TET Enzymes Research Articles

A hybrid residue based sequential encoding mechanism with XGBoost improved ensemble model for identifying 5-hydroxymethylcytosine modifications

RNA modifications play an important role in actively controlling recently created formation in cellular regulation mechanisms, which link them to gene expression and protein. The RNA modifications have numerous alterations, presenting broad glimpses of RNA’s operations and character. The modification process by the TET enzyme oxidation is the crucial change associated with cytosine hydroxymethylation. The effect of CR is an alteration in specific biochemical ways of the organism, such as gene expression and epigenetic alterations. Traditional laboratory systems that identify 5-hydroxymethylcytosine (5hmC) samples are expensive and time-consuming compared to other methods. To address this challenge, the paper proposed XGB5hmC, a machine learning algorithm based on a robust gradient boosting algorithm (XGBoost), with different residue based formulation methods to identify 5hmC samples. Their results were amalgamated, and six different frequency residue based encoding features were fused to form a hybrid vector in order to enhance model discrimination capabilities. In addition, the proposed model incorporates SHAP (Shapley Additive Explanations) based feature selection to demonstrate model interpretability by highlighting the high contributory features. Among the applied machine learning algorithms, the XGBoost ensemble model using the tenfold cross-validation test achieved improved results than existing state-of-the-art models. Our model reported an accuracy of 89.97%, sensitivity of 87.78%, specificity of 94.45%, F1-score of 0.8934%, and MCC of 0.8764%. This study highlights the potential to provide valuable insights for enhancing medical assessment and treatment protocols, representing a significant advancement in RNA modification analysis.

5-Formylcytosine is an activating epigenetic mark for RNA Pol III during zygotic reprogramming

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression invivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression.

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.

Distinct dynamics of parental 5-hydroxymethylcytosine during human preimplantation development regulate early lineage gene expression

The conversion of DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes represents a significant epigenetic modification, yet its role in early human embryos remains largely unknown. Here we showed that the early human embryo inherited a significant amount of 5hmCs from an oocyte, which unexpectedly underwent de novo hydroxymethylation during its growth. Furthermore, the generation of 5hmC in the paternal genome after fertilization roughly followed the maternal pattern, which was linked to DNA methylation dynamics and regions of sustained methylation. The 5hmCs persisted until the eight-cell stage and exhibited high enrichment at OTX2 binding sites, whereas knockdown of OTX2 in human embryos compromised the expression of early lineage genes. Specifically, the depletion of 5hmC affected the activation of embryonic genes, which was further evaluated by ectopically expressing mouse Tet3 in human early embryos. These findings revealed distinct dynamics of 5hmC and unravelled its multifaceted functions in early human embryonic development.

Molecular basis of an atypical dsDNA 5mC/6mA bifunctional dioxygenase CcTet from Coprinopsis cinerea in catalyzing dsDNA 5mC demethylation.

The eukaryotic epigenetic modifications 5-methyldeoxycytosine (5mC) and N6-methyldeoxyadenine (6mA) have indispensable regulatory roles in gene expression and embryonic development. We recently identified an atypical bifunctional dioxygenase CcTet from Coprinopsis cinerea that works on both 5mC and 6mA demethylation. The nonconserved residues Gly331 and Asp337 of CcTet facilitate 6mA accommodation, while D337F unexpectedly abolishes 5mC oxidation activity without interfering 6mA demethylation, indicating a prominent distinct but unclear 5mC oxidation mechanism to the conventional Tet enzymes. Here, we assessed the molecular mechanism of CcTet in catalyzing 5mC oxidation by representing the crystal structure of CcTet-5mC-dsDNA complex. We identified the distinct mechanism by which CcTet recognizes 5mC-dsDNA compared to 6mA-dsDNA substrate. Moreover, Asp337 was found to have a central role in compensating for the loss of a critical 5mC-stablizing H-bond observed in conventional Tet enzymes, and stabilizes 5mC and subsequent intermediates through an H-bond with the N4 atom of the substrates. These findings improve our understanding of Tet enzyme functions in the dsDNA 5mC and 6mA demethylation pathways, and provide useful information for future discovery of small molecular probes targeting Tet enzymes in DNA active demethylation processes.

Selective and Catalytic Conversion of Hydroxymethyl Cytosine to Formyl Cytosine by a Synthetic Model of TET Enzymes

The TET enzymes, known as the ten-eleven translocation enzymes, have become central figures in epigenetic regulation due to their remarkable ability to oxidize 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC),...

Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects.

In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.

TET enzyme driven epigenetic reprogramming in early embryos and its implication on long-term health.

Mammalian embryo development is initiated by the union of paternal and maternal gametes. Upon fertilization, their epigenome landscape is transformed through a series of finely orchestrated mechanisms that are crucial for survival and successful embryogenesis. Specifically, maternal or oocyte-specific reprogramming factors modulate germ cell specific epigenetic marks into their embryonic states. Rapid and dynamic changes in epigenetic marks such as DNA methylation and histone modifications are observed during early embryo development. These changes govern the structure of embryonic genome prior to zygotic genome activation. Differential changes in epigenetic marks are observed between paternal and maternal genomes because the structure of the parental genomes allows interaction with specific oocyte reprogramming factors. For instance, the paternal genome is targeted by the TET family of enzymes which oxidize the 5-methylcytosine (5mC) epigenetic mark into 5-hydroxymethylcytosine (5hmC) to lower the level of DNA methylation. The maternal genome is mainly protected from TET3-mediated oxidation by the maternal factor, STELLA. The TET3-mediated DNA demethylation occurs at the global level and is clearly observed in many mammalian species. Other epigenetic modulating enzymes, such as DNA methyltransferases, provide fine tuning of the DNA methylation level by initiating de novo methylation. The mechanisms which initiate the epigenetic reprogramming of gametes are critical for proper activation of embryonic genome and subsequent establishment of pluripotency and normal development. Clinical cases or diseases linked to mutations in reprogramming modulators exist, emphasizing the need to understand mechanistic actions of these modulators. In addition, embryos generated via in vitro embryo production system often present epigenetic abnormalities. Understanding mechanistic actions of the epigenetic modulators will potentially improve the well-being of individuals suffering from these epigenetic disorders and correct epigenetic abnormalities in embryos produced in vitro. This review will summarize the current understanding of epigenetic reprogramming by TET enzymes during early embryogenesis and highlight its clinical relevance and potential implication for assisted reproductive technologies.

Auto-suppression of Tet dioxygenases protects the mouse oocyte genome from oxidative demethylation.

DNA cytosine methylation plays a vital role in repressing retrotransposons, and such derepression is linked with developmental failure, tumorigenesis and aging. DNA methylation patterns are formed by precisely regulated actions of DNA methylation writers (DNA methyltransferases) and erasers (TET, ten-eleven translocation dioxygenases). However, the mechanisms underlying target-specific oxidation of 5mC by TET dioxygenases remain largely unexplored. Here we show that a large low-complexity domain (LCD), located in the catalytic part of Tet enzymes, negatively regulates the dioxygenase activity. Recombinant Tet3 lacking LCD is shown to be hyperactive in converting 5mC into oxidized species in vitro. Endogenous expression of the hyperactive Tet3 mutant in mouse oocytes results in genome-wide 5mC oxidation. Notably, the occurrence of aberrant 5mC oxidation correlates with a consequent loss of the repressive histone mark H3K9me3 at ERVK retrotransposons. The erosion of both 5mC and H3K9me3 causes ERVK derepression along with upregulation of their neighboring genes, potentially leading to the impairment of oocyte development. These findings suggest that Tet dioxygenases use an intrinsic auto-regulatory mechanism to tightly regulate their enzymatic activity, thus achieving spatiotemporal specificity of methylome reprogramming, and highlight the importance of methylome integrity for development.

Abstract C036: Investigating the phenotypic changes in in vitro and in vivo cancer models expressing isocitrate dehydrogenase 1 (IDH1) mutations R132H and R132Q

Abstract According to the National Cancer Institute, there were 25,050 new brain cancer cases in 2022, representing 1.3% of all cancer incidents in the United States. In the same year, the estimated death was 18,280 cases, representing 3.0% of all cancer deaths. Relative survival statistics between 2017 and 2018 showed that only 32.5% of patients would survive five years or more. Disparities in cancer incidence and outcomes, including brain cancers, can be due to biological, socioeconomic, and care access factors that often have roots in structural racism. Research shows lower three years survival rates of glioblastoma in the African American population by 28.8% compared to the Asian Pacific Islanders at 36.6% and Hispanic at 32.3%. Mutant isocitrate dehydrogenase 1 (IDH1) can lead to brain cancer, including > 80% of lower-grade gliomas and secondary glioblastomas. Mutant IDH1 catalyzes the NADPH-dependent conversion of α-ketoglutarate to D2-hydroxyglutarate (D2HG), an oncometabolite linked to many cancer types. IDH1 mutation occurs primarily at residue 132, and our lab has shown that the catalytic activity differs based on the amino acid substituted at that location. There is a lack of information describing the relation of D2HG produced to cancer cells’ phenotype severity. Understanding IDH1 mutation will clarify cancer progression, patients’ prognosis, and treatment responses. Here, we hypothesize that the toxic effect of D2HG will impair the viability of cells expressing mutant IDH1 and will cause a reduction in ROS and NADPH cellular levels. This work examines D2HG production efficiency in vivo and in vitro and the metabolic consequences of this oncometabolite. We created cell lines stably overexpressing IDH1-R132Q and IDH1-R132H to conduct this work. Our results showed higher levels of D2HG in cells expressing IDH1-R132Q compared to DH1-wild type and IDH1-R132H, supportive of our previous finding that IDH1-R132Q mutation has higher catalytic activities (higher efficiency in producing mutant IDH1). These higher D2HG levels caused cells to proliferate and migrate at lower rates. D2HG interferes with DNA demethylation by inhibiting the TET enzyme, and we observed higher levels of histone protein expression in IDH1-R132Q compared to the other modified cells. In in vivo studies, we identified elevated D2HG levels in mouse xenografts expressing IDH1-R132Q that affected tumor growth. Citation Format: Mowaffaq A.A. Adam, Mikella Robinson, An Hoang, Grace A. Wells, David A. Scott, Olga Zagnitko, Carrie House, Christal D. Sohl. Investigating the phenotypic changes in in vitro and in vivo cancer models expressing isocitrate dehydrogenase 1 (IDH1) mutations R132H and R132Q [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C036.

A Real-World Single-Institution Experience with Vitamin C Supplementation for Hypomethylated Drugs Combined with Venetoclax Versus “7+3” in Elderly AML Patients

Background: Vitamin C deficiency is frequently observed in AML patients compared to the normal population (Huijskens, et al. 2016; Liu, et al. 2016), and in vitro and in vivo studies have demonstrated that Vitamin C improves the response of AML patients to demethylated drug epigenetic therapy by directly regulating the activity of TET enzymes (Liu, et al. 2016; Zhao, et al. 2018). In addition, a number of in vitro and in vivo studies have shown that Vitamin C selectively kills cancer cells by generating ROS and can enhance the activity of chemotherapy drugs (Chen, et al. 2007; Verrax and Calderon 2009; Yun, et al. 2015). Today, optimal treatment for elder AML patients remains challenging, and treatment options include intensive chemotherapy (anthracyclines combined with cytarabine, known as “7+3”), hypomethylated drugs (HMA), and the addition of venetoclax to HMAs (azacytidine or decitabine) significantly improve median OS(DiNardo, et al. 2020; Ossenkoppele and Löwenberg 2015). However, it is unclear whether Vitamin C has a synergistic effect with chemotherapy and demethylated drug therapy in elderly patients with AML. Our objective was to investigate whether adding Vitamin C to “7+3” or VEN/HMA can improve the efficacy and survival of elderly AML patients. Patients and methods: A retrospective cohort study of newly diagnosed elderly AML patients treated with a “7+3” regimen or HMA/Ven from 2010 to 2023 at the First Affiliated Hospital of Anhui Medical University, following approval by an institutional ethics Review committee, was conducted. The analysis included 126 patients. Collect clinical, demographic and genomic data. In SPSS (v.26), bilateral Student's t test or one-way ANOVA were used for inter-group difference analysis, Chi-square test was used to analyze the composite complete remission rate, and Kaplan-Meier method was used for survival analysis. Result: The median age of patients in the “7+3” cohort was 65 years, and the median age of patients in the VEN/HMA cohort was 69 years. Compared with VEN/HMA cohort, patients in the “7+3” cohort had better physical status(p＜0.001)(Table 1). Compared with the patients in the “7+3” cohort, the cCR (CR+CRi) in the VEN/HMA cohort was not statistically significant (Table 1). The cCR (CR+CRi) rates of VEN/HMA + Vitamin C and “VEN/HMA” treatment after 1 cycle were 82% and 68% (p=0.69). Compare OS between queues, the median OS (mOS) was 400 days in the “7+3” + Vitamin C cohort and 276 days in the “7+3” cohort (Figure a, p=0.220). Additionally, VEN/HMA+Vitamin C patients demonstrated a superior, yet not statistically significant, median overall survival (OS) benefit compared to VEN/HMA (median OS not reached, Figure b, p = 0.083). Conclusion: VEN/HMA and “7+3” are effective frontline treatment options with similar response rates in newly diagnosed elderly AML. Additionally, the overall survival in VEN/HMA+Vitamin C appeared to be better, but did not reach statistical significance. Our data demonstrated that in the VEN/HMA cohort, VEN/HMA+Vitamin C the OS was longer and approached statistical significance. Future prospective trial designs or larger cohorts are needed to confirm these findings.

The Influence of TET2 and/or DNMT3A Knockout on the 5hmC Levels and Growth Characteristics of Modified Human Primary CD4 T Cells after Activation

Angioimmunoblastic T-cell lymphoma (AITL) has a unique immunohistological profile featuring malignant CD4+ T follicular helper cells (Tfh) inter-mixed with an extensive infiltrate of multiple lineages of immune cells. The AITL tumor microenvironment (TME) classically features small or large B cells (variably Epstein-Barr Virus (EBV) positive), reactive CD8+ T-cells, plasma cells and proliferative high endothelial venules (HEVs). Common genetic mutations have been identified within the malignant Tfh clone in AITL including epigenetic modifiers TET2, DNMT3A, and IDH2. DNMT3A mutations in AITL occur at a frequency of 20-38.5%and most of the DNMT3A mutations occur together with TET2 mutations. Tet2/ Dnmt3a double-knockout mice show synergistic effects on the expression of lineage-specifying transcription factor genes via epigenetic mechanisms. However, it is still not clear how DNMT3A mutations enhance the effect of TET2 mutations on promoting AITL development. To investigate how TET2 and DNMT3A mutations interact in AITL development, we first generated TET2 and/or DNMT3A knockout human primary CD4-T cells with CRISPR-Cas9 mediated genome editing. TET proteins could oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmc) in replication- independent DNA demethylation. Unmethylated cytosine base can be methylated by the DNA methylation machinery (DNMT3A/B) to form 5mC, which in turn provides the substrate for TET enzymes to produce 5hmC. TET and DNMT family members are thus needed to be dynamically modulate the epigenome to maintain cellular homeostasis or alter cellular function or development. We found that in response to CD3/CD28 stimulation, WT CD4-T cells exhibited division mediated dilutional reduction in 5 hmC which was later reconstitution. However, in TET2 or DKO cells, this process was compromised, showing delayed global 5hmc levels recovery (Fig. 1A). We then investigated whether the changes in methylation dynamics would alter cellular function. When using CD3/CD28 beads stimulation (10 days/cycle), we found that after two cycles of stimulation, these CD4-T cells with different gene editing started showing distinct behaviors with proliferation and apoptosis when exposed to CD3/CD28 beads again. The TET2/ DNMT3A double knockout (DKO) CD4-T cells showed more rapid response in cell division and higher proliferation rate than the other three groups (Fig. 1B). Compared with WT cells, consistently higher apoptotic cells were found in DNMT3A-KO group, but not with either TET2-KO or DKO cells (Fig. 1C). Interestingly, withdraw of beads at the end of the 10 days cycle, DKO cells kept higher baseline proliferation level than WT cells (Fig. 1D). We then used autologous or allogeneic EBV transformed B-cell for stimulation of the CD4 T-cells after one or two cycles of CD3/28 beads stimulation, and different response kinetics were observed among these prior activated CD4-T cells with TET2 and/or DNMT3A mutations. DKO cells showed more rapid response in cell division and elevated percentages of replicated cells to either autologous or allogeneic B-cells compared with WT cells (Fig. 1E). An interesting aspect of this B/T interaction is that, even the autologous B-cells could activate a large percentage of the T-cell population, particularly in the DKO cells, so it is not likely due to specific antigen recognition but rather a non-specific stimulation by the B-cells, probably via one or more co-stimulatory molecules (ICOSL, CD86 or 4-1BBL). This may be relevant to in vivo evolution of the mutated T-cells with B-cells/activated B-cells providing support.

Vitamin C activates young LINE-1 elements in mouse embryonic stem cells via H3K9me3 demethylation

BackgroundVitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells.ResultsHere we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism.ConclusionVitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.

A genetic screen identifies BEND3 as a regulator of bivalent gene expression and global DNA methylation.

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elementsand higher-order chromatin structures. Consequently, these chromatin marks are indispensable for mammalian development and alterations often lead to disease, such as cancer. Bivalent promoters are especially important during differentiation and development. Here we used a genetic screen to identify new regulators of a bivalent repressed gene. We identify BEND3 as a regulator of hundreds of bivalent promoters, some of which it represses, and some of which it activates. We show that BEND3 is recruited to a CpG-containg consensus site that is present in multiple copies in many bivalent promoters. Besides having direct effect on the promoters it binds, the loss of BEND3 leads to genome-wide gains of DNA methylation, which are especially marked at regions normally protected by the TET enzymes. DNA hydroxymethylation is reduced in Bend3 mutant cells, possibly as consequence of altered gene expression leading to diminished alpha-ketoglutarate production, thus lowering TET activity. Our results clarify the direct and indirect roles of an important chromatin regulator, BEND3, and, more broadly, they shed light on the regulation of bivalent promoters.

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.

DNA demethylation fine-tunes IL-2 production during thymic development and promotes Regulatory T cell differentiation

Abstract Regulatory T (T reg) cells developing in the thymus are essential to maintain tolerance and prevent fatal autoimmunity in mice and humans. Expression of the T reg lineage-defining transcription factor FoxP3, is critically dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that ten-eleven translocation (Tet) enzymes, which are DNA demethylases, are required early during double positive (DP) thymic T cell differentiation and prior to the upregulation of FoxP3 in CD4 single-positive (SP) thymocytes, to promote Treg differentiation. We show that Tet enzymes selectively control the development of CD25 −FoxP3 loCD4SPTreg cell precursors and are critical for optimal TCR-dependent IL-2 production, which drive chromatin remodeling at the FoxP3 locus as well as other Treg-effector gene loci in an autocrine/paracrine manner. Together, our results demonstrate a novel role for DNA demethylation in regulating the TCR response and promoting Treg cell differentiation. These findings highlight a novel epigenetic pathway to promote the generation of endogenous Treg cells for mitigation of autoimmune responses.

Partial erosion on under-methylated regions and chromatin reprogramming contribute to oncogene activation in IDH mutant gliomas

BackgroundIDH1/2 hotspot mutations are well known to drive oncogenic mutations in gliomas and are well-defined in the WHO 2021 classification of central nervous system tumors. Specifically, IDH mutations lead to aberrant hypermethylation of under-methylated regions (UMRs) in normal tissues through the disruption of TET enzymes. However, the chromatin reprogramming and transcriptional changes induced by IDH-related hypermethylation in gliomas remain unclear.ResultsHere, we have developed a precise computational framework based on Hidden Markov Model to identify altered methylation states of UMRs at single-base resolution. By applying this framework to whole-genome bisulfite sequencing data from 75 normal brain tissues and 15 IDH mutant glioma tissues, we identified two distinct types of hypermethylated UMRs in IDH mutant gliomas. We named them partially hypermethylated UMRs (phUMRs) and fully hypermethylated UMRs (fhUMRs), respectively. We found that the phUMRs and fhUMRs exhibit distinct genomic features and chromatin states. Genes related to fhUMRs were more likely to be repressed in IDH mutant gliomas. In contrast, genes related to phUMRs were prone to be up-regulated in IDH mutant gliomas. Such activation of phUMR genes is associated with the accumulation of active H3K4me3 and the loss of H3K27me3, as well as H3K36me3 accumulation in gene bodies to maintain gene expression stability. In summary, partial erosion on UMRs was accompanied by locus-specific changes in key chromatin marks, which may contribute to oncogene activation.ConclusionsOur study provides a computational strategy for precise decoding of methylation encroachment patterns in IDH mutant gliomas, revealing potential mechanistic insights into chromatin reprogramming that contribute to oncogenesis.

Epigenetic modifier alpha-ketoglutarate modulates aberrant gene body methylation and hydroxymethylation marks in diabetic heart

BackgroundDiabetic cardiomyopathy (DCM) is a leading cause of death in diabetic patients. Hyperglycemic myocardial microenvironment significantly alters chromatin architecture and the transcriptome, resulting in aberrant activation of signaling pathways in a diabetic heart. Epigenetic marks play vital roles in transcriptional reprogramming during the development of DCM. The current study is aimed to profile genome-wide DNA (hydroxy)methylation patterns in the hearts of control and streptozotocin (STZ)-induced diabetic rats and decipher the effect of modulation of DNA methylation by alpha-ketoglutarate (AKG), a TET enzyme cofactor, on the progression of DCM.MethodsDiabetes was induced in male adult Wistar rats with an intraperitoneal injection of STZ. Diabetic and vehicle control animals were randomly divided into groups with/without AKG treatment. Cardiac function was monitored by performing cardiac catheterization. Global methylation (5mC) and hydroxymethylation (5hmC) patterns were mapped in the Left ventricular tissue of control and diabetic rats with the help of an enrichment-based (h)MEDIP-sequencing technique by using antibodies specific for 5mC and 5hmC. Sequencing data were validated by performing (h)MEDIP-qPCR analysis at the gene-specific level, and gene expression was analyzed by qPCR. The mRNA and protein expression of enzymes involved in the DNA methylation and demethylation cycle were analyzed by qPCR and western blotting. Global 5mC and 5hmC levels were also assessed in high glucose-treated DNMT3B knockdown H9c2 cells.ResultsWe found the increased expression of DNMT3B, MBD2, and MeCP2 with a concomitant accumulation of 5mC and 5hmC, specifically in gene body regions of diabetic rat hearts compared to the control. Calcium signaling was the most significantly affected pathway by cytosine modifications in the diabetic heart. Additionally, hypermethylated gene body regions were associated with Rap1, apelin, and phosphatidyl inositol signaling, while metabolic pathways were most affected by hyperhydroxymethylation. AKG supplementation in diabetic rats reversed aberrant methylation patterns and restored cardiac function. Hyperglycemia also increased 5mC and 5hmC levels in H9c2 cells, which was normalized by DNMT3B knockdown or AKG supplementation.ConclusionThis study demonstrates that reverting hyperglycemic damage to cardiac tissue might be possible by erasing adverse epigenetic signatures by supplementing epigenetic modulators such as AKG along with an existing antidiabetic treatment regimen.

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.

Abstract 2226: Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression

Abstract Loss of function in DNA methylation regulators such as the Ten-eleven translocation (TET1-3) proteins, have been implicated in the progression of lymphoma. TET enzymes constitute a family of iron and αKG-dependent dioxygenases that oxidize 5-methylcytosine in DNA to promote demethylation and the reversal of gene silencing. The TET enzymes require vitamin C for their optimal catalytic activity, however, many patients with hematological malignancies are vitamin C deficient. Enhancing and restoring TET function with high-dose vitamin C has been shown to block the progression of solid tumors and blood malignancies. A combination of genetic and epigenetic diversity in lymphoma can result in the aberrant overexpression of oncogenes, loss of tumor suppressor function, and genomic instability. Our research models Bcl2-driven B cell lymphoma with Tet-deficiency using transgenic RNAi for the knockdown of Tet1 or Tet2. In vitro, our results have shown that this model shows increased self-renewal capacity of early B cell progenitors and precursors that can be suppressed by genetic restoration of Tet expression or upon treatment with vitamin C. In vivo, the B cell compartment of mice with Bcl2 overexpression and Tet1 or Tet2 deficiency exhibit a block in B cell maturation during a long period of pre-malignancy, and a decreased frequency of follicular B cells in the spleen from 3 months of age. Our aim is to determine mechanistically the effects of Tet-deficiency on B cell lymphoma progression and whether vitamin C can restore Tet activity to suppress lymphoma progression. Citation Format: Minh Quang Lam, Luisa Cimmino. Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2226.