The DNA Methylation Inhibitor Zebularine Controls CD4+ T Cell Mediated Intraocular Inflammation.
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ABSTRACT:
SUBMITTER: Zou Y
PROVIDER: S-EPMC6706956 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Through its role in the regulation of gene expression, DNA methylation can participate in the control of specialized metabolite production. We have investigated the link between DNA methylation and anthocyanin accumulation in grapevine using the hypomethylating drug, zebularine and Gamay Teinturier cell suspensions. In this model, zebularine increased anthocyanin accumulation in the light, and induced its production in the dark. To unravel the underlying mechanisms, cell transcriptome, metabolic content, and DNA methylation were analyzed. The up-regulation of stress-related genes, as well as a decrease in cell viability, revealed that zebularine affected cell integrity. Concomitantly, the global DNA methylation level was only slightly decreased in the light and not modified in the dark. However, locus-specific analyses demonstrated a decrease in DNA methylation at a few selected loci, including a CACTA DNA transposon and a small region upstream from the UFGT gene, coding for the UDP glucose:flavonoid-3-O-glucosyltransferase, known to be critical for anthocyanin biosynthesis. Moreover, this decrease was correlated with an increase in UFGT expression and in anthocyanin content. In conclusion, our data suggest that UFGT expression could be regulated through DNA methylation in Gamay Teinturier, although the functional link between changes in DNA methylation and UFGT transcription still needs to be demonstrated.
| S-EPMC9316115 | biostudies-literature
Project description:Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.
| S-EPMC2713825 | biostudies-literature
Project description:Cholangiocarcinoma (CCA) is a cancer arising from the neoplastic transformation of cholangiocytes. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on CCA cells. We demonstrate that zebularine exerts an antitumor effect on CCA cells. Zebularine treatment decreased the concentrations of DNA methyltransferase (DNMT) proteins, and DNMT1 knockdown led to apoptotic cell death in the CCA cell lines TFK-1 and HuCCT1. DNA methylation analysis demonstrated that zebularine induced DNA demethylation, and the GO Biological Process terms "hemophilic cell adhesion", "regulation of transcription, DNA-dependent" and "Wnt signaling pathway" were found to be significantly enriched in association with demethylated genes. Furthermore, we observed that zebularine treatment decreased β-catenin protein levels in TFK-1 and HuCCT1 cells. These results suggest that zebularine alters DNA methylation status, and that some aspect of DNA demethylation by zebularine induces suppression of the Wnt signaling pathway, which leads to apoptotic cell death in CCA. We previously reported a novel mechanism of zebularine-induced cell growth arrest and apoptosis in hepatocellular carcinoma via a DNA methylation-independent pathway. Together, our present and previous studies indicate that zebularine could function as both a DNMT inhibitor and a non-DNMT inhibitor reagent, and that, while the optimal usage of zebularine may depend on cancer type, zebularine may be useful for chemotherapy against cancer.
| S-EPMC4370694 | biostudies-literature
Project description:ObjectiveLifestyle factors associated with obesity may alter epigenome-regulated gene expression. Most studies examining epigenetic changes in obesity have analyzed DNA 5´-methylcytosine (5mC) in whole blood, representing a weighted average of several distantly related and regulated leukocyte classes. To examine leukocyte-specific differences associated with obesity, a pilot study examining 5mC in three distinct leukocyte types isolated from peripheral blood of women with normal weight and obesity was conducted.MethodsCD4+ T cells, CD8+ T cells, and CD16+ neutrophils were reiteratively isolated from blood, and 5mC levels were measured across >450,000 CG sites.ResultsNineteen CG sites were differentially methylated between women with obesity and with normal weight in CD4+ cells, 16 CG sites in CD8+ cells, and 0 CG sites in CD16+ neutrophils (q?<?0.05). There were no common differentially methylated sites between the T-cell types. The amount of visceral adipose tissue was strongly associated with the methylation level of 79 CG sites in CD4+ cells, including 4 CG sites in CLSTN1's promoter, which, this study shows, may regulate its expression.ConclusionsThe methylomes of various leukocytes respond differently to obesity and levels of visceral adipose tissue. Highly significant differentially methylated sites in CD4+ and CD8+ cells in women with obesity that have apparent biological relevance to obesity were identified.
| S-EPMC6107382 | biostudies-literature
Project description:Intraocular tuberculosis (IOTB) is amongst the leading causes of uveitis in tropical countries. Despite reports on involvement of proinflammatory cytokines, studies on innate immune responses in disease pathogenesis are lacking. Reports from animal models and patients with pulmonary tuberculosis indicate that defects in toll like receptor (TLR)2 and TLR9 signalling predispose them to tuberculosis. In this context, we investigated the role of TLR2, TLR4 and TLR9 in generation of CD4+ T effector (Teff) cell responses during IOTB. Firstly, the cells in vitreous fluids showed lower expression of TLR2 and TLR9 in IOTB as compared to non-uveitis and non-TB uveitis groups. Next, peripheral CD4+ Teff cells of subjects with IOTB showed decreased proliferative responses and lower induction of Tregs following TLR2 and TLR9 stimulation. Further, TLR9 ligation resulted in increased IFN-γ and IL-17a but decreased expression of IL-10 and TGF-β. Lastly, lower expression of genes involved in TLR9 signalling after direct TLR9 ligation was observed in IOTB. Collectively, our results show that a subdued response to direct TLR2 and TLR9 stimulation in CD4+ T cells is associated with increased proinflammatory responses in IOTB. These findings reveal an important link between innate immune signalling and ensuing adaptive immune responses in IOTB with implications in other forms of extrapulmonary tuberculosis.
| S-EPMC6138653 | biostudies-literature
Project description:Pediatric acute lung injury, usually because of pneumonia, has a mortality rate of more than 20% and an incidence that rivals that of all childhood cancers combined. CD4+ T-cells coordinate the immune response to pneumonia but fail to function robustly among the very young, who have poor outcomes from lung infection. We hypothesized that DNA methylation represses a mature CD4+ T-cell transcriptional program in neonates with pneumonia. Here, we found that neonatal mice (3-4 days old) aspirated with Escherichia coli bacteria had a higher mortality rate than juvenile mice (11-14 days old). Transcriptional profiling with an unsupervised RNA-Seq approach revealed that neonates displayed an attenuated lung CD4+ T-cell transcriptional response to pneumonia compared with juveniles. Unlike neonates, juveniles up-regulated a robust set of canonical T-cell immune response genes. DNA methylation profiling with modified reduced representation bisulfite sequencing revealed 44,119 differentially methylated CpGs, which preferentially clustered around transcriptional start sites and CpG islands. A methylation difference-filtering algorithm detected genes with a high likelihood of differential promoter methylation regulating their expression; these 731 loci encoded important immune response and tissue-protective T-cell pathway components. Disruption of DNA methylation with the hypomethylating agent decitabine induced plasticity in the lung CD4+ T-cell marker phenotype. Altogether, multidimensional profiling suggested that DNA methylation within the promoters of a core set of CD4+ T-cell pathway genes contributes to the hyporesponsive neonatal immune response to pneumonia. These findings also suggest that DNA methylation could serve as a mechanistic target for disease-modifying therapies in pediatric lung infection and injury.
| S-EPMC6066312 | biostudies-literature
Project description:DNA methylation (DNAm) is an important epigenetic process involved in the regulation of gene expression. While many studies have identified thousands of loci associated with age, few have differentiated between linear and non-linear DNAm trends with age. Non-linear trends could indicate early- or late-life gene regulatory processes. Using data from the Illumina 450K array on 336 human peripheral blood samples, we identified 21 CpG sites that associated with age (P<1.03E-7) and exhibited changing rates of DNAm change with age (P<1.94E-6). For 2 of these CpG sites (cg07955995 and cg22285878), DNAm increased with age at an increasing rate, indicating that differential DNAm was greatest among elderly individuals. We observed significant replication for both CpG sites (P<5.0E-8) in a second set of peripheral blood samples. In 8 of 9 additional data sets comprising samples of monocytes, T cell subtypes, and brain tissue, we observed a pattern directionally consistent with DNAm increasing with age at an increasing rate, which was nominally significant in the 3 largest data sets (4.3E-15<P<0.039). cg07955995 and cg22285878 reside in the promoter region of KLF14, which encodes a protein involved in immune cell differentiation via the repression of FOXP3. These findings may suggest a possible role for cg07955995 and cg22285878 in immunosenescence.
| S-EPMC5501198 | biostudies-literature
Project description:Hepatocellular carcinoma is one of the most common cancers worldwide. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-(D)-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on hepatocellular carcinoma cell line HepG2. We demonstrate that zebularine exhibits antitumor activity on HepG2 cells by inhibiting cell proliferation and inducing apoptosis, however, it has little effect on DNA methylation in HepG2 cells. On the other hand, zebularine treatment downregulated CDK2 and the phosphorylation of retinoblastoma protein (Rb), and upregulated p21(WAF/CIP1) and p53. We also found that zebularine treatment upregulated the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). These results suggest that the p44/42 MAPK pathway plays a role in zebularine-induced cell-cycle arrest by regulating the activity of p21(WAF/CIP1) and Rb. Furthermore, although the proapoptotic protein Bax levels were not affected, the antiapoptotic protein Bcl-2 level was downregulated with zebularine treatment. In addition, the data in the present study indicate that inhibition of the double-stranded RNA-dependent protein kinase (PKR) is involved in inducing apoptosis with zebularine. These results suggest a novel mechanism of zebularine-induced cell growth arrest and apoptosis via a DNA methylation-independent pathway in hepatocellular carcinoma.
| S-EPMC3540068 | biostudies-literature
Project description:Cholangiocarcinoma (CCA) is a cancer arising from the neoplastic transformation of cholangiocytes. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on CCA cells. We demonstrate that zebularine exerts an antitumor effect on CCA cells. Zebularine treatment decreased the concentrations of DNA methyltransferase (DNMT) proteins, and DNMT1 knockdown led to apoptotic cell death in the CCA cell lines TFK-1 and HuCCT1. DNA methylation analysis demonstrated that zebularine induced DNA demethylation, and the GO Biological Process terms “hemophilic cell adhesion”, “regulation of transcription, DNAdependent” and “Wnt signaling pathway” were found to be significantly enriched in association with demethylated genes. Furthermore, we observed that zebularine treatment decreased β-catenin protein levels in TFK-1 and HuCCT1 cells. These results suggest that zebularine alters DNA methylation status, and that some aspect of DNA demethylation by zebularine induces suppression of the Wnt signaling pathway, which leads to apoptotic cell death in CCA. We previously reported a novel mechanism of zebularine-induced cell growth arrest and apoptosis in hepatocellular carcinoma via a DNA methylation-independent pathway. Together, our present and previous studies indicate that zebularine could function as both a DNMT inhibitor and a non-DNMT inhibitor reagent, and that, while the optimal usage of zebularine may depend on cancer type, zebularine may be useful for chemotherapy against cancer.
2015-03-24 | GSE60446 | GEO
Project description:Background: Differences in DNA methylation have been reported in B and T lymphocyte populations, including CD4+ T cells, isolated from rheumatoid arthritis (RA) patients when compared to healthy controls. CD4+ T cells are a heterogeneous cell type with subpopulations displaying distinct DNA methylation patterns. In this study, we investigated DNA methylation using reduced representation bisulfite sequencing in two CD4+ T cell populations (CD4+ memory and naïve cells) in three groups: newly diagnosed, disease modifying antirheumatic drugs (DMARD) naïve RA patients (N = 11), methotrexate (MTX) treated RA patients (N = 18), and healthy controls (N = 9) matched for age, gender and smoking status. Results: Analyses of these data revealed significantly more differentially methylated positions (DMPs) in CD4+ memory than in CD4+ naïve T cells (904 vs. 19 DMPs) in RA patients compared to controls. The majority of DMPs (72%) identified in newly diagnosed and DMARD naïve RA patients with active disease showed increased DNA methylation (39 DMPs), whereas most DMPs (80%) identified in the MTX treated RA patients in remission displayed decreased DNA methylation (694 DMPs). Interestingly, we also found that about one third of the 101 known RA risk loci overlapped (±500 kb) with the DMPs. Notably, introns of the UBASH3A gene harbor both the lead RA risk SNP and two DMPs in CD4+ memory T cells. Conclusion: Our results suggest that RA associated DNA methylation differences vary between the two T cell subsets, but are also influenced by RA characteristics such as disease activity, disease duration and/or MTX treatment.
| S-EPMC7033478 | biostudies-literature