DNA Methylation Of Gene Promoter Research Articles

Abstract LB422: Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease

Abstract Introduction: Epstein-Barr virus positive (EBV+) central nervous system lymphoproliferative diseases (CNS-LPD) are aggressive clinical conditions with poor prognosis. We have previously reported use of a ganciclovir (GCV), zidovudine (AZT), rituximab and dexamethasone regimen (GARD) that induced complete and durable responses in patients with primary CNS post-transplant lymphoproliferative disease (PTLD). Responses were associated with detection of two GCV/AZT viral drug targets, the EBV protein kinases BGLF4 and BXLF1. These viral proteins are normally expressed in lytic phase EBV, and the molecular basis for expression in latently infected EBV+ CNS-LPD is unknown. Methods: RNA expression of EBV genes LMP1, BZLF1, BXLF1, and BGLF4 was measured in 12 CNS-LPD biopsies (n=8/12 DLBCL-type) via qRT-PCR. Mass spectrometry-based EpiTYPER assay was used to quantify DNA methylation in EBV gene promoters. Biopsies from 25 patients with systemic PTLD were used for comparison. The BGLF4 locus was investigated in detail using luciferase reporters and 5’RACE mapping of transcription start sites in 3 EBV-infected cell lines and 4 CNS-LPD biopsy samples. Overall EBV methylation was assessed in a screening assay covering 31 EBV loci. Mechanistic involvement of active demethylation via TET2 was studied in a HEK293 CRISPR-Cas9 gene knockout model under EBV (strain M81) infection. Results: Gene expression analysis of biopsy specimens showed expression of LMP1, BXLF1, and BGLF4, but not BZLF1. Expression of BXLF1 and BGLF4 was significantly higher in CNS-LPD when compared to systemic EBV+ PTLD (p=0.043 and p=0.00022, respectively). EpiTYPER data showed significantly decreased promoter methylation at BGLF4 (p=0.00013). Luciferase reporter analysis of the BGLF4 upstream sequence revealed 3 regions of promoter activity which were silenced by in vitro methylation of reporters. 5’RACE identified transcription start sites at all 3 BGLF4 promoters and detected their activity in EBV+ cell lines and CNS-LPD biopsies. By overlaying transcriptional activity, 5’RACE and DNA methylation data, we identified DNA methylation loss at single CpG dinucleotides correlated with BGLF4 expression (r2=0.73). These sites were specifically demethylated in CNS-LPD, while surrounding EBV methylation remained high. Knockout of TET2 followed by EBV infection revealed that active demethylation is required for demethylation at BGLF4 promoters. Conclusions: The molecular determinant for success of the GARD regimen in CNS-LPD has remained poorly understood, with immunohistochemical detection of BGLF4/BXLF1 as the only predictor of treatment response. Our results provide the molecular basis for expression of BGLF4 in CNS-LPD by showing decreased promotor DNA methylation. We identified specific BGLF4 methylation sites that hold promise as potential DNA biomarkers for EBV+ CNS-LPD. Citation Format: Christoph Weigel, Haley L. Klimaszewski, Selamawit Addissie, Sarah Schlotter, Fode Tounkara, James P. Dugan, Bradley M. Haverkos, Lynda Villagomez, Mark Lustberg, Pierluigi Porcu, Timothy Voorhees, Michael A. Caligiuri, Ginny Bumgardner, Christopher C. Oakes, Robert Baiocchi. Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB422.

Longitudinal analysis of influenza vaccination implicates regulation of RIG-I signaling by DNA methylation

Influenza virus infection alters the promoter DNA methylation of key immune response-related genes, including type-1 interferons and proinflammatory cytokines. However, less is known about the effect of the influenza vaccine on the epigenome. We utilized a targeted DNA methylation approach to study the longitudinal effects (day 0 pre-vaccination and day 28 post-vaccination) on influenza vaccination responses in peripheral blood mononuclear cells. We found that baseline, pre-vaccination methylation profiles are associated with pre-existing, protective serological immunity. Additionally, we identified 481 sites that were differentially methylated between baseline and day 28 post-vaccination. These were enriched for genes involved in the regulation of the RIG-I signaling pathway, an important regulator of viral responses. Our results suggest that DNA methylation changes to components of the RIG-I pathway are associated with vaccine effectiveness. Therefore, immunization strategies that target this pathway may improve serological responses to influenza vaccination.

TET2-mediated ECM1 hypomethylation promotes the neovascularization in active proliferative diabetic retinopathy

BackgroundStudies have shown that tet methylcytosine dioxygenase 2 (TET2) is highly expressed in diabetic retinopathy (DR), which reduces the DNA methylation of downstream gene promoters and activates the transcription. Abnormally expressed TET2 and downstream genes in a high-glucose environment are associated with retinal capillary leakage and neovascularization. Here, we investigated the downstream genes of TET2 and its potential association with neovascularization in proliferative diabetic retinopathy (PDR).MethodsGSE60436, GSE57362, and GSE158333 datasets were analyzed to identify TET2-related hypomethylated and upregulated genes in PDR. Gene expression and promoter methylation of these genes under high glucose treatment were verified. Moreover, TET2 knockdown was used to assess its impact on tube formation and migration in human retinal microvascular endothelial cells (HRMECs), as well as its influence on downstream genes.ResultsOur analysis identified three key genes (PARVB, PTPRE, ECM1) that were closely associated with TET2 regulation. High glucose-treated HRMECs exhibited increased expression of TET2 and ECM1 while decreasing the promoter methylation level of ECM1. Subsequently, TET2 knockdown led to decreased migration ability and tube formation function of HRMECs. We further found a decreased expression of PARVB, PTPRE, and ECM1, accompanied by an increase in the promoter methylation of ECM1.ConclusionsOur findings indicate the involvement of dysregulated TET2 expression in neovascularization by regulating the promoter methylation and transcription of downstream genes (notably ECM1), eventually leading to PDR. The TET2-induced hypomethylation of downstream gene promoters represents a potential therapeutic target and offers a novel perspective on the mechanism underlying neovascularization in PDR.Graphical

DNA Methylation of the IL-17A Gene Promoter Is Associated with Subclinical Atherosclerosis and Coronary Artery Disease: The Genetics of Atherosclerotic Disease Mexican Study.

The interleukin-17 (IL-17) has a crucial role during inflammation and has been associated with cardiovascular diseases, but its role in epigenetics is still poorly understood. Therefore, the aim of this study was to evaluate the DNA methylation status of the IL-17A gene promoter to establish whether it may represent a risk factor for subclinical atherosclerosis (SA) or clinical coronary artery disease (CAD). We included 38 patients with premature CAD (pCAD), 48 individuals with SA, and 43 healthy controls. Methylation in the CpG region of the IL-17A gene promoter was assessed via methylation-specific polymerase chain reaction (MSP). Individuals with SA showed increased methylation levels compared to healthy controls and pCAD patients, with p < 0.001 for both. Logistic regression analysis showed that high methylation levels represent a significant risk for SA (OR = 5.68, 95% CI = 2.38-14.03, p < 0.001). Moreover, low methylation levels of the IL-17A gene promoter DNA represent a risk for symptomatic pCAD when compared with SA patients (OR = 0.16, 95% CI = 0.06-0.41, p < 0.001). Our data suggest that the increased DNA methylation of the IL-17A gene promoter is a risk factor for SA but may be a protection factor for progression from SA to symptomatic CAD.

DNA methylation profiles in urothelial bladder cancer tissues and children with schistosomiasis from Eggua, Ogun State, Nigeria

BackgroundSquamous cell carcinoma has been attributed to chronic schistosomiasis and is the predominant type of bladder cancer in schistosomiasis endemic areas. The aim of this study was to assess early promoter DNA methylation in selected genes implicated in schistosomiasis-associated bladder cancer (SABC).MethodsA total of 159 urine samples were collected from school-aged children in Eggua Community of Ogun State and examined by microscopy for Schistosoma haematobium eggs. From this sample, a subset of 34 (21.1%) urine samples positive for S. haematobium, age and sex-matched with negative urine control samples, and 16 formalin-fixed paraffin-embedded bladder cancer tissues obtained from the University College Hospital were subjected to DNA isolation and bisulphite DNA conversion. Quantitative methylation-specific PCR was used to determine the methylation status of APC, RARβ2, RASSF1A, and TIMP3 in the samples.ResultsHigh degrees of methylation of RARβ2(67.7%), RASSF1A (38.2%), and TIMP3(52.9%) was more common in urogenital schistosomiasis (UGS)-positive urine samples than negative urine (control) samples and in bladder cancer tissues. Promoter DNA methylation in the positive urine samples was 1.4-fold, 13.3-fold, 3.4-fold, and 3.8-fold higher in APC, RARβ2, RASSF1A, and TIMP3, respectively, than in the matched controls. The odds of promoter methylation were likely to increase with age group for APC (OR: 1.615) and TIMP3(OR: 2.000); sex for TIMP3(OR: 2.644); and haematuria for RARβ2(OR: 1.094), RASSF1A (OR: 1.143), and TIMP3(OR: 1.842), although there were no significant associations. Conclusions: Gene promoter DNA methylation in tumour suppressor genes was observed in schistosomiasis cases. Hence, promoter DNA methylation may occur during active schistosomiasis in children. This result may serve as an early non-invasive biomarker to detect and hint at the risk of developing SABC later in life.

DNA methylation of ADAMTS7 gene promoter in direct oral anticoagulant-induced bleeding in patients with atrial fibrillation

Abstract Introduction Direct Oral Anticoagulants (DOACs) are recommended as first-line treatment in atrial fibrillation (AF) patients. Despite their benefits and effectiveness, several patients experience bleeding events. No studies have addressed how epigenetic modifications may affect DOAC treatment. To fill this gap, we are conducting a study aiming to follow in time changes of DNA methylation patterns in naïve AF patients starting DOAC therapy. Herein, we present our findings regarding DNA methylation analysis of ADAM Metallopeptidase with Thrombospondin Type 1 Motif 7 (ADAMTS7), a protease that acts on the cardiovascular system. Methods A total of 76 AF patients treated with dabigatran, rivaroxaban, or apixaban and 22 non-AF controls were included. Genomic DNA has been isolated at baseline (t0, controls and patients), and for patients at 7 (t1) and 28 (t2) days of DOAC treatment. Blood genomic DNA was isolated and bisulfite converted prior to methylation analysis. Promoter DNA methylation of ADAMTS7 was analyzed with qMSP-PCR. Results In our study, no major bleeding or thrombotic events were recorded. A total of 16 minor bleeding events occurred. The percentage of ADAMTS7 methylation at baseline did not differ between patients and controls (16,8% vs. 14,8%, p=0,138). In patient cohort, DOAC therapy did not alter ADAMTS7 methylation at different timepoints (16,8% at t0, 15,7% at t1 and 14,3% at t2, p=0,293 from t0 to t1, p=0,06 from t0 to t2 and p=0,205 from t0 to t1). When patients were categorized into experiencing bleeding events (cases) or not (controls), ADAMTS7 was demethylated from t0 to t2 (-3,7% in cases vs. -1.2% in controls, p=0.015) and from t1 to t2 (-2,1% in cases vs. -1,1% in controls, p=0.034). In adjusted regression analysis, bleeding was associated with ADAMTS7 demethylation (β=-2,973, 95%CI -5,481, -0,466, p=0,021). Conclusion This is the first study of DNA methylation on DOACs. Our findings suggest that the methylation of the promoter of ADAMTS7 is reduced when DOAC-related bleeding occurs, increasing its expression levels, and may subsequently promote hemostasis and endothelium remodeling through different pathways.

DNA Methylation Landscape of Peripheral T-Cell Lymphomas Not Otherwise Specified

DNA methylation is an epigenetic modification catalyzed by DNA methyltransferases that regulate gene transcription in mammalian cells. DNA methylation in gene promoters is associated with repressive histone marks and contributes to gene repression. This process is involved in various physiological processes, including development, X chromosome inactivation, genomic imprinting, differentiation, and hematopoiesis. The global, genome-wide deregulation of methylation patterns contributes to the pathogenesis of immune disorders, hematologic malignancies, and cancer. Because aberrations in DNA methylation can deregulate spatiotemporal gene expression in a cell type-specific manner, genome-wide changes in this epigenetic modification may contribute to tumor development. Specifically, the downregulation of tumor suppressors through promoter hypermethylation is a widely accepted mechanism in cancer progression. Due to its distinct patterns in cancer, DNA methylation can be used as a diagnostic and predictive biomarker. Peripheral T-cell lymphomas, Not Otherwise Specified (PTCL-NOS), are a group of heterogeneous T-cell malignancies with diverse cytogenic, molecular, and morphological phenotypes. Despite large-scale gene expression profiling, the molecular features-and DNA methylation in particular-of these rare but aggressive malignancies remain poorly understood. To better understand the molecular landscape of PTCL-NOS, we performed global methylation profiling via Whole Genome Bisulfite Sequencing on 10 lymphomas and compared those to patterns seen in normal T-cells and Anaplastic Large Cell Lymphoma (ALCL). Our analysis revealed large-scale deregulation of methylation in all tumors at levels of both differentially methylated cytosines and regions. The dominant molecular change was hypomethylation, which was, on average, 7 times more frequent than hypermethylation. Hypomethylation was not limited to repetitive elements but was also seen in all genomic elements, including promoters, enhancers, gene bodies, and exons. Out of 1,286 genes whose promoters were consistently hypomethylated in Peripheral T-cell lymphomas (PTCL), 39 genes were associated with an increase in expression, suggesting that the loss of methylation may have contributed to their deregulation. This hypomethylated and overexpressed signature contained genes that might be involved in tumor progression or maintenance, such as Rac GTPase activating protein 1 ( RACGAP1), regulator of chromosome condensation 1 ( RCC1), anillin actin-binding protein ( ANLN), and others. Out of 578 genes whose promoters were consistently hypermethylated in PTCL, the expression of 56 genes was decreased. These included genes with tumor suppressor function, such as splicing factor 3b subunit 1 ( SF3B1) and lymphoid enhancer-binding factor 1 ( LEF1). Our analysis also identified differential methylation between PTCL-NOS and ALCL in various genomic features. For example, 71 hypo- and 59 hypermethylated promoters were found only in ALCL but not PTCL-NOS. Conversely, 326 hypo- and 254 hypermethylated promoters were observed only in PTCL-NOS. Analysis of gene expression also revealed specific changes in both PTCL subtypes, manifested by the activation of different signaling pathways as identified by Ingenuity Pathway Analysis. PTCL-NOS was characterized by the suppression of the SUMOylation pathway, PTEN, and EIF2 Signaling, and the activation of NF-κB and Notch signaling. ALCLs were characterized by the suppression of the Role of CHK Proteins in Cell Cycle Control, Erythropoietin, ERBB signaling, the Coronavirus Pathogenesis pathway, IGF-1 signaling, and the activation of Glutathione-mediated Detoxification and Triacylglycerol Biosynthesis. Altogether, we identified recurrent specific methylation and expression changes that could serve as the basis for the development of tools facilitating the diagnosis and prognosis of PTCL-NOS. In addition, our data may contribute to the identification of epigenetically-regulated drivers of tumor initiation, progression, and maintenance.

METHYLATION PATTERN OF THE RUNX3 GENE AND ITS IMPACT ON RESPONSE TO IMATINIB THERAPY IN CHRONIC MYELOID LEUKEMIA

BackgroundChronic Myelogenous Leukemia (CML) is a clonal stem cell disease with deregulated tyrosine kinase activity of BCR-ABL. DNA methylation in gene promoters influences disease development, therapeutic response, and clinical outcome in various diseases.&#x0D; ObjectivesThis study aimed to investigate the methylation pattern in promoter regions of the runt-related transcription factor 3 (RUNX3) gene and its effect on the response to imatinib therapy, among CML patients.&#x0D; Patients and MethodsBlood samples were collected from 100 participants from November 2020 to February 2022, in two groups 75 samples of CML patients and 25 samples of healthy controls, genomic DNA was extracted from the samples by using the salting-out method, then Isolated DNA was treated with sodium bisulfite. By Methylation-Sensitive High-Resolution Melting (MS-HRM), the samples were analyzed.&#x0D; ResultsThe promoter sequence methylation level is significantly higher (P&lt;0.05) in DNA samples from CML patients than in the control group, the methylation level in most of the healthy control samples 96% was detected between 0-24% while in CML patients was 16%, in 25-49%, RUNX3 methylation levels in Health individuals were 4%, moreover in CML patients were 24% also, in 50-100% of methylation level, health control was 0%, in CML patient showed 60%. According to methylation detection of RUNX3 among Imatinib resistance and good response, the results of IM resistance were detected between 0-40% was 15.21%, Furthermore, in good response was 79.31%. In other hand at 41-100% RUNX3 methylation levels in IM resistance was 84.78%, while a good response was recorded at 20.68%.&#x0D; ConclusionThe study concluded that RUNX3 gene hypermethylation often occurs in CML patients. Additionally, there is a strong correlation between RUNX3 hypermethylation and poor imatinib respondents. We found that both IM resistance and CML patients have higher levels of RUNX3 gene hypermethylation. Moreover, research on a larger sample size is required to validate these findings, which may serve as possible indicators of disease progression and imatinib resistance.

CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny

Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.

Let-7a Downregulation Accompanied by KRAS Mutation Is Predictive of Lung Cancer Onset in Cigarette Smoke-Exposed Mice.

Let-7 is a tumor suppressor microRNA targeting the KRAS lung oncogene. Let-7a downregulation is reversible during the early stages of lung carcinogenesis but is irreversible in cancer cells. The aim of this study is to shed light on the relationship between oncogene (KRAS) mutation and let-7a downregulation in cigarette smoke (CS)-induced lung carcinogenesis. A total of 184 strain H Swiss albino mice were either unexposed (control) or exposed to CS for 2 weeks (short CS) or 8 months (long CS). After 8 months, the lungs were individually collected. The following end points have been evaluated: (a) DNA methylation of the let-7a gene promoter by bisulphite-PCR and pyrosequencing; (b) let-7a expression by qPCR; (c) KRAS mutation by DNA pyrosequencing; (d) cancer incidence by histopathological examination. let-7a expression decreased by 8.3% in the mice exposed to CS for two weeks (CS short) and by 33.4% (p ≤ 0.01) in the mice exposed to CS for 8 months (CS long). No significant difference was detected in the rate of let-7a-promoter methylation between the Sham-exposed mice (55.1%) and the CS short-(53%) or CS long (51%)-exposed mice. The percentage of G/T transversions in KRAS codons 12 and 13 increased from 2.3% (Sham) to 6.4% in CS short- and to 11.5% in CS long-exposed mice. Cancer incidence increased significantly in the CS long-exposed mice (11%) as compared to both the Sham (4%) and the CS short-exposed (2%) mice. In the CS long-exposed mice, the correlation between let-7a expression and the number of KRAS mutations was positive (R = +0.5506) in the cancer-free mice and negative (R = -0.5568) in the cancer-bearing mice. The effects of CS-induced mutations in KRAS are neutralized by the high expression of let-7a in cancer-free mice (positive correlation) but not in cancer-bearing mice where an irreversible let-7a downregulation occurs (negative correlation). This result provides evidence that both genetic (high load of KRAS mutation) and epigenetic alterations (let-7a irreversible downregulation) are required to produce lung cancer in CS-exposed organisms.

ZMYM2 is essential for methylation of germline genes and active transposons in embryonic development.

ZMYM2 is a transcriptional repressor whose role in development is largely unexplored. We found that Zmym2-/- mice show embryonic lethality by E10.5. Molecular characterization of Zmym2-/- embryos revealed two distinct defects. First, they fail to undergo DNA methylation and silencing of germline gene promoters, resulting in widespread upregulation of germline genes. Second, they fail to methylate and silence the evolutionarily youngest and most active LINE element subclasses in mice. Zmym2-/- embryos show ubiquitous overexpression of LINE-1 protein as well as aberrant expression of transposon-gene fusion transcripts. ZMYM2 homes to sites of PRC1.6 and TRIM28 complex binding, mediating repression of germline genes and transposons respectively. In the absence of ZMYM2, hypermethylation of histone 3 lysine 4 occurs at target sites, creating a chromatin landscape unfavourable for establishment of DNA methylation. ZMYM2-/- human embryonic stem cells also show aberrant upregulation and demethylation of young LINE elements, indicating a conserved role in repression of active transposons. ZMYM2 is thus an important new factor in DNA methylation patterning in early embryonic development.

Lineage plasticity and treatment resistance in prostate cancer: the intersection of genetics, epigenetics, and evolution.

Androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, and the development of castrate-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-related mortality. While CRPC typically develops through a gain in androgen receptor (AR) signaling, a subset of CRPC will lose reliance on the AR. This process involves genetic, epigenetic, and hormonal changes that promote cellular plasticity, leading to AR-indifferent disease, with neuroendocrine prostate cancer (NEPC) being the quintessential example. NEPC is enriched following treatment with second-generation anti-androgens and exhibits resistance to endocrine therapy. Loss of RB1, TP53, and PTEN expression and MYCN and AURKA amplification appear to be key drivers for NEPC differentiation. Epigenetic modifications also play an important role in the transition to a neuroendocrine phenotype. DNA methylation of specific gene promoters can regulate lineage commitment and differentiation. Histone methylation can suppress AR expression and promote neuroendocrine-specific gene expression. Emerging data suggest that EZH2 is a key regulator of this epigenetic rewiring. Several mechanisms drive AR-dependent castration resistance, notably AR splice variant expression, expression of the adrenal-permissive 3βHSD1 allele, and glucocorticoid receptor expression. Aberrant epigenetic regulation also promotes radioresistance by altering the expression of DNA repair- and cell cycle-related genes. Novel therapies are currently being developed to target these diverse genetic, epigenetic, and hormonal mechanisms promoting lineage plasticity-driven NEPC.

Hypoxic Preconditioning Modulates BDNF and Its Signaling through DNA Methylation to Promote Learning and Memory in Mice.

Hypoxic preconditioning (HPC) as an endogenous mechanism can resist hypoxia/ischemia injury and exhibit protective effects on neurological function including learning and memory. Although underlying molecular mechanisms remain unclear, HPC probably regulates the expression of protective molecules by modulating DNA methylation. Brain-derived neurotrophic factor (BDNF) activates its signaling upon binding to the tropomyosin-related kinase B (TrkB) receptor, which is involved in neuronal growth, differentiation, and synaptic plasticity. Therefore, this study focused on the mechanism by which HPC regulates BDNF and BDNF/TrkB signaling through DNA methylation to influence learning and memory. Initially, the HPC model was established by hypoxia stimulations on ICR mice. We found that HPC downregulated the expression of DNA methyltransferase (DNMT) 3A and DNMT3B. Then, the upregulation of BDNF expression in HPC mice was generated from a decrease in DNA methylation of the BDNF gene promoter detected by pyrophosphate sequencing. Subsequently, upregulation of BDNF activated BDNF/TrkB signaling and ultimately improved learning and spatial memory in HPC mice. Moreover, after mice were intracerebroventricularly injected with the DNMT inhibitor, the restraint of DNA methylation accompanied by an increase of BDNF and BDNF/TrkB signaling was also discovered. Finally, we observed that the inhibitor of BDNF/TrkB signaling prevented HPC from ameliorating learning and memory in mice. However, the DNMT inhibitor promoted spatial cognition in mice. Thus, we suggest that HPC may upregulate BDNF by inhibiting DNMTs and decreasing DNA methylation of the BDNF gene and then activate BDNF/TrkB signaling to improve learning and memory in mice. This may provide theoretical guidance for the clinical treatment of cognitive dysfunction caused by ischemia/hypoxia disease.

The combination of DNA methylation and positive regulation of anthocyanin biosynthesis by MYB and bHLH transcription factors contributes to the petal blotch formation in Xibei tree peony.

Xibei tree peony is a distinctive cultivar group that features red-purple blotches in petals. Interestingly, the pigmentations of blotches and non-blotches are largely independent of one another. The underlying molecular mechanism had attracted lots of attention from investigators, but was still uncertain. Our present work demonstrates the factors that are closely related to blotch formation in Paeonia rockii 'Shu Sheng Peng Mo'. Non-blotch pigmentation is prevented by the silencing of anthocyanin structural genes, among which PrF3H, PrDFR, and PrANS are the three major genes. We characterized two R2R3-MYBs as the key transcription factors that control the early and late anthocyanin biosynthetic pathways. PrMYBa1, which belongs to MYB subgroup 7 (SG7) was found to activate the early biosynthetic gene (EBG) PrF3H by interacting with SG5 member PrMYBa2 to form an 'MM' complex. The SG6 member PrMYBa3 interacts with two SG5 (IIIf) bHLHs to synergistically activate the late biosynthetic genes (LBGs) PrDFR and PrANS, which is essential for anthocyanin accumulation in petal blotches. The comparison of methylation levels of the PrANS and PrF3H promoters between blotch and non-blotch indicated a correlation between hypermethylation and gene silencing. The methylation dynamics of PrANS promoter during flower development revealed a potential early demethylating reaction, which may have contributed to the particular expression of PrANS solely in the blotch area. We suggest that the formation of petal blotch may be highly associated with the cooperation of transcriptional activation and DNA methylation of structural gene promoters.

The Effect of Metformin and Carbohydrate-Controlled Diet on DNA Methylation and Gene Expression in the Endometrium of Women with Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is an endocrine disease associated with infertility and metabolic disorders in reproductive-aged women. In this study, we evaluated the expression of eight genes related to endometrial function and their DNA methylation levels in the endometrium of PCOS patients and women without the disease (control group). In addition, eight of the PCOS patients underwent intervention with metformin (1500 mg/day) and a carbohydrate-controlled diet (type and quantity) for three months. Clinical and metabolic parameters were determined, and RT-qPCR and MeDIP-qPCR were used to evaluate gene expression and DNA methylation levels, respectively. Decreased expression levels of HOXA10, GAB1, and SLC2A4 genes and increased DNA methylation levels of the HOXA10 promoter were found in the endometrium of PCOS patients compared to controls. After metformin and nutritional intervention, some metabolic and clinical variables improved in PCOS patients. This intervention was associated with increased expression of HOXA10, ESR1, GAB1, and SLC2A4 genes and reduced DNA methylation levels of the HOXA10 promoter in the endometrium of PCOS women. Our preliminary findings suggest that metformin and a carbohydrate-controlled diet improve endometrial function in PCOS patients, partly by modulating DNA methylation of the HOXA10 gene promoter and the expression of genes implicated in endometrial receptivity and insulin signaling.

DNA methylation of selected tumor suppressor genes in endometrial hyperplasia.

To investigate DNA methylation of specific gene promoters in endometrial hyperplasia compared to normal endometrial tissue. To search for epigenetic events, methylation-specific multiplex ligation-dependent probe amplification was employed to compare the methylation status of 64 tissue samples with atypical endometrial hyperplasia, 60 tissue samples with endometrial hyperplasia without atypia, and 40 control tissue samples with normal endometrium. Differences in DNA methylation among the groups were found in PTEN, CDH13, and MSH6 promoters (PTEN: atypical hyperplasia 32%, benign hyperplasia 6.8%, normal endometrium 10%; P=0.004; CDH13: atypical hyperplasia, 50%; benign hyperplasia, 43%; normal endometrium 8.1%; P=0.003; MSH6 atypical hyperplasia 84%, benign hyperplasia, 62%; normal endometrium, 52%; P=0.008.) Higher rates of CDH13 promoter methylation were identified in the groups with both forms of endometrial hyperplasia when compared to the control group (atypical hyperplasia, P=0.003, benign hyperplasia, P=0.0002). A higher rate of DNA methylation of the PTEN and MSH6 promoters was observed in samples with atypical endometrial hyperplasia than in samples with benign endometrial hyperplasia (PTEN: P=0.02; MSH6: P=0.01) and samples with normal endometrial tissue (PTEN, P=0.04; MSH6, P=0.006). DNA methylation of CDH13, PTEN, and MSH6 appear to be involved in the development of endometrial hyperplasia.

DNMT3B overexpression downregulates genes with CpG islands, common motifs, and transcription factor binding sites that interact with DNMT3B

DNA methylation is a key epigenetic modification to regulate gene expression in mammalian cells. Abnormal DNA methylation in gene promoters is common across human cancer types. DNMT3B is the main de novo methyltransferase enhanced in several primary tumors. How de novo methylation is established in genes related to cancer is poorly understood. CpG islands (CGIs), common sequences, and transcription factors (TFs) that interact with DNMT3B have been associated with abnormal de novo methylation. We initially identified cis elements associated with DNA methylation to investigate the contribution of DNMT3B overexpression to the deregulation of its possible target genes in an epithelial cell model. In a set of downregulated genes (n = 146) from HaCaT cells with DNMT3B overexpression, we found CGI, common sequences, and TFs Binding Sites that interact with DNMT3B (we called them P-down-3B). PPL1, VAV3, IRF1, and BRAF are P-down-3B genes that are downregulated and increased their methylation in DNMT3B presence. Together these findings suggest that methylated promoters aberrantly have some cis elements that could conduce de novo methylation by DNMT3B.

New insight into the role of PTCH1 protein in serous ovarian carcinomas.

The Hedgehog (Hh) signaling pathway is essential for normal embryonic development, while its hyperactivation in the adult organism is associated with the development of various cancers. The role of the Hh signaling pathway in ovarian cancer has not been sufficiently investigated. Therefore, the present study investigated the role of protein patched homolog 1 (PTCH1), a component of the Hh signaling pathway, and changes in the promoter methylation status of the corresponding gene in a cohort of low-(LGSC) and high-grade serous ovarian carcinomas (HGSC) and HGSC cell lines (OVCAR8 and OVSAHO). PTCH1 protein expression level was analyzed using immunohistochemistry in tissue samples and immunofluorescence and western blotting in cell lines. DNA methylation patterns of the PTCH1 gene were analyzed using methylation-specific PCR. PTCH1 protein expression was significantly higher in HGSCs and LGSCs compared with controls (healthy ovaries and fallopian tubes). Similarly, ovarian cancer cell lines exhibited significantly higher PTCH1 protein expression compared with a normal fallopian tube non-ciliated epithelial cell line (FNE1). PTCH1 protein fragments of different molecular weights were detected in all cell lines, indicating possible proteolytic cleavage of this protein, resulting in the generation of soluble N-terminal fragments that are translocated to the nucleus. DNA methylation of the PTCH1 gene promoter was exclusively detected in a proportion of HGSC (13.5%) but did not correlate with protein expression. PTCH1 protein was highly expressed in serous ovarian carcinoma tissues and cell lines, while PTCH1 promoter methylation was only detected in HGSC. Further investigation is required to elucidate the possible mechanisms of PTCH1 activation in serous ovarian carcinomas.

Loss of Function of the RRMF Domain in OsROS1a Causes Sterility in Rice (Oryza sativa L.).

For crop seed production, the development of anthers and male fertility are the main agronomic traits and key biological processes for flowering plants. Active DNA demethylation regulates many plant developmental processes and is ensured by 5-meC DNA glycosylase enzymes. To find out the role of OsROS1a, OsROS1a gene editing mutants were generated using the CRISPR/Cas9 system. The osros1a mutants had shrink spikelets, smaller anthers and pollen grains, and were not stained by iodine staining showing a significant reduction in total soluble sugar and starch contents as compared to wildtype (WT), which caused complete male sterility. Similarly, the expression of genes involved in pollen and anther development was decreased in osros1a mutants as compared to WT. Furthermore, bisulfite sequencing showed that the CG and CHG methylation of the OsPKS2 gene promoter was significantly increased in the osros1a mutant, which caused a reduced expression of OsPKS2 in osros1a mutants. DNA methylation of the TDR gene promoter was similar between WT and osros1a mutants, indicating that the DNA methylation effect by OsROS1a was gene specific. The expression of OsROS1a in the mutants was not changed, but it produced a frame-shift mutation to truncate the Pem-CXXC and RRMF domains. Combined with previous studies, our findings suggested that the RRMF domain in OsROS1a is the functional domain and loss of RRMF for OsROS1a causes sterility in rice.

An integrative analysis of DNA methylation and gene expression to predict lung adenocarcinoma prognosis

Background: Abnormal DNA methylation of gene promoters is an important feature in lung adenocarcinoma (LUAD). However, the prognostic value of DNA methylation remains to be further explored. Objectives. We sought to explore DNA methylation characteristics and develop a quantifiable criterion related to DNA methylation to improve survival prediction for LUAD patients. Methods: Illumina Human Methylation450K array data, level 3 RNA-seq data and corresponding clinical information were obtained from TCGA. Cox regression analysis and the Akaike information criterion were used to construct the best-prognosis methylation signature. Receiver operating characteristic curve analysis was used to validate the prognostic ability of the DNA methylation-related feature score. qPCR was used to measure the transcription levels of the identified genes upon methylation. Results: We identified a set of DNA methylation features composed of 11 genes (MYEOV, KCNU1, SLC27A6, NEUROD4, HMGB4, TACR3, GABRA5, TRPM8, NLRP13, EDN3 and SLC34A1). The feature score, calculated based on DNA methylation features, was independent of tumor recurrence and TNM stage in predicting overall survival. Of note, the combination of this feature score and TNM stage provided a better overall survival prediction than either of them individually. The transcription levels of all the hypermethylated genes were significantly increased after demethylation, and the expression levels of 3 hypomethylated proteins were significantly higher in tumor tissues than in normal tissues, as indicated by immunohistochemistry data from the Human Protein Atlas. Our results suggested that these identified genes with prognostic features were regulated by DNA methylation of their promoters. Conclusion: Our studies demonstrated the potential application of DNA methylation markers in the prognosis of LUAD.