5hmC Expression Research Articles

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<0.05, log2FC>|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<0.05, delta>|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<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.

Abstract 6825: Epigenetic dysregulation in well-differentiated pancreatic neuroendocrine tumor (PNET): An immunohistochemistry (IHC) analysis

Abstract Epigenetic dysregulation through DNA Methyltransferase 1 (DNMT1) is integral to pancreatic neuroendocrine tumorigenesis. We aim to investigate the interplay between epigenetic modifiers and pathogenetic markers in the PNET microenvironment. 12 paraffin-embedded PNET specimens and 12 benign adjacent tissue specimens, used as controls, were obtained from Cooper University Hospital Pathology Department (IRB 23-130). IHC was performed using epigenetic markers, including DNMT1, DNMT3A, DNMT3B, and 5-Hydroxymethylcytosine (5-HMC), and pathogenic markers, including Multiple Endocrine Neoplasia 1 (MEN1), Protein-Tyrosine Phosphatase (PTEN), and Ras Association Domain Family 1 (RASSF1). Image J software was utilized for IHC scoring under the guidance of a board-certified pathologist. DNMT1 was upregulated in PNET tumor specimens with a mean density score of 72.02 +/- 31 versus 16.02 +/- 17 in adjacent benign tissue (p<.001). Grade 2 tumors displayed increased DNMT1 expression compared to Grade 1 tumors with a mean score of 37 +/-21 versus 71 +/- 23 (p<.05). Decreased expression of 5-HMC was noted in tumor specimens in comparison to benign tissue with a mean score of 3.92 +/- 5.05 versus 19.37 +/- 5.05 (p<.05). Loss of 5-HMC was observed in higher tumor grades with a mean score of .79 +/- 2 versus 8.2+/-2 (p<.01). DNMT3B was upregulated in PNET tumor specimens with a mean score of 13.16 +/- 3.94 versus 7.02+/- 2.553 (p<.01) in adjacent benign tissue and was positively correlated to DNMT1 expression. DNMT3A was not found to have a difference in expression between tumor and benign tissue. MEN1 was downregulated in PNET, with 45% of tumor specimens displaying loss of MEN1 expression. Mean MEN1 density in tumor tissue was 4.47 +/- 5.95 versus 11.41 +/- 3.53 in adjacent benign (p<.01). Increased expression of DNMT1 found to negatively correlate with MEN1 expression (R2=.23). Loss of RASSF1 expression was present in 70% of tumor specimens with a mean score of 1.32 +/- 1.99 versus 9.50+/- 6.89 (p<.001) in adjacent benign tissue. RASSF1 expression correlated negatively with DNMT1 (R2=.21). PTEN expression showed intratumoral homogeneity to benign tissue, and its expression was not correlated with any epigenetic markers. Our work provides a framework of epigenetic regulators involved in the pathobiology of PNET, and can refine prognostic evaluation while providing therapeutic benefits for patients with PNET. Citation Format: Zena Saleh, Yazid Ghanem, Hansa Joshi, Mathew C. Moccia, Helen Toma, Yahui Li, Upsana Joneja, Tao Gao, Young Hong. Epigenetic dysregulation in well-differentiated pancreatic neuroendocrine tumor (PNET): An immunohistochemistry (IHC) analysis [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 6825.

Epigenetic landscape of 5-hydroxymethylcytosine and associations with gene expression in placenta

ABSTRACT 5-hydroxymethylcystosine (5hmC), is an intermediate product in the DNA demethylation pathway, but may act as a functional epigenetic modification. We have conducted the largest study of site-specific 5hmC in placenta to date using parallel bisulphite and oxidative bisulphite modification with array-based assessment. Incorporating parallel RNA-sequencing data allowed us to assess associations between 5hmC and gene expression, using expression quantitative trait hydroxymethylation (eQTHM) analysis. We identified ~ 47,000 loci with consistently elevated (systematic) 5hmC proportions. Systematic 5hmC was significantly depleted (p < 0.0001) at CpG islands (CGI), and enriched (p < 0.0001) in ‘open sea’ regions (CpG >4 kb from CGI). 5hmC was most and least abundant at CpGs in enhancers and active transcription start sites (TSS), respectively (p < 0.05). We identified 499 significant (empirical-p <0.05) eQTHMs within 1 MB of the assayed gene. At most (75.4%) eQTHMs, the proportion of 5hmC was positively correlated with transcript abundance. eQTHMs were significantly enriched among enhancer CpGs and depleted among CpGs in active TSS (p < 0.05 for both). Finally, we identified 107 differentially hydroxymethylated regions (DHMRs, p < 0.05) across 100 genes. Our study provides insight into placental distribution of 5hmC, and sheds light on the functional capacity of this epigenetic modification in placenta.

Comparison of TERT and 5-Hydroxymethylcytocine immunohistochemistry in various thyroid carcinomas

Telomerase reverse transcriptase (TERT) promoter mutation is associated with an aggressive clinical course in thyroid carcinomas. Therefore, detection of TERT promoter mutation is essential for proper patient management. 5-Hydroxymethylcytosine (5hmC) is an epigenetic marker involved in the DNA demethylation pathway, and its loss has been observed in various tumors. Loss of 5hmC has also been reported in thyroid carcinomas and is presented as a possible predictive biomarker for TERT promoter mutation and worse prognosis. This study evaluated the expression of TERT and 5hmC by immunohistochemistry (IHC) in 105 patients (44 in the TERT mutant group and 61 in the TERT wild group) with various thyroid carcinomas. H-scores were calculated using an image analyzer. The median H-scores of TERT IHC were significantly higher in the TERT mutant group than in the TERT wild group (47.15 vs. 9.80). The sensitivity and specificity of TERT IHC for predicting TERT promoter mutations were 65.9 and 65.7 %, respectively. Regardless of TERT promoter mutation status, the 5hmC H-scores were markedly lower in all subtypes of thyroid carcinomas compared to those in their normal counterparts. Significant differences in 5hmC H-scores were observed between N0 and N1 in total thyroid carcinomas, but not within the papillary thyroid carcinoma subgroup. In conclusion, TERT and 5hmC IHC have limitations in predicting the presence of TERT promoter mutations. The expression of 5hmC was downregulated in various thyroid carcinomas compared to that in normal and benign lesions, but comprehensive further studies are required to elucidate the role of 5hmC in thyroid carcinomas.

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.

Distinctive Patterns of 5-Methylcytosine and 5-Hydroxymethylcytosine in Schizophrenia.

Schizophrenia is a highly heritable neuropsychiatric disorder characterized by cognitive and social dysfunction. Genetic, epigenetic, and environmental factors are together implicated in the pathogenesis and development of schizophrenia. DNA methylation, 5-methycytosine (5mC) and 5-hydroxylcytosine (5hmC) have been recognized as key epigenetic elements in neurodevelopment, ageing, and neurodegenerative diseases. Recently, distinctive 5mC and 5hmC pattern and expression changes of related genes have been discovered in schizophrenia. Antipsychotic drugs that affect 5mC status can alleviate symptoms in patients with schizophrenia, suggesting a critical role for DNA methylation in the pathogenesis of schizophrenia. Further exploring the signatures of 5mC and 5hmC in schizophrenia and developing precision-targeted epigenetic drugs based on this will provide new insights into the diagnosis and treatment of schizophrenia.

Alterations of the expression of TET2 and DNA 5-hmC predict poor prognosis in Myelodysplastic Neoplasms

BackgroundMyelodysplastic Neoplasms (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis and progression to acute myeloid leukemia, myelodysplasia-related (AML-MR). A major mechanism of pathogenesis of MDS is the aberration of the epigenetic landscape of the hematopoietic stem cells and/or progenitor cells, especially DNA cytosine methylation, and demethylation. Data on TET2, the predominant DNA demethylator of the hematopoietic system, is limited, particularly in the MDS patients from India, whose biology may differ since these patients present at a relatively younger age. We studied the expression and the variants of TET2 in Indian MDS and AML-MR patients and their effects on 5-hydroxymethyl cytosine (5-hmC, a product of TET2 catalysis) and on the prognosis of MDS patients.ResultsOf the 42 MDS patients, cytogenetics was available for 31 sub-categorized according to the Revised International Prognostic Scoring System (IPSS-R). Their age resembled that of the previous studies from India. Bone marrow nucleated cells (BMNCs) were also obtained from 13 patients with AML-MR, 26 patients with de-novo AML, and 11 subjects with morphologically normal bone marrow. The patients had a significantly lower TET2 expression which was more pronounced in AML-MR and the IPSS-R higher-risk MDS categories. The 5-hmC levels in higher-risk MDS and AML-MR correlated with TET2 expression, suggesting a possible mechanistic role in the loss of TET2 expression. The findings on TET2 and 5-hmC were also confirmed at the tissue level using immunohistochemistry. Pathogenic variants of TET2 were found in 7 of 24 patient samples (29%), spanning across the IPSS-R prognostic categories. One of the variants – H1778R – was found to affect local and global TET2 structure when studied using structural predictions and molecular dynamics simulations. Thus, it is plausible that some pathogenic variants in TET2 can compromise the structure of TET2 and hence in the formation of 5-hmC.ConclusionsIPSS-R higher-risk MDS categories and AML-MR showed a reduction in TET2 expression, which was not apparent in lower-risk MDS. DNA 5-hmC levels followed a similar pattern. Overall, a decreased TET2 expression and a low DNA 5-hmC level are predictors of advanced disease and adverse outcome in MDS in the population studied, i.e., MDS patients from India.

Targeting SIRT4/TET2 Signaling Alleviates Human Keratinocyte Senescence by Reducing 5-hydroxymethylcytosine Loss

Skin aging is characterized by wrinkle formation and increased frailty and laxity, leading to the risk of age-related skin diseases. Keratinocyte is an important component of the epidermis in skin structure, and keratinocyte senescence has been identified as a pivotal factor in skin aging development. Because epigenetic pathways play a vital role in the regulation of skin aging, we evaluated human skin samples for DNA hydroxymethylation (5-hydroxymethylcytosine; 5-hmC) and SIRT4 expressions. Results found that both 5-hmC and SIRT4 showed a significant decrease in aged human skin samples. To test the results in vitro, human keratinocytes were cultured in H2O2, which modulates skin aging in vivo. However, H2O2-induced keratinocytes showed senescence-associated protein expression and significant downregulation of 5-hmC and SIRT4 expressions. Moreover, 5-hmC-converting enzymes ten eleven translocation 2 (TET2) showed a decrease and enhanced TET2 acetylation level in H2O2-induced keratinocytes. However, the overexpression of SIRT4 in keratinocytes alleviates the senescence phenotype, such as senescence-associated protein expression, decreases the TET2 acetylation, but increases TET2 and 5-hmC expressions. Our results provide a novel relevant mechanism whereby the epigenetic regulation of keratinocytes in skin aging may be correlated with SIRT4 expression and TET2 acetylation in 5-hmC alteration. Our study may provide a potential strategy for antiskin aging, which targets the SIRT4/TET2 axis involving epigenetic modification in keratinocyte senescence.

Standardized Computer-Assisted Analysis of 5-hmC Immunoreactivity in Dysplastic Nevi and Superficial Spreading Melanomas.

5-Hydroxymethylcytosine (5-hmC) is an important intermediate of DNA demethylation. Hypomethylation of DNA is frequent in cancer, resulting in deregulation of 5-hmC levels in melanoma. However, the interpretation of the intensity and distribution of 5-hmC immunoreactivity is not very standardized, which makes its interpretation difficult. In this study, 5-hmC-stained histological slides of superficial spreading melanomas (SSM) and dysplastic compound nevi (DN) were digitized and analyzed using the digital pathology and image platform QuPath. Receiver operating characteristic/area under the curve (ROCAUC) and t-tests were performed. A p-value of <0.05 was used for statistical significance, and a ROCAUC score of >0.8 was considered a "good" result. In total, 92 5-hmC-stained specimens were analyzed, including 42 SSM (45.7%) and 50 DN (54.3%). The mean of 5-hmC-positive cells/mm2 for the epidermis and dermo-epidermal junction and the entire lesion differed significantly between DN and SSM (p = 0.002 and p = 0.006, respectively) and showed a trend towards higher immunoreactivity in the dermal component (p = 0.069). The ROCAUC of 5-hmC-positive cells of the epidermis and dermo-epidermal junction was 0.79, for the dermis 0.74, and for the entire lesion 0.76. These results show that the assessment of the epidermal with junctional expression of 5-hmC is slightly superior to dermal immunoreactivity in distinguishing between DN and SSM.

Dissection of transcriptomic and epigenetic heterogeneity of grade 4 gliomas: implications for prognosis

BackgroundGrade 4 glioma is the most aggressive and currently incurable brain tumor with a median survival of one year in adult patients. Elucidating novel transcriptomic and epigenetic contributors to the molecular heterogeneity underlying its aggressiveness may lead to improved clinical outcomes.MethodsTo identify grade 4 glioma -associated 5-hydroxymethylcytosine (5hmC) and transcriptomic features as well as their cross-talks, genome-wide 5hmC and transcriptomic profiles of tissue samples from 61 patients with grade 4 gliomas and 9 normal controls were obtained for differential and co-regulation/co-modification analyses. Prognostic models on overall survival based on transcriptomic features and the 5hmC modifications summarized over genic regions (promoters, gene bodies) and brain-derived histone marks were developed using machine learning algorithms.ResultsDespite global reduction, the majority of differential 5hmC features showed higher modification levels in grade 4 gliomas as compared to normal controls. In addition, the bi-directional correlations between 5hmC modifications over promoter regions or gene bodies and gene expression were greatly disturbed in grade 4 gliomas regardless of IDH1 mutation status. Phenotype-associated co-regulated 5hmC–5hmC modules and 5hmC–mRNA modules not only are enriched with different molecular pathways that are indicative of the pathogenesis of grade 4 gliomas, but also are of prognostic significance comparable to IDH1 mutation status. Lastly, the best-performing 5hmC model can predict patient survival at a much higher accuracy (c-index = 74%) when compared to conventional prognostic factor IDH1 (c-index = 57%), capturing the molecular characteristics of tumors that are independent of IDH1 mutation status and gene expression-based molecular subtypes.ConclusionsThe 5hmC-based prognostic model could offer a robust tool to predict survival in patients with grade 4 gliomas, potentially outperforming existing prognostic factors such as IDH1 mutations. The crosstalk between 5hmC and gene expression revealed another layer of complexity underlying the molecular heterogeneity in grade 4 gliomas, offering opportunities for identifying novel therapeutic targets.

Role of TET1-mediated epigenetic modulation in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder influenced by a complex interplay of environmental, epigenetic, and genetic factors. DNA methylation (5mC) and hydroxymethylation (5hmC) are DNA modifications that serve as tissue-specific and temporal regulators of gene expression. TET family enzymes dynamically regulate these epigenetic modifications in response to environmental conditions, connecting environmental factors with gene expression. Previous epigenetic studies have identified 5mC and 5hmC changes associated with AD. In this study, we performed targeted resequencing of TET1 on a cohort of early-onset AD (EOAD) and control samples. Through gene-wise burden analysis, we observed significant enrichment of rare TET1 variants associated with AD (p = 0.04). We also profiled 5hmC in human postmortem brain tissues from AD and control groups. Our analysis identified differentially hydroxymethylated regions (DhMRs) in key genes responsible for regulating the methylome: TET3, DNMT3L, DNMT3A, and MECP2. To further investigate the role of Tet1 in AD pathogenesis, we used the 5xFAD mouse model with a Tet1 KO allele to examine how Tet1 loss influences AD pathogenesis. We observed significant changes in neuropathology, 5hmC, and RNA expression associated with Tet1 loss, while the behavioral alterations were not significant. The loss of Tet1 significantly increased amyloid plaque burden in the 5xFAD mouse (p = 0.044) and lead to a non-significant trend towards exacerbated AD-associated stress response in 5xFAD mice. At the molecular level, we found significant DhMRs enriched in genes involved in pathways responsible for neuronal projection organization, dendritic spine development and organization, and myelin assembly. RNA-Seq analysis revealed a significant increase in the expression of AD-associated genes such as Mpeg1, Ctsd, and Trem2. In conclusion, our results suggest that TET enzymes, particularly TET1, which regulate the methylome, may contribute to AD pathogenesis, as the loss of TET function increases AD-associated pathology.

Identification and Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine on Random DNA Sequences by a Nanoconfined Electrochemiluminescence Platform.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are two of the most abundant epigenetic marks in mammalian genomes, and it has been proven that these dual epigenetic marks give a more accurate prediction of recurrence and survival in cancer than the individual mark. However, due to the similar structure and low expression of 5mC and 5hmC, it is challenging to distinguish and quantify the two methylation modifications. Herein, we employed the ten-eleven translocation family dioxygenases (TET) to convert 5mC to 5hmC via a specific labeling process, which realized the identification of the two marks based on a nanoconfined electrochemiluminescence (ECL) platform combined with the amplification strategy of a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system. Benefiting from the TET-mediated conversion strategy, a highly consistent labeling pathway was developed for identifying dual epigenetic marks on random sequence, which reduced the system error effectively. The ECL platform was established via preparing a carbonized polymer dot embedded SiO2 nanonetwork (CPDs@SiO2), which exhibited higher ECL efficiencies and more stable ECL performance compared to those of the scattered emitters due to the nanoconfinement-enhanced ECL effect. The proposed bioanalysis strategy could be employed for the identification and quantification of 5mC and 5hmC in the range from 100 aM to 100 pM, respectively, which provides a promising tool for early diagnosis of diseases associated with abnormal methylation.

Decrease of 5-hydroxymethylcytosine in hepatitis B virus-related hepatocellular carcinoma: A cross-sectional study.

Many epigenetic studies had found the decrease of 5-hydroxymethylcytosine (5-hmC) in various tumor tissues. However, limited information is available for hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC). The present study aimd to investigate whether the decrease also existed in tumor tissues of HBV-related HCC and, if possible, to disclose its mechanism. We used immunohistochemistry and Image Pro Plus 6.0 Image Analysis Software to quantify the expression of 5-hmC, 5-methylcytosine, 10-eleven translocation (TET), isocitrate dehydrogenase (IDH) in pathological sections of tumor tissues and its para cancerous tissues of 40 HBV-related HCC patients. Our results showed that 5-hmC was decreased while 5-methylcytosine was increased in tumor tissues. We also detected TET1 and IDH2 were decreased in the tumor tissues and the decrease were positively correlated with the 5-hmC. The results suggested that the deficiency of 5-hmC was an epigenetic characteristic of HBV-related HCC and was mainly caused by the decrease of TET1 and IDH2.

The Tet2-Upf1 complex modulates mRNA stability under stress conditions.

Introduction: Environmental stress promotes epigenetic alterations that impact gene expression and subsequently participate in the pathological processes of the disorder. Among epigenetic regulations, ten-eleven Translocation (Tet) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA and RNA and function as critical players in the pathogenesis of diseases. Our previous results showed that chronic stress increases the expression of cytoplasmic Tet2 in the hippocampus of mice exposed to chronic mild stress (CMS). Whether the cytoplasmic Tet2 alters RNA 5hmC modification in chronic stress-related processes remains largely unknown. Methods: To explore the role of cytoplasmic Tet2 under CMS conditions, we established CMS mice model and detected the expression of RNA 5hmC by dot blot. We verified the interaction of Tet2 and its interacting protein by co-immunoprecipitation combined with mass spectrometry and screened downstream target genes by cluster analysis of Tet2 and upstream frameshift 1 (Upf1) interacting RNA. The expression of protein was detected by Western blot and the expression of the screened target genes was detected by qRT-PCR. Results: In this study, we found that increased cytoplasmic Tet2 expression under CMS conditions leads to increase in total RNA 5hmC modification. Tet2 interacted with the key non-sense-mediated mRNA decay (NMD) factor Upf1, regulated the stability of stress-related genes such as Unc5b mRNA, and might thereby affect neurodevelopment. Discussion: In summary, this study revealed that Tet2-mediated RNA 5hmC modification is involved in stress-related mRNA stability regulation and may serve as a potential therapeutic target for chronic stress-related diseases such as depression.

Methylation of TET2 Promoter Is Associated with Global Hypomethylation and Hypohydroxymethylation in Peripheral Blood Mononuclear Cells of Systemic Lupus Erythematosus Patients.

(1) Background: It is widely accepted that aberrant methylation patterns contribute to the development of systemic lupus erythematosus (SLE). Ten-eleven translocation (TET) methylcytosine dioxygenase is an essential enzyme of which there are three members, TET1, 2, and 3, involved in hydroxymethylation, a newly uncovered mechanism of active DNA methylation. The epigenomes of gene transcription are regulated by 5-hydroxymethylcytocine (5-hmC) and TETs, leading to dysregulation of the immune system in SLE. The purpose of this study was to investigate the global hydroxymethylation status in SLE peripheral blood mononuclear cells (PBMCs) and to explore the role of TETs in changing the patterns of methylation. (2) Methods: We collected PBMCs from 101 SLE patients and 100 healthy donors. TaqMan real-time polymerase chain-reaction assay was performed for the detection of 5-methylcytosine (5-mC), 5-hmC, and TET2 mRNA expression and single-nucleotide polymorphism genotyping. The methylation rates in different CpG sites of TET2 promoters were examined using next-generation sequencing-based deep bisulfite sequencing. Putative transcription factors were investigated using the UCSC Genome Browser on the Human Dec. 2013 (GRCh38/hg38) Assembly. (3) Results: 5-mC and 5-hmC were both decreased in SLE. The mRNA expression level of TET2 was notably high and found to be correlated with the levels of immunologic biomarkers that are indicative of SLE disease activity. The analysis of methylation rates in the TET2 promoter revealed that SLE patients had significantly higher and lower rates of methylation in TET2 105146072-154 and TET2 105146218-331, respectively. (4) Conclusions: TET2 may play an important role in 5-mC/5-hmC dynamics in the PBMCs of SLE patients. The epigenetic modification of TET2 promoters could contribute to the pathogenesis of SLE and the intensity of the immunologic reaction.

ROS attenuates TET2-dependent ZO-1 epigenetic expression in cerebral vascular endothelial cells

AimsTo investigate whether DNA active demethylase TET regulates the expression of tight junction proteins in endothelial cells of the blood–brain barrier (BBB).MethodsCorrelations between TET2 activity (indicated by its catalytic product 5hmC) and the expression of BBB tight junction proteins were examined in Tet2 knockout mice and post-mortem human brain tissues. In cultured endothelial cells, the impact of changes of TET activity on the expression of tight junction protein, ZO-1, was studied. BBB permeability assays were performed in Tet2 knockout mice.ResultsIt was found that the level of 5hmC decreased in brain microvascular endothelial cells of aging mice. In Tet2 knockout mice, the level of 5hmC in endothelial cells was weaker and significantly correlated with the reduced expression of tight junction protein ZO-1. In cultured endothelial cells, H2O2 significantly decreased the expression of 5hmC and ZO-1. Tet2 knock-down using siRNA significantly downregulated the expression of ZO-1 in endothelial cells. hMeChip-PCR showed that H2O2 decreased the level of 5hmC in the ZO-1 promoter region, which was rescued by N-acetyl cysteine (NAC). Consistently, Tet2 knock-down using siRNA significantly downregulated the level of 5hmC in the ZO-1 promoter region. It was also found that the level of 5hmC decreased in endothelial cells of aging human brains compared with that of adult brains, and the level of ZO-1 was positively correlated with that of 5hmC in microvascular endothelial cells.ConclusionsThese findings suggest that TET activity is essential in regulating ZO-1 expression of BBB. It might be a potential target for neuroprotection during aging and in diverse neurological conditions.

Abstract P2054: Epigenetic Regulation Of TLR2 By Gut Microbial Antigens Aggravates Myocardial Fibrosis Through Activating Myd88/IRAK4/NF-kB Signaling In Type 2 Diabetes

Background: Type 2 diabetic (T2D) patients are 40% more likely to develop cardiovascular diseases compared to healthy individuals. Previously, we have shown that circulating gut peptides such as peptidoglycan (PGN) aggravate myocardial fibrosis through DNA hypomethylation-induced activation of TLR2; however, the underlying molecular mechanisms responsible remain elusive. Hypothesis: Gut microbial antigens activate MyD88/IRAK4/NF-κB signaling through TLR2 in cardiac tissue, resulting in myocardial fibrosis and cardiac dysfunction in T2D. Methods: Gut barrier integrity, cardiac inflammation, and DNA hypomethylation (5hmC) were measured in cardiac tissue via IHC, qPCR, ELISA, and western blot analysis in db/db mice, a murine model of T2D with 4 months of diabetes and age-matched controls. PGN-treated cardiac fibroblasts were analyzed via in-vitro studies with and without IRAK4 and NF-κB inhibitors. Results: db/db mice exhibited cardiac dysfunction and myocardial fibrosis as measured by reduced LVEF and LVFS with increased LV mass and collagen synthesis. These mice also exhibited higher gut permeability as observed by reduced tight junctional proteins (ZO-1, p120-catenin, and VE-cadherin, YAP, PV1) and increased levels of plasma FABP-2. The levels of PGN were higher in plasma (p&lt;0.003) and cardiac tissue (p&lt;0.001) of db/db mice. The expression of TLR2 (p&lt;0.003) and pro-inflammatory cytokines IL-1β (p&lt;0.008) and TNF-α (p&lt;0.0004) were significantly up-regulated in diabetic hearts compared to WT. Expression of 5hmC was significantly higher (p&lt;0.001) in the hearts of diabetic mice. Increased expression of 5hmC, ten-eleven translocation 1-3 (TET-1, 2, 3), IL-1β, IL-6, and TNF-α were also observed in PGN-treated cardiac fibroblasts. IRAK4 (PF-06650883; 10μM) and NF-κB (CAPE; 50 μM) inhibition reduced the PGN-mediated fibroblast activation, resulting in reduced inflammation. Conclusions: Increased circulating gut microbial antigens by induction of systemic and cardiac inflammatory pathways result in damage to the myocardium of db/db mice. Therapies designed to restore the gut barrier and reduce TLR2 activation may potentially minimize the risk of diabetes-induced cardiovascular complications.

Diagnostic utility of PRAME, p53 and 5-hmC immunostaining for distinguishing melanomas from naevi, neurofibromas, scars and other histological mimics

High expression of PRAME (PReferentially expressed Antigen in MElanoma) and p53 (a proposed marker of desmoplastic melanoma) and low expression of 5-hydroxymethylcytosine (5-hmC) have each been reported in melanoma. However, their combined diagnostic utility for distinguishing melanomas, including uncommon variants, from histological mimics is unknown. This study sought to determine the utility of PRAME, p53 and 5-hmC immunostains for diagnosing melanocytic tumours. A total of 333 cutaneous melanocytic tumours (melanoma n=280, naevi n=53), 20 cutaneous neurofibromas, and 15 scars were evaluated using multiplex immunofluorescence (n=313) or single-plex chromogenic immunohistochemical staining (n=55). Immunostaining for PRAME and 5-hmC were each scored using a previously described semiquantitative scale 0 (absent) to 4+ (diffuse). p53 was scored using a previously described immunoreactive score (range 0-300). PRAME expression was significantly higher in melanomas than in naevi (p<0.0001), with the lowest PRAME expression found in low-grade desmoplastic melanomas compared to the other melanoma subtypes. In non-desmoplastic melanomas, 38% showed 4+ staining (>75% positive tumour cells) and 70% showed 3+ or 4+(>50% positive tumour cells). Conversely, 96% of naevi showed 0, 1+ or 2+ expression. 5-hmC expression was significantly lower in melanomas than in naevi (p<0.0001). However, acral melanomas were not significantly associated with loss of 5-hmC expression (p=0.84). Compared with using PRAME in isolation, combining PRAME and 5-hmC scores increased sensitivity (64%-84%) for detecting melanoma. With respect to desmoplastic melanoma compared to scar or neurofibroma, strong PRAME or p53 staining were almost exclusively found in high-grade desmoplastic melanomas; low-grade desmoplastic melanomas, neurofibromas and scars were negative. 5-hmC was not useful in distinguishing desmoplastic melanomas from neurofibromas or scars. Our data support the use of PRAME as a highly specific ancillary investigation in the diagnosis of melanoma, however PRAME should be considered 'positive' if there is 3+ or 4+ staining (rather than the widely recommended 4+ threshold). 5-hmC, PRAME and p53 appear to have a limited role in the diagnosis of low-grade desmoplastic melanomas.

LB964 TET2 participates in the dysregulation of skin barrier in psoriasis

Psoriasis is a chronic inflammatory skin disease. Dysregulation of the epidermal barrier in psoriasis has been demonstrated and epigenetic modifications have been found to be associated with psoriasis. TET2 has dioxygenase activity and oxidizes 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and is a key regulator of DNA demethylation. There is a lack of studies on the effect of TET2 on the epidermal barrier in psoriasis. We observed reduced expression of TET2 and 5hmc in the lesions of psoriasis patients. In imiquimod psoriatic dermatitis mice, we obtained similar results by immunohistochemistry. We constructed epidermal TET2 conditional knockout mice (TET2CKO). In the imiquimod psoriasis dermatitis model, TET2CKO mice presented more severe erythema, scaling and skin thickness. The TEWL assay was performed on imiquimod-treated mice and was elevated in the TET2CKO group. This indicates that epidermal TET2 deficiency exacerbates psoriatic dermatitis in mice. We next examined the expression of K14, K10, filaggrin, involucrin, which represent proliferation and differentiation, TET2CKO mice showed K14, involucrin increased and K10, filaggrin decreased. TET2 absence may accelerate the proliferation and inhibit differentiation of keratinocytes. We subsequently observed that intercellular linker molecules Claudin-1 and E-cadherin were both downregulated in TET2CKO mice. In conclusion, deletion of epidermal TET2 is involved in dysregulation of the skin barrier. By RNAseq, we found that FLG mRNA is reduced after TET2 knockdown. Deficiency of filaggrin encoded by the FLG gene, leads to impaired epidermal barrier. Filaggrin expression reduced but mutations in the FLG gene have been reported to be rarely observed in psoriasis, therefore we suppose that FLG gene expression might be regulated by TET2, which is an alternative mechanism for barrier dysregulation in non-mutant FLG patients.

TET3 Mediates 5hmC Level and Promotes Tumorigenesis by Activating AMPK Pathway in Papillary Thyroid Cancer.

Thyroid cancer is the most common endocrine malignant tumor. The accurate risk stratification and prognosis assessment is particularly important for patients with thyroid cancer, which can reduce the tumor recurrence rate, morbidity, and mortality effectively. DNA methylation is one of the most widely studied epigenetic modifications. Many studies have shown that 5hmC-mediated demethylation played an important role in tumors. The hydroxylation of 5mC is catalyzed by ten-eleven translocation dioxygenase (TET). In this study, we first found that the abnormal expression of 5hmC was closely related to microcarcinoma, multifocal, extraglandular invasion and lymph node metastasis of thyroid carcinoma. Then, we identified TET3 was differentially expressed in thyroid cancers and normal tissues from the TET family. TET3 can promote the proliferation, migration, and invasion of thyroid cancer. TET3-mediated 5hmC can regulate the transcription of AMPK pathway-related genes to activate the AMPK pathway and autophagy and therefore promote PTC proliferation. These findings provide a preclinical rationale for the design of novel therapeutic strategies for this target to improve the clinical outcome of patients with PTC.