaths) in PR H/L men. PR H/L men also have significantly higher PCa-specific mortality than non-Hispanic Whites (NHW) and non-Hispanic Blacks (NHB). So far, little is known about higher mortality in PR H/L men. It is believed that epigenetic changes of key genes play a critical role in aggressive tumors. Therefore, our study was aimed to identify key 5-hydroxymethylcytosine (5hmC) changes in PR H/L men with aggressive PCa. Methods: We prepared sequencing libraries using the enriched 5hmC DNA from 42 prostate tumor and 44 adjacent normal FFPE samples. We performed sequencing on an Illumina NextSeq 500 with 75bp single end. We used DESeq2 package to identify differentially methylated genes (DMGs). We also downloaded additional 5hmC data (Database: EGAS00001004942) of 51 localized PCa and 7 adjacent normal for confirmation analysis. We used Gleason score (GS) as a cut-off to identify DMGs in aggressive PCa patients (GS- 7 (3+4) or less, Non-aggressive; GS- 7 (4+3) and above, Aggressive). Results: We identified 808 DMGs in tumors compared to adjacent normal tissues (FDR&amp;lt;0.05, log2FC&amp;gt;|0.4|). Pathway analysis of DMGs demonstrated that cell-cycle, cell division, and DNA repair related pathways were most upregulated in tumors. Since 5hmC abundance is highly correlated with gene expression levels, we further investigated 808 DMGs with TCGA prostate cancer gene expression data. This analysis identified 59/73 DMGs (80.1%, (FDR&amp;lt;0.05, delta&amp;gt;|1|) with significant 5hmC and gene expression changes in the same direction. We also performed differential methylation analysis in another dataset and observed that 129 DMGs were common with our results. Finally, we demonstrated 111 DMGs (p&amp;lt;0.05) in aggressive tumors (n=7) compared to non-aggressive tumors (n=15). These 111 DMGs include important 5hmC hypomethylated genes (BCCIP and KLK10) and hypermethylated genes (COX6C, ARMC2, PVT1) in aggressive PCa patients. Conclusions: Our study identifies 59 genes having coordinated epigenetic and transcriptomic changes in PR H/L men and 111 DMGs in aggressive tumors. The coordinated 59 genes include tumor suppressor genes such as DKK3 and PRDM8 with downregulated 5hmC and gene expression levels. Also, previous studies reported downregulation of BCCIP and KLK10 and upregulation of COX6C, ARMC2, PVT1 expression in PCa. The coordinated changes in 5hmC could be critical in aggressive PCa biology, hence in future, these genes will be validated as candidate biomarkers and targets for aggressive PCa among PR H/L men. Citation Format: Manishkumar S. Patel, Muosa Almubarak, Jaime Matta, Carmen Ortiz-Sánchez, Jarline Encarnacion, Gilberto Ruiz-Deya, Julie Dutil, Jasreman Dhillon, Kosj Yamoah, Anders Berglund, Hyun Park, Liang Wang, Jong Y. Park. 5hmc-sequencing identifies candidate genes in Puerto Rican Hispanic/Latino men with aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7007.

Association of tet methylcytosine dioxygenase 2 and 5-hydroxymethylcytosine in endometrioid adenocarcinoma and its clinical significance

BackgroundAberrant DNA methylation is a vital molecular alteration commonly detected in type I endometrial cancers (EC), and tet methylcytosine dioxygenase 2 (TET2) and 5-hydroxymethylcytosine (5hmC) play significant roles in DNA demethylation. However, little is known about the function and correlation of TET2 and 5hmC co-expressed in EC. This study intended to investigate the clinical significance of TET2 and 5hmC in EC.MethodsThe levels of TET2 and 5hmC were detected in 326 endometrial tissues by immumohistochemistry, and the correlation of their level was detected by Pearson analysis. The association between the levels of TET2 and 5hmC and clinicopathologic characteristics was analyzed. Prognostic value of TET2 and 5hmC was explored by Kaplan–Meier analysis. The Cox proportional hazard regression model was used for univariate and multivariate analyses.ResultsBased on the analysis results, TET2 protein level was positively correlated with 5hmC level in EC tissues (r = 0.801, P < 0.001). TET2+5hmC+ (high TET2 and high 5hmC) association was significantly associated with well differentiation, myometrial invasion, negative lymph node metastasis, and tumor stage in EC. Association of TET2 and 5hmC was confirmed as a prognostic factor (HR = 2.843, 95%CI = 1.226–3.605, P = 0.007) for EC patients, and EC patients with TET2−5hmC− level had poor overall survival.ConclusionsIn summary, the association of TET2 and 5hmC was downregulated in EC tissues, and may be a potential poor prognostic indicator for EC patients. Combined detection of TET2 and 5hmC may be valuable for the diagnosis and prognosis of EC.

Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal

Background5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time. ResultsWe develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by β-glucosyltransferase (β-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10−14 to 1 × 10−8 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells. SignificanceThis biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.

High estrogen during ovarian stimulation induced loss of maternal imprinted methylation that is essential for placental development via overexpression of TET2 in mouse oocytes.

Ovarian stimulation (OS) during assisted reproductive technology (ART) appears to be an independent factor influencing the risk of low birth weight (LBW). Previous studies identified the association between LBW and placenta deterioration, potentially resulting from disturbed genomic DNA methylation in oocytes caused by OS. However, the mechanisms by which OS leads to aberrant DNA methylation patterns in oocytes remains unclear. Mouse oocytes and mouse parthenogenetic embryonic stem cells (pESCs) were used to investigate the roles of OS in oocyte DNA methylation. Global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels were evaluated using immunofluorescence or colorimetry. Genome-wide DNA methylation was quantified using an Agilent SureSelectXT mouse Methyl-Seq. The DNA methylation status of mesoderm-specific transcript homologue (Mest) promoter region was analyzed using bisulfite sequencing polymerase chain reaction (BSP). The regulatory network between estrogen receptor alpha (ERα, ESR1) and DNA methylation status of Mest promoter region was further detected following the knockdown of ERα or ten-eleven translocation 2 (Tet2). OS resulted in a significant decrease in global 5mC levels and an increase in global 5hmC levels in oocytes. Further investigation revealed that supraphysiological β-estradiol (E2) during OS induced a notable decrease in DNA 5mC and an increase in 5hmC in both oocytes and pESCs of mice, whereas inhibition of estrogen signaling abolished such induction. Moreover, Tet2 may be a direct transcriptional target gene of ERα, and through the ERα-TET2 axis, supraphysiological E2 resulted in the reduced global levels of DNA 5mC. Furthermore, we identified that MEST, a maternal imprinted gene essential for placental development, lost its imprinted methylation in parthenogenetic placentas originating from OS, and ERα and TET2 combined together to form a protein complex that may promote Mest demethylation. In this study, a possible mechanism of loss of DNA methylation in oocyte caused by OS was revealed, which may help increase safety and reduce epigenetic abnormalities in ART procedures.

Cell-Free DNA 5-Hydroxymethylcytosine Signatures for Lung Cancer Prognosis.

Accurate prognostic markers are essential for guiding effective lung cancer treatment strategies. The level of 5-hydroxymethylcytosine (5hmC) in tissue is independently associated with overall survival (OS) in lung cancer patients. We explored the prognostic value of cell-free DNA (cfDNA) 5hmC through genome-wide analysis of 5hmC in plasma samples from 97 lung cancer patients. In both training and validation sets, we discovered a cfDNA 5hmC signature significantly associated with OS in lung cancer patients. We built a 5hmC prognostic model and calculated the weighted predictive scores (wp-score) for each sample. Low wp-scores were significantly associated with longer OS compared to high wp-scores in the training [median 22.9 versus 8.2 months; p = 1.30 × 10-10; hazard ratio (HR) 0.04; 95% confidence interval (CI), 0.00-0.16] and validation (median 18.8 versus 5.2 months; p = 0.00059; HR 0.22; 95% CI: 0.09-0.57) sets. The 5hmC signature independently predicted prognosis and outperformed age, sex, smoking, and TNM stage for predicting lung cancer outcomes. Our findings reveal critical genes and signaling pathways with aberrant 5hmC levels, enhancing our understanding of lung cancer pathophysiology. The study underscores the potential of cfDNA 5hmC as a superior prognostic tool for guiding more personalized therapeutic strategies for lung cancer patients.

Literary Identification of Differentially Hydroxymethylated DNA Regions for Type 2 Diabetes Mellitus: A Scoping Minireview.

Type 2 diabetes mellitus (T2DM) is a public health condition where environmental and genetic factors can intersect through hydroxymethylation. It was unclear which blood DNA regions were hydroxymethylated in human T2DM development. We aimed to identify the regions from the literature as designed in the ongoing Twins Discordant for Incident T2DM Study. A scoping review was performed using Medical Subject Headings (MeSH) and keyword methods to search PubMed for studies published in English and before 1 August 2022, following our registered protocol. The keyword and MeSH methods identified 12 and 3 records separately, and the keyword-identified records included all from the MeSH. Only three case-control studies met the criteria for the full-text review, including one MeSH-identified record. Increased global levels of 5-hydroxymethylated cytosine (5hmC) in T2DM patients versus healthy controls in blood or peripheral blood mononuclear cells were consistently reported (p < 0.05 for all). Among candidate DNA regions related to the human SOCS3, SREBF1, and TXNIP genes, only the SOCS3 gene yielded higher 5hmC levels in T2DM patients with high poly-ADP-ribosylation than participants combined from those with low PARylation and healthy controls (p < 0.05). Hydroxymethylation in the SOCS3-related region of blood DNA is promising to investigate for its mediation in the influences of environment on incident T2DM.

Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate.

Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1-3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1-3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design.

The Role of 5-Hydroxymethylcytosine as a Potential Epigenetic Biomarker in a Large Series of Thyroid Neoplasms.

Cytosine modifications at the 5-carbon position play a critical role in gene expression regulation and have been implicated in cancer development. 5-Hydroxymethylcytosine (5hmC), arising from 5-methylcytosine (5-mC) oxidation, has shown promise as a potential malignancy marker due to its depletion in various human cancers. However, its significance in thyroid tumors remains underexplored, primarily due to limited data. In our study, we evaluated 5hmC expression levels by immunohistochemistry in a cohort of 318 thyroid tumors. Our analysis revealed significant correlations between 5hmC staining extension scores and nodule size, vascular invasion, and oncocytic morphology. Nuclear 5hmC staining intensity demonstrated associations with focality, capsule status, extrathyroidal extension, vascular invasion, and oncocytic morphology. Follicular/oncocytic adenomas exhibited higher 5hmC expression than uncertain malignant potential (UMP) or noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), as well as malignant neoplasms, including papillary thyroid carcinomas (PTCs), oncocytic carcinomas (OCAs), follicular thyroid carcinomas (FTCs), and invasive encapsulated follicular variants of PTC (IEFV-PTC). TERT promoter mutation cases showed notably lower values for the 5hmC expression, while RAS (H, N, or K) mutations, particularly HRAS mutations, were associated with higher 5hmC expression. Additionally, we identified, for the first time, a significant link between 5hmC expression and oncocytic morphology. However, despite the merits of these discoveries, we acknowledge that 5hmC currently cannot segregate minimally invasive from widely invasive tumors, although 5hmC levels were lower in wi-FPTCs. Further research is needed to explore the potential clinical implications of 5hmC in thyroid tumors.

Association of DNA methylation/demethylation with the functional outcome of stroke in a hyperinflammatory state.

JOURNAL/nrgr/04.03/01300535-202410000-00024/figure1/v/2024-02-06T055622Z/r/image-tiff Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.

Exploring the epigenetic landscape: The role of 5-hydroxymethylcytosine in neurodevelopmental disorders.

Recent advances in genetic and epigenetic research have underscored the significance of 5-hydroxymethylcytosine (5hmC) in neurodevelopmental disorders (NDDs), such as autism spectrum disorder (ASD) and intellectual disability (ID), revealing its potential as both a biomarker for early detection and a target for novel therapeutic strategies. This review article provides a comprehensive analysis of the role of 5hmC in NDDs by examining both animal models and human studies. By examining mouse models, studies have demonstrated that prenatal environmental challenges, such as maternal infection and food allergies, lead to significant epigenetic alterations in 5hmC levels, which were associated with NDDs in offspring, impacting social behavior, cognitive abilities and increasing ASD-like symptoms. In human studies, researchers have linked alterations in 5hmC levels NDDs through studies in individuals with ASD, fragile X syndrome, TET3 deficiency and ID, specifically identifying significant epigenetic modifications in genes such as GAD1, RELN, FMR1 and EN-2, suggesting that dysregulation of 5hmC played a critical role in the pathogenesis of these disorders and highlighted the potential for targeted therapeutic interventions. Moreover, we explore the implications of these findings for the development of epigenetic therapies aimed at modulating 5hmC levels. The review concludes with a discussion on future directions for research in this field, such as machine learning, emphasizing the need for further studies to elucidate the complex mechanisms underlying NDDs and to translate these findings into clinical practice. This paper not only advances our understanding of the epigenetic landscape of NDDs but also opens up new avenues for diagnosis and treatment, offering hope for individuals affected by these conditions.

Intrauterine arsenic exposure induces glucose metabolism disorders in adult offspring by targeting TET2-mediated DNA hydroxymethylation reprogramming of HNF4α in developing livers, an effect alleviated by ascorbic acid

Exposure to arsenic during gestation has lasting health-related effects on the developing fetus, including an increase in the risk of metabolic disease later in life. Epigenetics is a potential mechanism involved in this process. Ten-eleven translocation 2 (TET2) has been widely considered as a transferase of 5-hydroxymethylcytosine (5hmC). Here, mice were exposed, via drinking water, to arsenic or arsenic combined with ascorbic acid (AA) during gestation. For adult offspring, intrauterine arsenic exposure exhibited disorders of glucose metabolism, which are associated with DNA hydroxymethylation reprogramming of hepatic nuclear factor 4 alpha (HNF4α). Further molecular structure analysis, by SEC-UV-DAD, SEC-ICP-MS, verified that arsenic binds to the cysteine domain of TET2. Mechanistically, arsenic reduces the stability of TET2 by binding to it, resulting in the decrease of 5hmC levels in Hnf4α and subsequently inhibiting its expression. This leads to the disorders of expression of its downstream key glucose metabolism genes. Supplementation with AA blocked the reduction of TET2 and normalized the 5hmC levels of Hnf4α, thus alleviating the glucose metabolism disorders. Our study provides targets and methods for the prevention of offspring glucose metabolism abnormalities caused by intrauterine arsenic exposure.

Aerobic exercise alters DNA hydroxymethylation levels in an experimental rodent model of temporal lobe epilepsy

The therapeutic potential of aerobic exercise in mitigating seizures and cognitive issues in temporal lobe epilepsy (TLE) is recognized, yet the underlying mechanisms are not well understood. Using a rodent TLE model induced by Kainic acid (KA), we investigated the impact of a single bout of exercise (i.e., acute) or 4 weeks of aerobic exercise (i.e., chronic). Blood was processed for epilepsy-associated serum markers, and DNA methylation (DNAme), and hippocampal area CA3 was assessed for gene expression levels for DNAme-associated enzymes. While acute aerobic exercise did not alter serum Brain-Derived Neurotrophic Factor (BDNF) or Interleukin-6 (IL-6), chronic exercise resulted in an exercise-specific decrease in serum BDNF and an increase in serum IL-6 levels in epileptic rats. Additionally, whole blood DNAme levels, specifically 5-hydroxymethylcytosine (5-hmC), decreased in epileptic animals following chronic exercise. Hippocampal CA3 5-hmC levels and ten-eleven translocation protein (TET1) expression mirrored these changes. Furthermore, immunohistochemistry analysis revealed that most 5-hmC changes in response to chronic exercise were neuron-specific within area CA3 of the hippocampus. Together, these findings suggest that DNAme mechanisms in the rodent model of TLE are responsive to chronic aerobic exercise, with emphasis on neuronal 5-hmC DNAme in the epileptic hippocampus.

Exposure of children to brominated flame retardants and heavy metals in Morocco: Urine and blood levels in association with global cytosine and adenine methylation

Persistent pollutants, namely brominated flame retardants (BFRs) and heavy metals, are compounds that are added to a wide range of products and materials for preventing ignition, increasing the functionality of materials or improving their performance, e.g. electric conductivity. The exposure of children might consequently be inferred, through indoor dust and hand-to-mouth or toy-chewing behaviors. The current study is aimed at assessing the exposure of Moroccan children to BFRs and heavy metal elements, and evaluating their associations with global DNA methylation. First, parents responded to a questionnaire pertaining to children’s lifestyle, then blood and urine samples were collected from (n = 93) children aged between 5 and 11 years for biomonitoring and DNA methylation analysis. BFRs were detected in 54.84% of samples with a median concentration of 0.01 nmol/mL (range: 0.004–0.051 nmol/mL) while metal elements were detected in more than 90% of samples. BFRs showed no variations with global DNA methylation, unlike metal elements, which revealed significant associations with global DNA methylation markers, namely 5-mC, 5-hmC and N⁶-mA levels. Moroccan children may be exposed to flame retardants and heavy metals through several routes. Further research is required to assess the exposure and the health impacts of environmental pollutants and ultimately protect the Moroccan population by the prevention of adverse health effects.

Quantification of cytosine modifications in the aged mouse brain.

Quantifying cytosine modifications in various brain regions provides important insights into the gene expression regulation and pathophysiology of neuropsychiatric disorders. In this study, we quantified 5-methylcytosine (5-mC), 5-hydroxymethylation (5-hmC), and 5-formylcytosine (5-fC) levels in five brain regions (the frontal lobe, cerebral cortical region without frontal lobe, hippocampus, basal ganglia, and the cerebellum) and the heart at three developmental periods (12, 48, and 101 weeks). We observed significant regional variations in cytosine modification. Notably, regional variations were generally maintained throughout development, suggesting that epigenetic regulation is unique to each brain region and remains relatively stable with age. The 5-mC and 5-hmC levels were positively correlated, although the extent of the correlations seemed to differ in different brain regions. On the contrary, 5-fC levels did not correlate with 5-mC or 5-hmC levels. Additionally, we observed an age-dependent decrease in 5-fC levels in the basal ganglia, suggesting a unique epigenetic regulation mechanism. Further high-resolution studies using animal models of neuropsychiatric disorders as well as postmortem brain evaluation are warranted.

Molecular imaging of aging-related 5-hydroxymethylcytosine in cell-free DNA at the single-copy level

In cell-free DNA (cfDNA), 5-hydroxymethylcytosine (5hmC) is a vital epigenetic modification closely related to aging. Although many methods have been developed to quantify 5hmC in DNA extracted from cells, few have been successfully applied to detect 5hmC in cfDNA due to their insufficient sensitivity. In our work, we quantified the ratio of 5hmC modified cfDNA in the circulating blood of volunteers at different ages using an ultrasensitive single-molecule immunofluorescent imaging method. Among the results, the ratio of 5hmC modified cfDNA in the old volunteers was significantly higher than that in the young volunteers. In addition, the sensitivity of this method was greatly improved with the minimal input of 0.5 pg cfDNA, compared to the traditional dot blot assay. Moreover, old volunteers with geriatric syndromes exhibited a significantly higher level of 5hmC in cfDNA than their counterparts without such syndromes. Last, in a single-molecule photobleaching experiment, we demonstrated that the higher ratio of 5hmC modified cfDNA among the old volunteers may result from the enrichment of 5hmC in a single cfDNA strand. Taken together, 5hmC in circulating cfDNA may play multiple roles in aging-related physical changes, especially in geriatric syndromes, and could be a biomarker for evaluating such syndromes.

Gender dimorphism in hepatocarcinogenesis-DNA methylation modification regulated X-chromosome inactivation escape molecule XIST.

Sex disparities constitute a significant issue in hepatocellular carcinoma (HCC). However, the mechanism of gender dimorphism in HCC is still not completely understood. 5-Hydroxymethylcytosine (5hmC)-Seal technology was utilised to detect the global 5hmC levels from four female and four male HCC samples. Methylation of XIST was detected by Sequenom MassARRAY methylation profiling between HCC tissues (T) and adjacent normal liver tissues (L). The role of Tet methylcytosine dioxygenase 2 (TET2) was investigated using diethylnitrosamine (DEN)-administered Tet2-/- female mice, which regulated XIST in hepatocarcinogenesis. All statistical analyses were carried out by GraphPad Prism 9.0 and SPSS version 19.0 software. The results demonstrated that the numbers of 5hmC reads in the first exon of XIST from female HCC tissues (T) were remarkably lower than that in female adjacent normal liver tissues (L). Correspondingly, DNA methylation level of XIST first exon region was significantly increased in female T than in L. By contrast, no significant change was observed in male HCC patients. Compared to L, the expression of XIST in T was also significantly downregulated. Female patients with higher XIST in HCC had a higher overall survival (OS) and more extended recurrence-free survival (RFS). Moreover, TET2 can interact with YY1 binding to the promoter region of XIST and maintain the hypomethylation state of XIST. In addition, DEN-administered Tet2-/- mice developed more tumours than controls in female mice. Our study provided that YY1 and TET2 could interact to form protein complexes binding to the promoter region of XIST, regulating the methylation level of XIST and then affecting the expression of XIST. This research will provide a new clue for studying sex disparities in hepatocarcinogenesis. XIST was significantly downregulated in HCC tissues and had gender disparity. Methylation levels in the XIST first exon were higher in female HCC tissues, but no significant change in male HCC patients. The TET2-YY1 complex regulate XIST expression in female hepatocytes. Other ways regulate XIST expression in male hepatocytes.