Increase In 5hmC Research Articles

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

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

TET1-mediated epigenetic regulation of tumor necrosis factor-α in trigeminal ganglia contributes to chronic temporomandibular joint pain

Chronic temporomandibular joint (TMJ) pain profoundly affects patients' quality of life. Trigeminal tumor necrosis factor-α (TNFα) plays a pivotal role in mediating TMJ pain in mice, yet the underlying epigenetic mechanisms remain enigmatic. To unravel these epigenetic intricacies, we employed a multifaceted approach. Hydroxymethylated DNA immunoprecipitation (hMeDIP) and chromatin immunoprecipitation (ChIP) followed by qPCR were employed to investigate the demethylation of TNFα gene (Tnfa) and its regulation by ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in a chronic TMJ pain mouse model. The global levels of 5-hydroxymethylcytosine (5hmc) and percentage of 5hmc at the Tnfa promoter region were measured in the trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) following complete Freund's adjuvant (CFA) or saline treatment. TET1 knockdown and pain behavioral testing were conducted to ascertain the role of TET1-mediated epigenetic regulation of TNFα in the pathogenesis of chronic TMJ pain. Our finding revealed an increase in 5hmc at the Tnfa promoter region in both TG and Sp5C of CFA-treated mice. TET1 was upregulated in the mouse TG, and the ChIP result showed TET1 direct binding to the Tnfa promoter, with higher efficiency in the CFA-treated group. Immunofluorescence revealed the predominant expression of TET1 in trigeminal neurons. TET1 knockdown in the TG significantly reversed CFA-induced TNFα upregulation and alleviated chronic TMJ pain. In conclusion, our study implicates TET1 as a vital epigenetic regulator contributing to chronic inflammatory TMJ pain via trigeminal TNFα signaling. Targeting TET1 holds promise for epigenetic interventions in TMJ pain management.

Triclosan (TCS) affects the level of DNA methylation in the human oral squamous cell carcinoma (SCC-15) cell line in a nontoxic concentration

The oral cancer is presumably caused by genetic factors and exposure to substances derived from cosmetics and disinfectants. Triclosan (TCS) is widely spread in many consumer products and oral care products. Since TCS can affect DNA methylation, which is one of the key mechanisms of gene expression that may lead to cancerogenesis, it is necessary to study this mechanism in oral cell carcinoma. The aim of the present study was to evaluate the impact of TCS on metabolic parameters, oxidative stress, gene expression, and DNA methylation and hydroxymethylation in the SCC-15 cell line. The experiments have shown TCS toxicity to SCC-15 cells only in the highest concentrations of 50 and 100 µM. TCS in a wide range of concentrations increases ROS production and caspase-3 activity. Our experiments have shown that TCS in the nontoxic concentrations of 10 µM exerts an impact on SOD2 mRNA expression and SOD activity in the SCC-15 cell line. Finally, our experiments have demonstrated that 6-h treatment with TCS decreases the mRNA expression of DNMT3A and DNMT3B. After 72-h exposure to TCS, an increased level of 5-methylcytosine and 5-hydroxymethylcytosine was observed in the SCC-15 cell line, but it was abolished by the NAC treatment. However, it is very likely that these results can be an effect of TET enzyme activity, especially in the case of the decrease in 5mC and the increase in 5hmC after the 48-h exposure to TCS, which was accompanied with a decrease in the mRNA expression of DNMT3A and DNMT3B.

Organization of the Cytoskeleton in Ectopic Foci of the Endometrium with Rare Localization.

(1) Background: Endometriosis is a common pathology of the female reproductive system, often accompanied by pain and decreased fertility. However, its pathogenesis has not been sufficiently studied regarding the role of the cytoskeleton. In this study, we describe two clinical cases involving rare localization of extragenital endometriosis (umbilicus) and compare them with genital endometriosis of different localization (ovaries and uterus), as well as eutopic endometrium obtained with separate diagnostic curettage without confirmed pathology. (2) Methods: The relative content of actin and tubulin cytoskeleton proteins was determined by Western blotting, and the expression of genes encoding these proteins was determined by RT-PCR in the obtained intraoperative biopsies. The content of 5hmC was estimated by dot blot experiments, and the methylase/demethylase and acetylase/deacetylase contents were determined. (3) Results: The obtained results indicate that the content of the actin-binding protein alpha-actinin1 significantly increased (p < 0.05) in the groups with endometriosis, and this increase was most pronounced in patients with umbilical endometriosis. In addition, both the mRNA content of the ACTN1 gene and 5hmC content increased. It can be assumed that the increase in 5hmC is associated with a decrease in the TET3 demethylase content. Moreover, in the groups with extragenital endometriosis, alpha- and beta-tubulin content was decreased (p < 0.05) compared to the control levels. (4) Conclusions: In analyzing the results, further distance of ectopic endometrial foci from the eutopic localization may be associated with an increase in the content of alpha-actinin1, probably due to an increase in the expression of its gene and an increase in migration potential. In this case, a favorable prognosis can be explained by a decrease in tubulin content and, consequently, a decrease in the rate of cell division.

High fat diet-induced- and genetically inherited- obesity differential alters DNA demethylation pathways in the germline of adult male rats

Obesity is a multifactorial condition with predominantly genetic and environmental causes and is an emerging risk factor for male infertility/subfertility. Epigenetic mechanisms are vulnerable to genetic and environmental changes. Our earlier studies have shown differential effects of genetically inherited (GIO) - and diet-induced- obesity (DIO) on DNA methylation in male germline. Contrary to DNA methylation is DNA demethylation, which also regulates the gene expression by activating transcription. The present study aimed to delineate the effects of obesity on the DNA demethylation pathway using two rat models: GIO (WNIN/Ob) and DIO (high-fat diet). We observed differential alterations in enzymes involved in DNA demethylation by oxidation (Tet1-3) pathway in testis in both groups. An increase in Tets in DIO group and a decrease in GIO group were noted. Analysis of oxidation pathway intermediates (5-hmC, 5-fC, and 5-caC) did not show any effect on testis in DIO group but an increase in 5-hmC and decrease in 5-caC levels in GIO group was observed. Analysis of transcript levels of enzymes related to deamination pathway in testis showed an increase (Gadd45a, Aicda, and Tdg) in DIO group and a decrease (Gadd45a, Aicda, and Tdg) in GIO group. Also, 5-hmC levels were differentially altered in the spermatozoa of both groups without any changes in Tet enzyme levels. These findings highlight differences in effects of GIO and DIO on DNA demethylation mechanisms in male germline, which could be due to differences in endocrine and metabolic profile as well as white fat distribution observed earlier in two groups.

Regulation of Rac1 transcription by histone and DNA methylation in diabetic retinopathy

Cytosolic ROS, generated by NADPH oxidase 2 (Nox2) in diabetes, damage retinal mitochondria, which leads to the development of retinopathy. A small molecular weight G-protein essential for Nox2 activation, Rac1, is also transcriptionally activated viaactive DNA methylation-hydroxymethylation. DNA methylation is a dynamic process, and can also be regulated by histone modifications; diabetes alters retinal histone methylation machinery. Our aim is to investigate the role of histone methylation (H3K9me3) of Rac1 promoter in dynamic DNA methylation- transcriptional activation. Using human retinal endothelial cells in 20 mM D-glucose, H3K9me3 at Rac1 promoter was quantified by chromatin-Immunoprecipitation technique. Crosstalk between H3K9me3 and DNA methylation was examined in cells transfected with siRNA of histone trimethyl-transferase, Suv39H1, or Dnmt1, exposed to high glucose. Key parameters were confirmed in retinal microvessels from streptozotocin-induced diabetic mice, with intravitreally administered Suv39H1-siRNA or Dnmt1-siRNA. Compared to cells in normal glucose, high glucose increased H3K9me3 and Suv39H1 binding at Rac1 promoter, and Suv39H1-siRNA prevented glucose-induced increase 5 hydroxy methyl cytosine (5hmC) and Rac1 mRNA. Similarly, in diabetic mice, Suv39H1-siRNA attenuated increase in 5hmC and Rac1 mRNA. Thus, H3K9me3 at Rac1 promoter assists in active DNA methylation-hydroxymethylation, activating Rac1 transcription. Regulation of Suv39H1-H3K9 trimethylation could prevent further epigenetic modifications, and prevent diabetic retinopathy.

The Synergy of Vitamin C with Decitabine Activates TET2 in Leukemic Cells and Significantly Improves Overall Survival in Elderly Patients with Acute Myeloid Leukemia

Background: Decitabine is widely used in the treatment of acute myeloid leukemia (AML) in elderly patients. Low-dose ascorbic acid (Vitamin C) has also been indicated to induce DNA demethylation at the cellular level. Our previous study demonstrated its combined effect and safety with low-dose decitabine prior to aclarubicin andcytarabine (DCAG) in elderly patients with AML, in which it appeared to achieve a complete remission (CR) rate of 76.3% and a median overall survival of 10 months. Pharmacological concentrations of Vitamin C (10-1,000μM) induced a time- and dose-dependent increase in 5-hmC. However, little is known whether low-dose Vitamin C has a synergistic effect with decitabine in clinic. Methods: The effect of combined low-dose Vitamin C and decitabine on cell proliferation, the cell cycle, apoptosis and the expression level and activity of TET2 was investigated in HL-60 and NB-4 human leukemic cells. Additionally, we analyzed the clinical outcomes of 73 elderly AML patients who received A-DCAG (intravenous Vitamin C [IVC] plus DCAG [ n=39]) or DCAG (n=34) treatment. Patients received one of the two induction courses: DCAG or A-DCAG. DCAG consisted of 15mg/m2 of decitabine (intravenously, days 1-5) and 300 μg/day of granulocyte colony-stimulating factor (G-CSF, days 0-9) for priming its combination with 10 mg/m2 of cytarabine (q12h, days 3-9), 8 mg/m2 of aclarubicin (days 3-6, DCAG), or IVC (50-80 mg/kg/day, days 0-9; A-DCAG). The G-CSF priming was discontinued when white blood count (WBC) was ≥20×109/L. Results: We found that low-dose Vitamin C and decitabine has a synergistic efficacy on proliferation, apoptosis and TET2 activity, compared to drug-alone treatment in HL60 and NB4 cell lines in vitro . In clinic, feasibility and safety evaluations revealed that patients who received A-DCAG regimen have a higher complete remission (CR) rate than those who received the DCAG regimen (79.92 % vs. 44.11%; P=0.004) after one cycle of chemotherapy. IVC admission decision appeared to beat the discretion of attending physicians. The median overall survival (OS) was better in the A-DCAG group compared with the DCAG group (15.3 months vs. 9.3 months, P=0.039). Patients with adverse cytogenetics did benefit from CR. There was no clinically significant additional toxicity observed with the addition of IVC. Conclusion: On the basis of these results, the addition of IVC at low doses to DCAG appeared to improve CR and prolong OS, compared with DCAG, in elderly patients with AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2046: Inhibiting IDH mutations in low-grade glioma alters cellular function and the immune environment

Abstract Somatic mutations in the genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 occur in many cancers, including glioma, cholangiocarcinoma, and acute myeloid leukemia. Mutant IDH (mIDH) proteins have a gain-of-function enzyme activity, catalyzing the reduction of alpha-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (2-HG). 2-HG competitively inhibits α-KG-dependent enzymes, leading to epigenetic dysregulation and tumorigenesis. TET2 is an α-KG-dependent enzyme mediating the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which leads to DNA demethylation. In mIDH tumors, TET2 activity is inhibited by 2-HG, leading to DNA hypermethylation. In addition, IDH mutations are associated with immune evasion in gliomas. IDH mutations are common in low-grade gliomas (LGG; WHO grade 2/3) and include mutations in IDH1 (~80%) and IDH2 (~4%). Ivosidenib (AG-120, IVO) is a first-in-class, oral inhibitor of mIDH1, and vorasidenib (AG-881, VOR) is an oral, potent, brain-penetrant inhibitor of both mIDH1 and mIDH2. In an ongoing perioperative study (NCT03343197), IVO and VOR demonstrated brain penetrance and &amp;gt;90% suppression of 2-HG in resected mIDH1 gliomas after pre-operative treatment for approximately 4 weeks. In this analysis, we examined the cellular mechanisms underlying mIDH inhibition in LGG using resected tumor tissues collected following treatment with IVO and VOR. Optimal 2-HG suppression (defined by post-treatment 2-HG levels equal to or lower than the upper range of IDH wild-type tumor 2-HG levels) in IVO- or VOR-treated samples (n=17) led to a ~2-fold increase in 5hmC compared with untreated controls and banked reference samples (n=35; P=0.04). Furthermore, immunohistochemistry (IHC) analysis of the proliferation marker Ki-67 showed a ~3-fold decrease in the percentage of Ki-67-positive cells in IVO- or VOR-treated samples with optimal 2-HG suppression (n=21) compared with untreated controls (n=5; P=0.02). Tumor immune microenvironment analyses were also conducted. Comparing IVO- or VOR-treated samples (n=21) with untreated controls and banked reference samples (n=33), RNAseq analysis showed a ~2-fold decrease in the expression of tumor-associated macrophage markers CD68 (P=0.01) and CD163 (P=0.03) in mIDH oligodendrogliomas. In addition, IHC analysis from paired pre-treatment (archival) and post-treatment samples showed an increase of CD3+ T-cell infiltration (P=0.03) and CD8+ T-cell infiltration (P=0.01) in IVO- or VOR-treated samples with optimal 2-HG suppression (n=21). Single-cell RNAseq was conducted for a subset of samples, and the results will be presented. Overall, these data suggest that mIDH inhibition decreases DNA hypermethylation and tumor cell proliferation, and activates the immune microenvironment in mIDH LGG. In conclusion, these data elucidate the mechanisms underlying mIDH inhibition and support further development of mIDH inhibitors in mIDH LGG. Page 1 of 1 Citation Format: Min Lu, Ingo K. Mellinghoff, Aaron Diaz, Jennie W. Taylor, Sung Choe, Ania Tassinari, Dongwei Zhu, Katie Sellers, Kha Le, Feng Tai, Islam Hassan, Shuchi S. Pandya, Lori Steelman, Bin Wu. Inhibiting IDH mutations in low-grade glioma alters cellular function and the immune environment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2046.

Sarcosine Suppresses Epileptogenesis in Rats With Effects on Hippocampal DNA Methylation.

Epileptogenesis is a common consequence of brain insults, however, the prevention or delay of the epileptogenic process remains an important unmet medical challenge. Overexpression of glycine transporter 1 (GlyT1) is proposed as a pathological hallmark in the hippocampus of patients with temporal lobe epilepsy (TLE), and we previously demonstrated in rodent epilepsy models that augmentation of glycine suppressed chronic seizures and altered acute seizure thresholds. In the present study we evaluated the effect of the GlyT1 inhibitor, sarcosine (aka N-methylglycine), on epileptogenesis and also investigated possible mechanisms. We developed a modified rapid kindling model of epileptogenesis in rats combined with seizure score monitoring to evaluate the antiepileptogenic effect of sarcosine. We used immunohistochemistry and Western blot analysis for the evaluation of GlyT1 expression and epigenetic changes of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the epileptogenic hippocampi of rats, and further evaluated expression changes in enzymes involved in the regulation of DNA methylation, ten-eleven translocation methylcytosine dioxygenase 1 (TET1), DNA-methyltransferase 1 (DNMT1), and DNMT3a. Our results demonstrated: (i) experimental evidence that sarcosine (3 g/kg, i.p. daily) suppressed kindling epileptogenesis in rats; (ii) the sarcosine-induced antiepileptogenic effect was accompanied by a suppressed hippocampal GlyT1 expression as well as a reduction of hippocampal 5mC levels and a corresponding increase in 5hmC; and (iii) sarcosine treatment caused differential expression changes of TET1 and DNMTs. Together, these findings suggest that sarcosine has unprecedented disease-modifying properties in a kindling model of epileptogenesis in rats, which was associated with altered hippocampal DNA methylation. Thus, manipulation of the glycine system is a potential therapeutic approach to attenuate the development of epilepsy.

Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor

A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies.

A MYC Driven and Metabolite Executed Axis Controls the Subcellular Localization and Activity of TET DNA Hydroxylases and RNA Demethylases in B Cell Lymphomas

Mitochondria play a role in epigenetic remodeling by generating intermediate metabolites (αKG, 2-HG, succinate, etc.) and modulating the activity of enzymes that control DNA, RNA and histone demethylation. The mitochondrial dehydrogenases D2HGDH and L2HGDH regulate 2-HG/αKG homeostasis by catalyzing the oxidation of D2-HG and L2-HG, respectively, into αKG. In humans, loss of either enzyme causes neuro-metabolic syndromes and early mortality, pointing to their essential role in physiology. Yet, the regulation of D2HGDH and L2HGDH remains to be characterized. To address this issue, we mapped the D2HGDH and L2HGDH promoter regions. Using reporter and ChIP assays, lymphoma cell lines and a mouse model, we discovered that MYC binds to E-boxes in these promoters and directly induce D2HGDH and L2HGDH transcription. We created CRISPR KOs of D2HGDH or L2HGDH in MYC-inducible P493-6 cells, and quantified αKG, D2-HG and L2-HG by LC/MS. We found that turning MYC expression ON led to significantly higher accumulation of αKG (and decrease in 2-HG) in cells expressing D2/L2HGDH than in the KO models, confirming that MYC's transcriptional activation of D2/L2HGDH directly influences 2-HG/αKG balance. Then, we tested whether the metabolic output of the MYC-D2/L2HGDH axis influenced the activity of the αKG-dependent TET enzymes, and of the RNA demethylases FTO and ALKBH5. In P493-6 cells, B cells from Eμ-Myc mice and DLBCL cell lines, MYC expression led to a significant increase in 5hmC, decrease in 5mC DNA, and reduction in m6A RNA marks. These MYC-induced changes were blunted in D2HGDH or L2HGDH KO cells. In addition, the effects of MYC on 5hmC/5mC and m6A were blocked in cells exposed to cell-permeable D2-HG or L2-HG, while synthetic αKG rescued D2HGDH and L2HGDH KO cells. Next, we quantified TET and FTO/ALKBH5 activity in the nuclear lysates of our models. In all instances, MYC significantly elevated TET/FTO/ALKBH5 activity in D2HGDH/L2HGDH WT cells, an outcome that was significantly dampened in the KO models. These data suggested that the MYC-D2/L2HGDH axis increases the activity of these enzymes by generating αKG. To examine whether additional layers of control were also operational, we quantified the expression of TET1-3, FTO and ALKBH5, and of members of the RNA methyltransferase complex, METLL3, METLL14 and WTAP, and confirmed that their global expression were unmodified by MYC or other genetic and metabolic perturbations. Strikingly, however, when examining subcellular localization, we found that MYC, in a D2/L2HGDH dependent manner, promoted the nuclear localization of TET1-3, FTO and ALKBH5 (but not METTL3/14). Remarkably, these effects were readily recapitulated by αKG, whereas 2-HG retained these proteins in the cytoplasm, effectively reducing their ability to chemically modify DNA and RNA. Lastly, to test if the MYC-driven increase of TET activity was also present in primary tumors, we explored the DLBCL TCGA dataset. We mapped all enhancers that are active in DLBCL and found that enhancer hypomethylation (a putative consequence of TET activity) significantly correlated with MYC expression (r=0.38, p=0.009) or activity (ssGSEA-derived MYC activity score, r=0.46, p=0.0013); this finding remained significant after excluding potentially cofounding TET2-mutant DLBCLs. We next tested if this MYC-driven/TET-mediated enhancer demethylation was functionally relevant by correlating it to the expression of the gene closest to, or overlapping with, the enhancer. A biologically coherent negative correlation between enhancer methylation and target transcript expression was found for multiple genes, including several products previously implicated in lymphoma pathogenesis, including, FOXP1, PIM1, ATF5, KLHL14 and BRD2. In summary, we showed that D2HGDH and L2HGDH are transcriptional targets of MYC, and that the control of αKG levels by the MYC-D2/L2HGDH axis activates TETs and RNA demethylases. We discovered that MYC and intermediate metabolites control the sub-cellular localization of these enzymes, possibly via covalent modifications, thus adding an new layer of complexity to the remodeling of the epigenome and epitranscriptome in cancer. We showed in primary DLBCLs that MYC expression/activity correlates with hypomethylated/active oncogenic enhancers. Thus, we postulate that downstream to MYC TET enzymes may in specific contexts function as oncogenes. Disclosures No relevant conflicts of interest to declare.

Epigenetics and Regulation of Oxidative Stress in Diabetic Retinopathy.

PurposeOxidative stress plays a central role in the development of diabetic retinopathy, and in the pathogenesis of this blinding disease, activation of NADPH oxidase 2 (Nox2)-mediated cytosolic reactive oxygen species (ROS) production precedes mitochondrial damage. The multicomponent cytosolic Nox2 has an obligatory component, Ras-related C3 botulinum toxin substrate 1 (Rac1); in diabetes, Rac1 is functionally and transcriptionally active. Diabetes also facilitates many epigenetic modifications, and activates both DNA methylating (Dnmts) and hydroxymethylating (Tets) enzymes. Our aim was to investigate the role of epigenetics in Rac1 regulation in diabetes.MethodsUsing human retinal endothelial cells, exposed to high glucose, 5-methyl cytosine (5mC) and 5-hydroxy methyl cytosine (5hmC) levels, and binding of Dnmt and Tets were quantified at the Rac1 promoter. The effect of inhibition of Dnmts/Tets (pharmacological inhibitors or short interfering RNA [siRNA]) on glucose-induced activation of Rac1-ROS production was evaluated. Results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice receiving intravitreally Dnmt1-siRNA.ResultsDespite high glucose-induced increased binding of Dnmt1, 5mC levels remained subnormal at Rac1 promoter. But, at the same site, 5hmC levels and transcription factor nuclear factor (NF)-kB binding were increased. Inhibition of Dnmts/Tets prevented increase in 5hmC and NF-kB binding, and attenuated Rac1 activation. Similarly, in mouse retinal microvessels, Dnmt1-siRNA ameliorated diabetes-induced increase in Rac1 transcripts and activity, and decreased ROS levels.ConclusionsThus, despite Dnmts activation, concomitant increase in Tets rapidly hydroxymethylates 5mC, allowing NF-κB to bind and activate Rac1. These results imply a critical role of an active DNA methylation in cytosolic ROS regulation in the development of diabetic retinopathy.

Abstract 5325: 5-hydroxymethylcytosine profiling identifies differential targeting in IDH1 mutant versus IDH1 wild-type high-grade gliomas

Abstract Glioblastoma demonstrates significant epigenetic dysregulation with subgroup differences highlighted by the Glioma CpG-Island Methylator Phenotype (G-CIMP) seen in IDH1 mutant tumors. 5-hydroxymethylcytosine (5hmC) has been implicated in IDH1 wild-type gliomas, however its role in IDH1 mutant tumors remains incompletely understood. We examined 5hmC profiles in high grade (WHO III/IV) IDH1 mutant (n = 12) and IDH1 wild-type (n = 9) tumors through parallel processing of samples using bisulfite (BS) and oxidative bisulfite (OxBS) conversion, with subsequent analysis on the Illumina MethylationEPIC Beadchip platform. Hydroxymethylation profiles were correlated with gene expression measured using the Affymetrix Human Gene 2.0 ST array platform. Cumulative density plots for mean 5hmC β-value across IDH1 mutant and wild-type tumors demonstrated 5hmC accumulation predominantly within the top 10th percentile of probes. No significant difference in global 5hmC levels between IDH1 mutant versus wild-type tumors was evident. Mean 5hmC β-values were 4.6%% and 3.8% for IDH1 mutant and wild-type tumors across all probes, respectively. To focus on regions with greatest 5hmC abundance, we assessed probes among the top 1% mean 5hmC β-values. In addition to probes with high 5hmC abundance, differentially hydroxymethylated (DHMR) regions were identified using the R package ChAMP, comparing IDH1 mutant to wild-type tumors. At the probe level, top 1% and DHMR probes demonstrated a relative increase in 5hmC among IDH1 mutant tumors versus IDH1 wild-type. Top 1% and DHMR probes were enriched for enhancer and super-enhancer regions in both IDH1 mutant and IDH1 wild-type tumors, however enhancer and super-enhancers targeted only partially overlapped. Pathway enrichment analysis for top 1% probe gene targets and DHMR-associated genes identified pathways implicated in glioma pathogenesis. Among genes characteristically ‘hypermethylated' in G-CIMP + tumors, 28/50 were methylated while 22/50 were hypermethylated in our IDH1 mutant cohort, suggesting that 5hmC contributes to overall methylation of G-CIMP target genes. 5hmC marked the most highly expressed genes in our tumor cohort, with increased expression associated with gene body hydroxymethylation. Among 1367 genes differentially expressed between IDH1 mutant and wild-type tumors, 48 demonstrated a positive significant Spearman correlation between probe 5hmC β-value and gene expression (r ≥ 0.5, p &amp;lt; 0.05), including genes implicated in gliomagenesis as well as novel candidates. This correlation was most evident for genes upregulated in the IDH1 mutant cohort. Citation Format: Wioletta Glowacka, Harshika Jain, Makiko Okura, Abulizi Maimaitiming, Romina Nejad, Mamatjan Yasin, Hamza Farooq, Kenneth Aldape, Paul Kongkham. 5-hydroxymethylcytosine profiling identifies differential targeting in IDH1 mutant versus IDH1 wild-type high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5325.

DNA Hydroxymethylation Regulates Beta-Cell Maturation and Expansion

Epigenetic mechanisms play a central role in governing functional beta-cell mass. In particular, the methylation of DNA to generate 5-methylcytosine (5mC), directed by DNA methyltransferases, has been shown to regulate the establishment and maintenance of beta-cell identity and function. While the contribution of DNA methylation to beta-cell homeostasis and diabetes pathogenesis has been studied in detail, not much is known about the role of DNA demethylation in beta-cells. Oxidative conversion of 5mC to 5-hydroxymethylcytosine (5hmC) by Ten Eleven Translocation (Tet) enzymes constitutes a key step towards active DNA demethylation. In the present study, we provide evidence that the 5hmC landscape is dynamic, and undergoes specific changes during embryonic and postnatal islet development, islet adaptation, and in diabetes. We report that the Pdx1-positive pancreatic progenitors are marked by very low 5hmC, and beta-cell differentiation and functional maturation is accompanied by a progressive increase in 5hmC. However, under conditions during adaptive beta-cell expansion as well as in diabetes 5hmC levels decline in part due to the oxidative stress. These changes in 5hmC pattern are conserved in mouse and human pancreas, and are accompanied by the changes in Tet2 levels. We show that deletion of Tet2 in the pancreatic lineage results in improved glucose tolerance and beta-cell function. Together, our data show the importance of Tet2-dependent 5hmC patterning in maintaining a differentiated beta-cell phenotype, and highlight the remodeling of beta-cell epigenetic landscape as a key component of diabetes pathogenesis. Disclosure T. Gurlo: None. S. Georgia: None. S. Dhawan: None.

A cross-omics approach to investigate temporal gene expression regulation by 5-hydroxymethylcytosine via TBH-derived oxidative stress showed involvement of different regulatory kinases

Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.

Histone and DNA methylation control by H3 serine 10/threonine 11 phosphorylation in the mouse zygote

BackgroundIn the mammalian zygote, epigenetic reprogramming is a tightly controlled process of coordinated alterations of histone and DNA modifications. The parental genomes of the zygote show distinct patterns of histone H3 variants and distinct patterns of DNA and histone modifications. The molecular mechanisms linking histone variant-specific modifications and DNA methylation reprogramming during the first cell cycle remain to be clarified.ResultsHere, we show that the degree and distribution of H3K9me2 and of DNA modifications (5mC/5hmC) are influenced by the phosphorylation status of H3S10 and H3T11. The overexpression of the mutated histone variants H3.1 and 3.2 at either serine 10 or threonine 11 causes a decrease in H3K9me2 and 5mC and a concomitant increase in 5hmC in the maternal genome. Bisulphite sequencing results indicate an increase in hemimethylated CpG positions following H3.1T10A overexpression suggesting an impact of H3S10 and H3T11 phosphorylation on DNA methylation maintenance.ConclusionsOur data suggest a crosstalk between the cell-cycle-dependent control of S10 and T11 phosphorylation of histone variants H3.1 and H3.2 and the maintenance of the heterochromatic mark H3K9me2. This histone H3 “phospho-methylation switch” also influences the oxidative control of DNA methylation in the mouse zygote.

Dynamic DNA methylation of matrix metalloproteinase-9 in the development of diabetic retinopathy

Diabetes elevates matrix metalloproteinase-9 (MMP-9) in the retina and its capillary cells, and activated MMP-9 damages mitochondria, accelerating retinal capillary cell apoptosis, a phenomenon which precedes the development of retinopathy. Diabetes also favors epigenetic modifications regulating the expression of many genes. DNA methylation is maintained by methylating–hydroxymethylating enzymes, and retinal DNA methyltransferase (Dnmt) is activated in diabetes. Our aim is to investigate the role of DNA methylation in MMP-9 regulation. The effect of high glucose on 5-methylcytosine (5mC) and 5-hydroxymethyl cytosine (5hmC), and binding of Dnmt1 and hydroxymethylating enzyme (Tet2) on MMP-9 promoter were quantified in retinal endothelial cells. Specific role of Tet2 in MMP-9 activation was validated using Tet2-siRNA. The results were confirmed in the retina from streptozotocin-induced diabetic mouse. Although glucose increased Dnmt1 binding at MMP-9 promoter, it decreased 5mC levels. At the same promoter site, Tet2 binding and 5hmC levels were elevated. Tet2-siRNA ameliorated increase in 5hmC and MMP-9 transcription, and protected mitochondrial damage. Diabetic mice also presented similar dynamic DNA methylation changes in the retinal MMP-9 promoter. Thus, in diabetes transcription of retinal MMP-9 is maintained, in part, by an active DNA methylation–hydroxymethylation process, and regulation of this machinery should help maintain mitochondrial homeostasis and inhibit the development/progression of diabetic retinopathy.

Enhanced MAPK signaling drives ETS1-mediated induction of miR-29b leading to downregulation of TET1 and changes in epigenetic modifications in a subset of lung SCC.

Non-small-cell lung cancer is the leading cause of cancer death worldwide and is comprised of several histological subtypes, the two most common being adenocarcinoma (AC) and squamous cell carcinoma (SCC). Targeted therapies have successfully improved response rates in patients with AC tumors. However, the majority of SCC tumors lack specific targetable mutations, making development of new treatment paradigms for this disease challenging. In the present study, we used iterative non-negative matrix factorization, an unbiased clustering method, on mRNA expression data from the cancer genome atlas (TCGA) and a panel of 24 SCC cell lines to classify three disease segments within SCC. Analysis of gene set enrichment and drug sensitivity identified an immune-evasion subtype that showed increased sensitivity to nuclear factor-κB and mitogen-activated protein kinase (MAPK) inhibition, a replication-stress associated subtype that showed increased sensitivity to ataxia telangiectasia inhibition, and a neuroendocrine-associated subtype that showed increased sensitivity to phosphoinositide 3-kinase and fibroblast growth factor receptor inhibition. Additionally, each of these subtypes exhibited a unique microRNA expression profile. Focusing on the immune-evasion subtype, bioinformatic analysis of microRNA promoters revealed enrichment for binding sites for the MAPK-driven ETS1 transcription factor. Indeed, we found that knockdown of ETS1 led to upregulation of eight microRNAs and downregulation of miR-29b in the immune-evasion subtype. Mechanistically, we found that miR-29b targets the DNA-demethylating enzyme, TET1, for downregulation resulting in decreased 5-hmC epigenetic modifications. Moreover, inhibition of MAPK signaling by gefitinib led to decreased ETS1 and miR-29b expression with a corresponding increase in TET1 expression and increase in 5-hmC. Collectively, our work identifies three subtypes of lung SCC that differ in drug sensitivity and shows a novel mechanism of miR-29b regulation by MAPK-driven ETS1 expression which leads to downstream changes in TET1-mediated epigenetic modifications.

Robust quantitative assessments of cytosine modifications and changes in the expressions of related enzymes in gastric cancer.

The rediscovery of 5-hydroxymethylcytosine, the ten-eleven translocation (TET) family, thymine-DNA glycosylase (TDG) and isocitrate dehydrogenase (IDH) have opened new avenues in the study of DNA demethylation pathways in gastric cancer (GC). We performed a comprehensive and robust analysis of these genes and modified cytosines in gastric cancer. Liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) was used to assess 5-methyldeoxycytidine (5-mC), 5-hydroxymethyldeoxycytidine (5-hmC), 5-formyldeoxycytidine (5-fC) and 5-carboxyldeoxycytidine (5-caC) quantitatively in tumorous and non-tumorous regions of GCs; [D2]-5-hmC was used as an internal standard. Expression levels of the genes TET1, TET2, TET3, TDG, IDH1 and IDH2 were measured using a real-time reverse transcription polymerase chain reaction (RT-PCR) and were compared to the clinical attributes of each case. Using HEK293T cells the effects of introducing plasmids containing full-length TET1, TET2, and TET3 and 7 variants of the TET2 catalytic domain were evaluated in terms of their effect on cytosine demethylation. LC-MS/MS showed that 5-hmC was significantly decreased in tumorous portions. 5-mC was also moderately decreased in tumors, while 5-fC and 5-caC were barely detectable. The expressions of TET1, TET2, TET3, TDG and IDH2, but not IDH1, were notably decreased in GCs, compared with the adjacent non-tumor portion. TET1 expression and the 5-hmC levels determined using LC-MS/MS had a significantly positive correlation and TET1 protein had a greater effect on the increase in 5-hmC than TET2 and TET3 in HEK293T cells. The loss of 5-hmC and the down-regulation of TET1-3, TDG and IDH2 were found in GCs. The loss of 5-hmC in GCs was mainly correlated with the down-regulation of TET1.

Highlights from the latest articles in epigenomics

ISSN 1750-1911 Epigenomics (2014) 6(1), 17–19 Regulation of memory acquisition and extinction by TET1 Evaluation of: Kaas GA, Zhong C, Eason DE et al. TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation. Neuron 79, 1086–1093 (2013); Rudenko A, Dawlaty MM, Seo J et al. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109–1122 (2013). Accumulating evidence indicates that altered gene expression in response to changes in the environment underlies the plasticity in the brain, including learning and memory. Epigenetic modifications, such as DNA methylation and histone acetylation, have been shown to critically regulate gene transcription, providing a candidate mechanism for synaptic and experience-dependent behavioral plasticity. The dynamic changes of epigenetic modifications (e.g., histone acetylation and deacetylation) play key roles in memory formation and extinction [1]. Generation of methylated cytosine (5mC) in DNA was once considered as a stable modification to silence gene transcription. However, recent studies indicated that DNA methylation may undergo rapid changes in vivo. The teneleven translocation (TET) family enzymes, TET1, TET2 and TET3, catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and thus participate in active DNA demethylation [2,3]. Guo et al. found that in response to neuronal activation, TET1 mediates demethylation of the Fgf1 and the Bdnf promoters in the brain [4]. However, the potential role of TET1 as well as TET1mediated DNA demethylation in memory formation is not clear. Recent work by Kaas et al. revealed that TET1 exerts function in active DNA demethylation, gene transcription and memory formation [5]. They showed that TET1 is expressed in neurons throughout the hippocampus in the brain and stimulation of neuronal activity by KCl or seizure downregulates its transcription in hippocampal neurons. Overexpression of TET1 in the dorsal hippocampus result in a significant decrease in global level of 5mC and an increase of 5hmC, indicating that TET1 effectively promotes DNA demethylation by converting 5mC to 5hmC. In parallel, upregulation of activity-dependent genes involved in neuroplasticity, such as Fos, Arc, Egr1, Homer1 and Ner4a2 was also detected in the hippocampus overexpressing TET1. Moreover, Kaas et al. investigated the behavioral effects of TET1. Overexpression of TET1 in the dorsal hippocampus does not affect locomotion and anxietylike behavior, but significantly impairs long-term conditioned fear memory. Interestingly, the expression of a catalytically inactive mutant of TET1 also affected gene expression and memory formation, raising the question that if TET1 regulates gene expression and memory via both hydroxylase activity-dependent and hydroxylase activity-independent mechanisms. Earlier research by Zhang and colleagues showed that Tet1-knockout mice exhibit impaired hippocampal neurogenesis and hippocampus-dependent spatial memory [6]. Rudenko et al. recently examined another stain of Tet1-knockout mice generated by depleting exon 4 [7]. They found that ablation of TET1 does not change the level of 5mC but leads to a reduction of Highlights from the latest articles in epigenomics