5hmC Levels Research Articles

Epigenetic regulation of myogenesis by vitamin C.

The micronutrient vitamin C is essential for the maintenance of skeletal muscle health and homeostasis. The pro-myogenic effects of vitamin C have long been attributed to its role as a general antioxidant agent, as well as its role in collagen matrix synthesis and carnitine biosynthesis. Here, we show that vitamin C also functions as an epigenetic compound, facilitating chromatin landscape transitions during myogenesis through its activity as an enzymatic cofactor for histone H3 and DNA demethylation. Utilizing C2C12 myoblast cells to investigate the epigenetic effects of vitamin C on myogenesis, we observe that treatment of cells with vitamin C decreases global H3K9 methylation and increases 5-hmC levels. Furthermore, vitamin C treatment enhances myoblast marker gene expression and myotube formation during differentiation. We identify KDM7A as a histone lysine demethylase markedly upregulated during myogenesis. Accordingly, knockdown of Kdm7a prevents the pro-myogenic effects of vitamin C. Chromatin immunoprecipitation analysis showed that KDM7A occupies the promoter region of the myogenic transcription factor MyoD1 where it facilitates histone demethylation. We also confirm that the methylcytosine dioxygenases TET1 and TET2 are required for myogenic differentiation and that their loss blunts stimulation of myogenesis by vitamin C. In conclusion, our data suggest that an epigenetic mode of action plays a major role in the myogenic effects of vitamin C.

Loss of TET Activity in the Postnatal Mouse Brain Perturbs Synaptic Gene Expression and Impairs Cognitive Function.

Conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins leads to the accumulation of 5hmC in the central nervous system; however, the role of 5hmC in the postnatal brain and how its levels and target genes are regulated by TETs remain elusive. We have generated mice that lack all three Tet genes specifically in postnatal excitatory neurons. These mice exhibit significantly reduced 5hmC levels, altered dendritic spine morphology within brain regions crucial for cognition, and substantially impaired spatial and associative memories. Transcriptome profiling combined with epigenetic mapping reveals that a subset of genes, which display changes in both 5hmC/5mC levels and expression patterns, are involved in synapse-related functions. Our findings provide insight into the role of postnatally accumulated 5hmC in the mouse brain and underscore the impact of 5hmC modification on the expression of genes essential for synapse development and function.

Genome-wide characterization of dynamic DNA 5-hydroxymethylcytosine and TET2-related DNA demethylation during breast tumorigenesis

BackgroundBreast tumorigenesis is a complex and multistep process accompanied by both genetic and epigenetic dysregulation. In contrast to the extensive studies on DNA epigenetic modifications 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in malignant breast tumors, their roles in the early phases of breast tumorigenesis remain ambiguous.ResultsDNA 5hmC and 5mC exhibited a consistent and significant decrease from usual ductal hyperplasia to atypical ductal hyperplasia and subsequently to ductal carcinoma in situ (DCIS). However, 5hmC showed a modest increase in invasive ductal breast cancer compared to DCIS. Genomic analyses showed that the changes in 5hmC and 5mC levels occurred around the transcription start sites (TSSs), and the modification levels were strongly correlated with gene expression levels. Meanwhile, it was found that differentially hydroxymethylated regions (DhMRs) and differentially methylated regions (DMRs) were overlapped in the early phases and accompanied by the enrichment of active histone marks. In addition, TET2-related DNA demethylation was found to be involved in breast tumorigenesis, and four transcription factor binding sites (TFs: ESR1, FOXA1, GATA3, FOS) were enriched in TET2-related DhMRs/DMRs. Intriguingly, we also identified a certain number of common DhMRs between tumor samples and cell-free DNA (cfDNA).ConclusionsOur study reveals that dynamic changes in DNA 5hmC and 5mC play a vital role in propelling breast tumorigenesis. Both TFs and active histone marks are involved in TET2-related DNA demethylation. Concurrent changes in 5hmC signals in primary breast tumors and cfDNA may play a promising role in breast cancer screening.

Integrated analyses of 5 mC, 5hmC methylation and gene expression reveal pathology-associated AKT3 gene and potential biomarkers for Alzheimer's disease

Aims5 mC methylation and hydroxymethylation (5hmC) are associated with Alzheimer's disease (AD). However, previous studies were limited by the absence of a 5hmC calculation. This study aims to find AD associated predictors and potential therapeutic chemicals using bioinformatics approach integrating 5 mC, 5hmC, and expression changes, and an AD mouse model. MethodsGene expression microarray and 5 mC and 5hmC sequencing datasets were downloaded from GEO repository. 142 AD and 52 normal entorhinal cortex specimens were enrolled. Data from oxidative bisulfite sequencing (oxBS)-treated samples, which represent only 5 mC, were used to calculate 5hmC level. Functional analyses, random forest supervised classification and methylation validation were applied. Potential chemicals were predicted by CMap. Morris water maze, Y maze and novel object recognition behavior tests were performed using FAD4T AD mice model. Cortex and hippocampus tissues were isolated for immunohistochemical staining. ResultsC1QTNF5, UBD, ZFP106, NEDD1, AKT3, and MBP genes involving 13 promoter CpG sites with 5mc, 5hmC methylation and expression difference were identified. AKT3 and MBP were down-regulated in both patients and mouse model. Three CpG sites in AKT3 and MBP showed significant methylation difference on validation. FAD4T AD mice showed recession in brain functions and lower AKT3 expression in both cortex and hippocampus. Ten chemicals were predicted as potential treatments for AD. ConclusionsAKT3 and MBP may be associated with AD pathology and could serve as biomarkers. The ten predicted chemicals might offer new therapeutic approaches. Our findings could contribute to identifying novel markers and advancing the understanding of AD mechanisms.

Tet1-mediated 5hmC regulates hippocampal neuroinflammation via wnt signaling as a novel mechanism in obstructive sleep apnoea leads to cognitive deficit

BackgroundObstructive sleep apnoea (OSA) is a sleep-disordered breathing characterized by intermittent hypoxia (IH) that may cause cognitive dysfunction. However, the impact of IH on molecular processes involved in cognitive function remains unclear.MethodsC57BL / 6 J mice were exposed to either normoxia (control) or IH for 6 weeks. DNA hydroxymethylation was quantified by hydroxymethylated DNA immunoprecipitation (hMeDIP) sequencing. ten-eleven translocation 1 (Tet1) was knocked down by lentivirus. Specifically, cognitive function was assessed by behavioral experiments, pathological features were assessed by HE staining, the hippocampal DNA hydroxymethylation was examined by DNA dot blot and immunohistochemical staining, while the Wnt signaling pathway and its downstream effects were studied using qRT-PCR, immunofluorescence staining, and Luminex liquid suspension chip analysis.ResultsIH mice showed pathological changes and cognitive dysfunction in the hippocampus. Compared with the control group, IH mice exhibited global DNA hydroxylmethylation in the hippocampus, and the expression of three hydroxylmethylases increased significantly. The Wnt signaling pathway was activated, and the mRNA and 5hmC levels of Wnt3a, Ccnd2, and Prickle2 were significantly up-regulated. Further caused downstream neurogenesis abnormalities and neuroinflammatory activation, manifested as increased expression of IBA1 (a marker of microglia), GFAP (a marker of astrocytes), and DCX (a marker of immature neurons), as well as a range of inflammatory cytokines (e.g. TNFa, IL3, IL9, and IL17A). After Tet1 knocked down, the above indicators return to normal.ConclusionActivation of Wnt signaling pathway by hippocampal Tet1 is associated with cognitive dysfunction induced by IH.Graphical

Alterations in DNA 5-hydroxymethylation patterns in the hippocampus of an experimental model of chronic epilepsy

Temporal lobe epilepsy (TLE) is a type of focal epilepsy characterized by spontaneous recurrent seizures originating from the hippocampus. The epigenetic reprogramming hypothesis of epileptogenesis suggests that the development of TLE is associated with alterations in gene transcription changes resulting in a hyperexcitable network in TLE. DNA 5-methylcytosine (5-mC) is an epigenetic mechanism that has been associated with chronic epilepsy. However, the contribution of 5-hydroxymethylcytosine (5-hmC), a product of 5-mC demethylation by the Ten-Eleven Translocation (TET) family proteins in chronic TLE is poorly understood. 5-hmC is abundant in the brain and acts as a stable epigenetic mark altering gene expression through several mechanisms. Here, we found that the levels of bulk DNA 5-hmC but not 5-mC were significantly reduced in the hippocampus of human TLE patients and in the kainic acid (KA) TLE rat model. Using 5-hmC hMeDIP-sequencing, we characterized 5-hmC distribution across the genome and found bidirectional regulation of 5-hmC at intergenic regions within gene bodies. We found that hypohydroxymethylated 5-hmC intergenic regions were associated with several epilepsy-related genes, including Gal, SV2, and Kcnj11 and hyperdroxymethylation 5-hmC intergenic regions were associated with Gad65, TLR4, and Bdnf gene expression. Mechanistically, Tet1 knockdown in the hippocampus was sufficient to decrease 5-hmC levels and increase seizure susceptibility following KA administration. In contrast, Tet1 overexpression in the hippocampus resulted in increased 5-hmC levels associated with improved seizure resiliency in response to KA. These findings suggest an important role for 5-hmC as an epigenetic regulator of epilepsy that can be manipulated to influence seizure outcomes.

71 Exploring DNA-methylation and DNA-hydroxymethylation in clear cell renal cell carcinoma using Dirichlet multinomial regression

Abstract Background DNA methylation alterations have been found related to clear cell renal cell carcinoma (ccRCC). In addition, DNA methylation has been used to identify cell heterogeneity at the tumor level using cell deconvolution methods. Other cytosine modifications are less studied in the field. In this study, we aim to explore the use of compositional method, Dirichlet multinomial regression1, to simultaneously explore DNA methylation and DNA hydroxymethylation in ccRCC. Methods A total of 243 clear cell renal cell carcinoma (ccRCC) samples were collected from the Dartmouth Renal Tumor Biobank. DNA methylation analysis was conducted using the Infinium MethylationEPIC BeadChip, using tandem bisulfite (BS) and oxidative bisulfite (oxBS) treatments to differentiate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) states. Beta values were calculated for all the arrayed samples. Quality control was performed using the ENmix pipeline to ensure the inclusion of high-quality samples using a stringent pOOBHA <0.05. The oxBS maximum likelihood estimate (oxBS-MLE) method was applied to assess 5mC and 5hmC levels based on paired BS and oxBS datasets. To determine the inclusion threshold for 5hmC beta values in the Dirichlet analysis, the R package oxBSCut was used to mask values close to zero2. Missing 5hmC values and those values masked below the detection limit were imputed using a compositional approach and a Tobit regression model to retain the model properties. The Dirichlet model was applied to analyze three methylation states: unmethylated, methylated, and hydroxymethylated, providing comprehensive insights into the methylation landscape of ccRCC samples. Results Here, we analyzed the top 1% most variable methylated sites (n=3020). When comparing tumor vs normal adjacent samples we tested the cytosine modifications in the same model adjusting for sex, age at diagnosis, tumor stage, and grade. For this exploratory analysis, we selected those results that passed an FDR<0.01 and an absolute difference >0.2 for further exploration. Four sites were hyperhydroxymethylated related to MYO5A, DLG2, ANKRD33B, and one open sea site. A total of 23 sites were significantly hypermethylated. Using eForge-TF, these sites tracked to transcription factors targeting estrogen receptors, retinoic acid receptors alpha and gamma, and FOXC1 among others3. Conclusions In this preliminary analysis, using a new modeling approach to interrogate cytosine modifications simultaneously, we found some intriguing results consistent with previous literature. We will expand this analysis to evaluate the relation between extreme changes in hydroxymethylation in particular in relation to alternative splicing alterations. Reference 1. Tsagris, M., Stewart, C. A Dirichlet Regression Model for Compositional Data with Zeros. Lobachevskii J Math39, 398–412 (2018). https://doi.org/10.1134/S1995080218030198 2. Zhang Z, Lee MK, Perreard L, Kelsey KT, Christensen BC, Salas LA. Navigating the hydroxymethylome: experimental biases and quality control tools for the tandem bisulfite and oxidative bisulfite Illumina microarrays. Epigenomics. 2022;14(3):139-152. doi:10.2217/epi-2021-0490 3. Charles E Breeze, Alex P Reynolds, Jenny van Dongen, Ian Dunham, John Lazar, Shane Neph, Jeff Vierstra, Guillaume Bourque, Andrew E Teschendorff, John A Stamatoyannopoulos, Stephan Beck, eFORGE v2.0: updated analysis of cell type-specific signal in epigenomic data, Bioinformatics, Volume 35, Issue 22, November 2019, Pages 4767–4769, https://doi.org/10.1093/bioinformatics/btz456

TET3 downregulation and low 5-hydroxymethylcytosine are epigenetic signatures of head and neck carcinoma.

Ten-eleven translocases (TETs) are enzymes responsible for demethylation processes, playing a crucial role in maintaining the body's methylation balance. Dysregulation of TET expression can lead to abnormal methylation levels. Isocitrate dehydrogenases (IDH) are upstream genes involved in Kreb cycle responsible for production of α-ketoglutarate (α-KG). α-KG and vitamin C are cofactors of TET3 enzyme. There is limited data on the relationship between TET3 and its cofactor Vitamin C in head and neck carcinoma (H&NC). In this study, we have investigated the expression of the TET3 gene along with IDH1/2 genes involved in the Krebs cycle in the peripheral blood of 32H&NC patients compared to 32 healthy controls. We estimated serum levels of TET3 protein and vitamin C and 5-hydroxymethylcytosine (5-hmC) percentage in DNA isolated from EDTA blood samples. Our findings revealed that TET3 and IDH1/2 were downregulated in H&NC patients compared to healthy controls. Serum levels of TET3 and Vitamin C were low in H&NC patients compared to healthy controls. Diminished levels of percentage 5-hmC were detected in EDTA blood samples of H&NC patients compared to controls. Spearman correlation analysis revealed a significant positive correlation between TET3 levels, vitamin C levels and 5-hmC percentage. The low levels of Vitamin C are believed to contribute to decreased activity of the TET3 gene and less conversion of 5-methylcytosine (5-mC) to 5-hmC. Dietary supplementation of Vitamin C may increase TET3 activity.

Maternal high-fat diet alters Tet-mediated epigenetic regulation during heart development

Imbalanced dietary intake, such as a high-fat diet (HFD) during pregnancy, has been associated with adverse offspring outcomes. Metabolic stress from imbalanced food intake alters the function of epigenetic regulators, resulting in abnormal transcriptional outputs in embryos to cause congenital disorders. We report herein that maternal HFD exposure causes metabolic changes in pregnant mice and non-compaction cardiomyopathy (NCC) in E15.5 embryos, accompanied by decreased 5-hydroxymethylcytosine (5hmC) levels and altered chromatin accessibility in embryonic heart tissues. Remarkably, maternal vitamin C supplementation mitigates these detrimental effects, likely by restoring iron, a cofactor for Tet enzymes, in a reduced state. Using a genetic approach, we further demonstrated that the cardioprotective benefits of vitamin C under HFD conditions are attributable to enhanced Tet activity. Our results highlight an interaction between maternal diet, specifically HFD or vitamin C, and epigenetic modifications during early heart development, emphasizing the importance of balanced maternal nutrition for healthy embryonic development.

Selective Ligase-Based Sample Processing-Free Discrimination and Detection of Site-Specific DNA 5-Hydroxymethylcytosine.

Accurate detection of site-specific 5-hydroxymethylcytosine (5hmC) in genomic DNA is of great significance, but it is technically challenging to directly distinguish very low levels of 5hmC from their abundant cytosine/5-methylcytosine (C/5mC) analogues. Herein, we wish to propose a selective ligase-mediated mechanism (SLim) that can directly discriminate 5hmC from C/5mC with a high specificity without the use of any sample processing protocol. In this new design, we discovered that HiFi Taq DNA Ligase can well tolerate the mismatched 5hmC/A base-pairing and then effectively ligate the associated nicking site while the mismatched 5mC/A or C/A pairs cannot be recognized by HiFi Taq DNA Ligase, providing a new way for direct and selective discriminating 5hmC from its similar analogues. Ultrasensitive and selective quantification of site-specific 5hmC is realized by coupling the SLim with polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP).

DNA methylation, but not microRNA expression, is affected by in vitro THC exposure in bovine granulosa cells

BackgroundA global increase in cannabis use has led to questions about its effects on fertility. The rise in consumption amongst women of reproductive age is a growing concern, as this group is vulnerable in terms of reproductive health. Ample evidence suggests that the psychoactive component of cannabis, Δ9-Tetrahydrocannabinol (THC), interacts with the endocannabinoid system (ECS), that helps regulate mammalian reproduction. This study aimed to research the epigenetic effects of THC in bovine granulosa cells (GCs) by (1) investigating global DNA methylation via measuring 5-mC and 5-hmC levels; (2) measuring key methylation regulators, including the methylating enzymes DNMT1, DNMT3a, DNMT3b and the demethylases TDG and TET1/2/3; and (3) assessing fertility-associated miRNAs key in developmental competency, including miR-21, -155, -33b, -324 and -346.MethodsBovine GCs were used as a translational model for reproductive toxicity in humans. To determine THC effects, GCs were isolated from Cumulus-Oocyte-Complexes (COCs) from bovine ovaries, cultured in vitro for 7 days, or until confluent, and cryopreserved at passage 1 (P1). For experimentation, cells were thawed, cultured until passage 2 (P2), serum restricted for 24-h and treated for 24-h in one of five groups: control, vehicle (1:1:18 ethanol: tween: saline) and three clinically relevant THC doses (0.032, 0.32 and 3.2 μM). Global methylation was assessed by measuring 5-mC and 5-hmC levels with flow cytometry. To assess mRNA and protein expression of methylation regulators and miRNA profiles, qPCR and Western Blotting were utilized. Shapiro-Wilk test was used to determine normality within datasets. One-way ANOVA was applied to determine statistical significance using GraphPad Prism 6.0.0.ResultsResults indicate a significant decrease (p = 0.0435) in 5-mC levels following low THC exposure, while no changes were observed in 5-hmC levels. A significant increase in DNMT1 following high THC exposure at the RNA level (p < 0.05) and a significant increase following low THC exposure at the protein level (p = 0.0048) were also observed. No significant differences were observed in DNMT3a/3b, TDG, TET1/2/3 mRNAs or in any of the miRNAs analyzed.ConclusionsThis research suggests that THC mainly affects DNA methylation, but not miRNA profiles, ultimately altering gene expression and likely impairing oocyte competence, maturation, and fertilization potential.

Decoding the Epigenetic Landscape: Insights into 5mC and 5hmC Patterns in Mouse Cortical Cell Types.

The DNA modifications, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), represent powerful epigenetic regulators of temporal and spatial gene expression. Yet, how the cooperation of these genome-wide, epigenetic marks determine unique transcriptional signatures across different brain cell populations is unclear. Here we applied Nanopore sequencing of native DNA to obtain a complete, genome-wide, single-base resolution atlas of 5mC and 5hmC modifications in neurons, astrocytes and microglia in the mouse cortex (99% genome coverage, 40 million CpG sites). In tandem with RNA sequencing, analysis of 5mC and 5hmC patterns across cell types reveals astrocytes drive uniquely high brain 5hmC levels and support two decades of research regarding methylation patterns, gene expression and alternative splicing, benchmarking this resource. As such, we provide the most comprehensive DNA methylation data in mouse brain as an interactive, online tool (NAM-Me, https://olsenlab.shinyapps.io/NAMME/) to serve as a resource dataset for those interested in the methylome landscape.

TET2 regulates extranodal NK/T cell lymphoma progression through regulation of DNA methylation.

The biological role of Ten-11 translocation 2 (TET2) and the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in the development of extra-nodal natural killer/T-cell lymphoma (ENKTL) remains unclear. The level of 5mC and 5hmC was detected in 112 cases of ENKTL tissue specimens by immunohistochemical (IHC) staining. Subsequently, TET2 knockdown and the overexpression cell models were constructed in ENKTL cell lines. Biochemical analyses were used to assess proliferation, apoptosis, cell cycle and monoclonal formation in cells treated or untreated with L-Ascorbic acid sodium salt (LAASS). Dot-Blots were used to detect levels of genome 5mC and 5hmC. Additionally, the ILLUMINA 850k methylation chip was used to analyze the changes of TET2 regulatory genes. RNA-Seq was used to profile differentially expressed genes regulated by TET2. The global level of 5hmC was significantly decreased, while 5mC was highly expressed in ENKTL tissue. TET2 protein expression was negatively correlated with the ratio of 5mC/5hmC (p<0.0001). The 5mC/5hmC status were related to the site of disease, clinical stage, PINK score and Ki-67 index, as well as the 5-year OS. TET2 knockdown prolonged the DNA synthesis period, increased the cloning ability of tumor cells, increased the level of 5mC and decreased the level of 5hmC in ENKTL cells. While overexpression of TET2 presented the opposite effect. Furthermore, treatment of ENKTL cells with LAASS significantly induced ENKTL cell apoptosis. These results suggest that TET2 plays an important role in ENKTL development via regulation of 5mC and 5hmC and may serve as a novel therapeutic target for ENKTL.

DNMT1 can induce primary germ layer differentiation through de novo DNA methylation.

DNA methyltransferases and Ten-Eleven Translocation (TET) proteins regulate the DNA methylation and demethylation cycles during mouse embryonic development. Although DNMT1 mainly plays a role in the maintenance of DNA methylation after DNA replication, it is also reported to possess de novo methyltransferase capacity. However, its physiological significance remains unclear. Here, we demonstrate that full-length DNMT1 (FL) and a mutant lacking the N-terminus necessary for its maintenance activity (602) confer the differentiation potential of mouse Dnmt1, Dnmt3a, and Dnmt3b (Dnmts-TKO) embryonic stem cells (ESCs). Both FL and 602 inhibit the spontaneous differentiation of Dnmts-TKO ESCs in the undifferentiated state. Dnmts-TKO ESCs showed loss of DNA methylation and de-repression of primitive endoderm-related genes, but these defects were partially restored in Dnmts-TKO + FL and Dnmts-TKO + 602 ESCs. Upon differentiation, Dnmts-TKO + FL ESCs show increased 5mC and 5hmC levels across chromosomes, including pericentromeric regions. In contrast, Dnmts-TKO + 602 ESCs didn't accumulate 5mC, and sister chromatids showed 5hmC asynchronously. Furthermore, in comparison with DNMT1_602, DNMT1_FL effectively promoted commitment to the epiblast-like cells and beyond, driving cell-autonomous mesendodermal and germline differentiation through embryoid body-based methods. With precise target selectivity achieved by its N-terminal region, DNMT1 may play a role in gene regulation leading to germline development.

Wnt/β-catenin-YAP axis in the pathogenesis of primary intraosseous carcinoma NOS, deriving from odontogenic keratocyst

Odontogenic tumors (OGTs), which originate from cells of odontogenic apparatus and their remnants, are rare entities. Primary intraosseous carcinoma NOS (PIOC), is one of the OGTs, but it is even rarer and has a worse prognosis. The precise characteristics of PIOC, especially in immunohistochemical features and its pathogenesis, remain unclear. We characterized a case of PIOC arising from the left mandible, in which histopathological findings showed a transition from the odontogenic keratocyst to the carcinoma. Remarkably, the tumor lesion of this PIOC prominently exhibits malignant attributes, including invasive growth of carcinoma cell infiltration into the bone tissue, an elevated Ki-67 index, and lower signal for CK13 and higher signal for CK17 compared with the non-tumor region, histopathologically and immunohistopathologically. Further immunohistochemical analyses demonstrated increased expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C) (accompanying expression of β-catenin in the nucleus) and yes-associated protein (YAP) in the tumor lesion. On the other hand, YAP was expressed and the expression of ARL4C was hardly detected in the non-tumor region. In addition, quantitative RT-PCR analysis using RNAs and dot blot analysis using genomic DNA showed the activation of Wnt/β-catenin signaling and epigenetic alterations, such as an increase of 5mC levels and a decrease of 5hmC levels, in the tumor lesion. A DNA microarray and a gene set enrichment analysis demonstrated that various types of intracellular signaling would be activated and several kinds of cellular functions would be altered in the pathogenesis of PIOC. Experiments with the GSK-3 inhibitor revealed that β-catenin pathway increased not only mRNA levels of ankyrin repeat domain1 (ANKRD1) but also protein levels of YAP and transcriptional co-activator with PDZ-binding motif (TAZ) in oral squamous cell carcinoma cell lines. These results suggested that further activation of YAP signaling by Wnt/β-catenin signaling may be associated with the pathogenesis of PIOC deriving from odontogenic keratocyst in which YAP signaling is activated.

Ultrasensitive detection of 5-hydroxymethylcytosine in genomic DNA using a graphene-based sensor modified with biotin and gold nanoparticles

Ten-eleven translocation (TET) proteins orchestrate deoxyribonucleic acid (DNA) methylation-demethylation dynamics by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and are frequently inactivated in various cancers. Due to the significance of 5hmC as an epigenetic biomarker for cancer diagnosis, pathogenesis, and treatment, its rapid and precise quantification is essential. Here, we report a highly sensitive electrochemical method for quantifying genomic 5hmC using graphene sheets that were electrochemically exfoliated and functionalized with biotin and gold nanoparticles (Bt-AuNPs) through a single-step electrical method. The attachment of Bt-AuNPs to graphene enhances the specificity of 5hmC-containing DNA and augments the oxidation of 5hmC to 5-formylcytosine in DNA. When coupled to a gold electrode, the Bt-AuNP-graphene-based sensor exhibits exceptional sensitivity and specificity for detecting 5hmC, with a detection limit of 63.2 fM. Furthermore, our sensor exhibits a remarkable capacity to measure 5hmC levels across a range of biological samples, including preclinical mouse tissues with varying 5hmC levels due to either TET gene disruption or oncogenic transformation, as well as human prostate cancer cell lines. Therefore, our sensing strategy has substantial potential for cancer diagnostics and prognosis.

Smoking-Induced DNA Hydroxymethylation Signature Is Less Pronounced than True DNA Methylation: The Population-Based KORA Fit Cohort.

Despite extensive research on 5-methylcytosine (5mC) in relation to smoking, there has been limited exploration into the interaction between smoking and 5-hydroxymethylcytosine (5hmC). In this study, total DNA methylation (5mC+5hmC), true DNA methylation (5mC) and hydroxymethylation (5hmC) levels were profiled utilizing conventional bisulphite (BS) and oxidative bisulphite (oxBS) treatment, measured with the Illumina Infinium Methylation EPIC BeadChip. An epigenome-wide association study (EWAS) of 5mC+5hmC methylation revealed a total of 38,575 differentially methylated positions (DMPs) and 2023 differentially methylated regions (DMRs) associated with current smoking, along with 82 DMPs and 76 DMRs associated with former smoking (FDR-adjusted p < 0.05). Additionally, a focused examination of 5mC identified 33 DMPs linked to current smoking and 1 DMP associated with former smoking (FDR-adjusted p < 0.05). In the 5hmC category, eight DMPs related to current smoking and two DMPs tied to former smoking were identified, each meeting a suggestive threshold (p < 1 × 10-5). The substantial number of recognized DMPs, including 5mC+5hmC (7069/38,575, 2/82), 5mC (0/33, 1/1), and 5hmC (2/8, 0/2), have not been previously reported. Our findings corroborated previously established methylation positions and revealed novel candidates linked to tobacco smoking. Moreover, the identification of hydroxymethylated CpG sites with suggestive links provides avenues for future research.

Predicting gene expression state and prioritizing putative enhancers using 5hmC signal

BackgroundLike its parent base 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) is a direct epigenetic modification of cytosines in the context of CpG dinucleotides. 5hmC is the most abundant oxidized form of 5mC, generated through the action of TET dioxygenases at gene bodies of actively-transcribed genes and at active or lineage-specific enhancers. Although such enrichments are reported for 5hmC, to date, predictive models of gene expression state or putative regulatory regions for genes using 5hmC have not been developed.ResultsHere, by using only 5hmC enrichment in genic regions and their vicinity, we develop neural network models that predict gene expression state across 49 cell types. We show that our deep neural network models distinguish high vs low expression state utilizing only 5hmC levels and these predictive models generalize to unseen cell types. Further, in order to leverage 5hmC signal in distal enhancers for expression prediction, we employ an Activity-by-Contact model and also develop a graph convolutional neural network model with both utilizing Hi-C data and 5hmC enrichment to prioritize enhancer-promoter links. These approaches identify known and novel putative enhancers for key genes in multiple immune cell subsets.ConclusionsOur work highlights the importance of 5hmC in gene regulation through proximal and distal mechanisms and provides a framework to link it to genome function. With the recent advances in 6-letter DNA sequencing by short and long-read techniques, profiling of 5mC and 5hmC may be done routinely in the near future, hence, providing a broad range of applications for the methods developed here.

Genome-wide 5-hydroxymethylation mapping and epigenetic pathways in multiple myeloma.

7566 Background: Multiple myeloma (MM), a B-cell neoplasm characterized by bone marrow infiltration of malignant plasma cells, is the second most common blood malignancy, with an estimated number of ~35,000 new patients annually in the United States. The molecular pathogenesis of MM is complex and involves various genetic and epigenetic alterations. Key molecular mechanisms, including chromosomal abnormalities such as translocations involving the immunoglobulin heavy chain locus and various oncogenes (e.g., MMSET, FGFR3, CCND1, MAF), alterations in epigenetic regulators (e.g., EZH2, DNMT3A), dysregulation of cell cycle control, and aberrant activation of signaling pathways (e.g., NF-kB, PI3K/AKT, and JAK/STAT), play critical roles in MM pathogenesis. However, epigenetic pathways implicated in MM has not been comprehensively investigated, partly due to technical limitations that cannot distinguish major cytosine modification types. Methods: Using the 5hmC-Seal, a highly sensitive chemical labeling technique, we profiled genome-wide 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA) from a population-based case-control study of MM (cases, n = 313; controls, n = 317) conducted in Canada. Results: The 5hmC modification levels were summarized for various genomic features, showing an enrichment in gene bodies and enhancer markers, consistent with the putative role of gene regulation for 5hmC modification. A genome-wide scan of gene bodies identified 771 differential features between cases and controls, adjusting for age, sex, smoking status, education, and first two principal components, at a permutation-based empirical p-value cutoff of 10-4. For instance, IL1RAP, a component of the interleukin-1 signaling cascade, may impact tumor progression and immune system evasion. Furthermore, functional analysis indicated canonical pathways associated with MM pathology and treatment, such as calcium signaling, and synthesis and secretion of cortisol/aldosterone, were enriched in the differential 5hmC features between cases and controls. Notably, the calcium signaling pathway, integral to Ca2+ transport and involved in various physiological and pathological processes, plays a critical role in MM pathogenesis. Within this pathway, the CAMK1D gene, which has been identified as a crucial regulator of tumor-intrinsic immune resistance, showed differential 5hmC level between cases and controls, highlighting a vital connection between epigenetic modifications and immune evasion mechanisms in MM. Conclusions: Leveraging a state-of-the-art technique, we identified novel epigenetic modifications and pathways in the risk of MM. This approach establishes a solid foundation for further investigating etiology of MM, deepening our understanding of the disease, and advancing the discovery of biomarkers, which could potentially guide preventive strategies.

Loci cg06256735 and cg15815843 in the MFAP5 gene regulatory regions are hypomethylated in varicose veins apparently due to active demethylation.

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril-associated protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, tunica (t.) intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.