methyl-CpG-binding Domain 2 Research Articles

MBD2 regulates the progression and chemoresistance of cholangiocarcinoma through interaction with WDR5

BackgroundCholangiocarcinoma (CCA) is a highly malignant, rapidly progressing tumor of the bile duct. Owing to its chemoresistance, it always has an extremely poor prognosis. Therefore, detailed elucidation of the mechanisms of chemoresistance and identification of therapeutic targets are still needed.MethodsWe analyzed the expression of MBD2 (Methyl-CpG-binding domain 2) in CCA and normal bile duct tissues using the public database and immunohistochemistry (IHC). The roles of MBD2 in CCA cell proliferation, migration, and chemoresistance ability were validated through CCK-8, plate cloning assay, wound healing assays and xenograft mouse model. In addition, we constructed a primary CCA mouse model to further confirm the effect of MBD2. RNA-seq and co-IP-MS were used to identify the mechanisms by how MBD2 leads to chemoresistance.ResultsMBD2 was upregulated in CCA. It promoted the proliferation, migration and chemoresistance of CCA cells. Mechanistically, MBD2 directly interacted with WDR5, bound to the promoter of ABCB1, promoted the trimethylation of H3K4 in this region through KMT2A, and activated the expression of ABCB1. Knocking down WDR5 or KMT2A blocked the transcriptional activation of ABCB1 by MBD2. The molecular inhibitor MM-102 targeted the interaction of WDR5 with KMT2A. MM-102 inhibited the expression of ABCB1 in CCA cells and decreased the chemoresistance of CCA to cisplatin.ConclusionMBD2 promotes the progression and chemoresistance of CCA through interactions with WDR5. MM-102 can effectively block this process and increase the sensitivity of CCA to cisplatin.

Kynurenine acts as a signaling molecule to attenuate pulmonary fibrosis by enhancing the AHR-PTEN axis

IntroductionPulmonary fibrosis (PF) is a fatal fibrotic lung disease without any options to halt disease progression. Feasible evidence suggests that aberrant metabolism of amino acids may play a role in the pathoetiology of PF. However, the exact impact of kynurenine (Kyn), a metabolite derived from tryptophan (Trp) on PF is yet to be addressed. ObjectivesThis study aims to elucidate the role of kynurenine in both the onset and advancement of PF. MethodsLiquid chromatography–tandem mass spectrometry was employed to assess Kyn levels in patients with idiopathic PF and PF associated with Sjögren’s syndrome. Additionally, a mouse model of PF induced by bleomycin was utilized to study the impact of Kyn administration. Furthermore, cell models treated with TGF-β1 were used to explore the mechanism by which Kyn inhibits fibroblast functions. ResultsWe demonstrated that high levels of Kyn are a clinical feature in both idiopathic PF patients and primary Sjögren syndrome associated PF patients. Further studies illustrated that Kyn served as a braking molecule to suppress fibroblast functionality, thereby protecting mice from bleomycin-induced lung fibrosis. The protective effects depend on AHR, in which Kyn induces AHR nuclear translocation, where it upregulates PTEN expression to blunt TGF-β mediated AKT/mTOR signaling in fibroblasts. However, in fibrotic microenviroment, the expression of AHR is repressed by methyl-CpG-binding domain 2 (MBD2), a reader interpreting the effect of DNA methylation, which results in a significantly reduced sensitivity of Kyn to fibroblasts. Therefore, exogenous administration of Kyn substantially reversed established PF. ConclusionOur studies not only highlighted a critical role of Trp metabolism in PF pathogenesis, but also provided compelling evidence suggesting that Kyn could serve as a promising metabolite against PF.

The main bioactive compound of Panax notoginseng, notoginsenoside R1, alleviates pulmonary fibrosis via MBD2/SHIP and STAT3 pathway

Notoginsenoside R1 is known as the main bioactive compound of the traditional Chinese medicine and functional food, Panax notoginseng. Network pharmacology prediction indicated that notoginsenoside R1 might be effective in the treatment of pulmonary fibrosis. This study investigated the effectiveness and pharmacological mechanism of notoginsenoside R1 on attenuating pulmonary fibrosis in mice. The results showed that notoginsenoside R1 alleviated the inflammatory response in the lung by down-regulating the polarization of M1 macrophages via the SOCS3/STAT3 pathway. Meanwhile, notoginsenoside R1 promoted the transcription of SH2 domain containing inositol 5′-phosphatase (SHIP) by down-regulating methyl-CpG binding domain 2 (MBD2) expression, thus attenuated PI3K/AKT-mediated polarization of M2 macrophages, and finally regulated the TGF-β1/Smad3 signaling pathway in order to attenuate pulmonary fibrosis. This study presented some possible mechanism of notoginsenoside R1 in the treatment of pulmonary fibrosis, suggesting that P. notoginseng may be served as potential medicined diet in preventing and treating such diseases.

MBD2 facilitates tumor metastasis by mitigating DDB2 expression

Despite past extensive studies, the pathoetiologies underlying tumor metastasis remain poorly understood, which renders its treatment largely unsuccessful. The methyl-CpG-binding domain 2 (MBD2), a “reader” to interpret DNA methylome-encoded information, has been noted to be involved in the development of certain types of tumors, while its exact impact on tumor metastasis remains elusive. Herein we demonstrated that patients with LUAD metastasis were highly correlated with enhanced MBD2 expression. Therefore, knockdown of MBD2 significantly attenuated the migration and invasion of LUAD cells (A549 and H1975 cell lines) coupled with attenuated epithelial–mesenchymal transition (EMT). Moreover, similar results were observed in other types of tumor cells (B16F10). Mechanistically, MBD2 selectively bound to the methylated CpG DNA within the DDB2 promoter, by which MBD2 repressed DDB2 expression to promote tumor metastasis. As a result, administration of MBD2 siRNA-loaded liposomes remarkably suppressed EMT along with attenuated tumor metastasis in the B16F10 tumor-bearing mice. Collectively, our study indicates that MBD2 could be a promising prognostic marker for tumor metastasis, while administration of MBD2 siRNA-loaded liposomes could be a viable therapeutic approach against tumor metastasis in clinical settings.

Blockade of Mbd2 by siRNA-loaded liposomes protects mice against OVA-induced allergic airway inflammation via repressing M2 macrophage production.

ObjectiveTo address the role of methyl-CpG-binding domain 2 (MBD2) in the pathogenesis of asthma and its potential as a target for the asthmatic therapy.MethodsStudies were conducted in asthmatic patients and macrophage-specific Mbd2 knockout mice to dissect the role of MBD2 in asthma pathogenesis. Additionally, RNAi-based therapy with Mbd2 siRNA-loaded liposomes was conducted in an ovalbumin (OVA)-induced allergic airway inflammation mouse model.ResultsAsthmatic patients and mice challenged with OVA exhibited upregulated MBD2 expression in macrophages, especially in alternatively activated (M2) macrophages. In particular, macrophage-specific knockout of Mbd2 protected mice from OVA-induced allergic airway inflammation and suppressed the M2 program. Notably, intratracheal administration of liposomes carrying Mbd2 siRNA decreased the expression of Mbd2 and prevented OVA-induced allergic airway inflammation in mice, as indicated by the attenuated airway inflammation and mucus production.ConclusionsThe above data indicate that Mbd2 implicates in the pathogenesis of asthma predominantly by regulating the polarization of M2 macrophages, which supports that Mbd2 could be a viable target for treatment of asthma in clinical settings.

The methyl-CpG-binding domain 2 facilitates pulmonary fibrosis by orchestrating fibroblast to myofibroblast differentiation.

Although DNA methylation has been recognised in the pathogenesis of idiopathic pulmonary fibrosis (IPF), the exact mechanisms are yet to be fully addressed. Herein, we demonstrate that lungs originated from IPF patients and mice after bleomycin (BLM)-induced pulmonary fibrosis are characterised by altered DNA methylation along with overexpression in myofibroblasts of methyl-CpG-binding domain 2 (MBD2), a reader responsible for interpreting DNA methylome-encoded information. Specifically, depletion of Mbd2 in fibroblasts or myofibroblasts protected mice from BLM-induced pulmonary fibrosis coupled with a significant reduction of fibroblast differentiation. Mechanistically, transforming growth factor (TGF)-β1 induced a positive feedback regulatory loop between TGF-β receptor I (TβRI), Smad3 and Mbd2, and erythroid differentiation regulator 1 (Erdr1). TGF-β1 induced fibroblasts to undergo a global DNA hypermethylation along with Mbd2 overexpression in a TβRI/Smad3 dependent manner, and Mbd2 selectively bound to the methylated CpG DNA within the Erdr1 promoter to repress its expression, through which it enhanced TGF-β/Smad signalling to promote differentiation of fibroblast into myofibroblast and exacerbate pulmonary fibrosis. Therefore, enhancing Erdr1 expression strikingly reversed established pulmonary fibrosis. Collectively, our data support that strategies aimed at silencing Mbd2 or increasing Erdr1 could be viable therapeutic approaches for prevention and treatment of pulmonary fibrosis in clinical settings.

MBD2 acts as a repressor to maintain the homeostasis of the Th1 program in type 1 diabetes by regulating the STAT1-IFN-\u03b3 axis

The methyl-CpG-binding domain 2 (MBD2) interprets DNA methylome-encoded information through binding to the methylated CpG DNA, by which it regulates target gene expression at the transcriptional level. Although derailed DNA methylation has long been recognized to trigger or promote autoimmune responses in type 1 diabetes (T1D), the exact role of MBD2 in T1D pathogenesis, however, remains poorly defined. Herein, we generated an Mbd2 knockout model in the NOD background and found that Mbd2 deficiency exacerbated the development of spontaneous T1D in NOD mice. Adoptive transfer of Mbd2−/− CD4 T cells into NOD.scid mice further confirmed the observation. Mechanistically, Th1 stimulation rendered the Stat1 promoter to undergo a DNA methylation turnover featured by the changes of DNA methylation levels or patterns along with the induction of MBD2 expression, which then bound to the methylated CpG DNA within the Stat1 promoter, by which MBD2 maintains the homeostasis of Th1 program to prevent autoimmunity. As a result, ectopic MBD2 expression alleviated CD4 T cell diabetogenicity following their adoptive transfer into NOD.scid mice. Collectively, our data suggest that MBD2 could be a viable target to develop epigenetic-based therapeutics against T1D in clinical settings.

MBD2 serves as a viable target against pulmonary fibrosis by inhibiting macrophage M2 program.

Despite past extensive studies, the mechanisms underlying pulmonary fibrosis (PF) still remain poorly understood. Here, we demonstrated that lungs originating from different types of patients with PF, including coronavirus disease 2019, systemic sclerosis-associated interstitial lung disease, and idiopathic PF, and from mice following bleomycin (BLM)-induced PF are characterized by the altered methyl-CpG-binding domain 2 (MBD2) expression in macrophages. Depletion of Mbd2 in macrophages protected mice against BLM-induced PF. Mbd2 deficiency significantly attenuated transforming growth factor-β1 (TGF-β1) production and reduced M2 macrophage accumulation in the lung following BLM induction. Mechanistically, Mbd2 selectively bound to the Ship promoter in macrophages, by which it repressed Ship expression and enhanced PI3K/Akt signaling to promote the macrophage M2 program. Therefore, intratracheal administration of liposomes loaded with Mbd2 siRNA protected mice from BLM-induced lung injuries and fibrosis. Together, our data support the possibility that MBD2 could be a viable target against PF in clinical settings.

Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells

BackgroundCell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window.MethodsWe performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry.ResultsHuman somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes.ConclusionsHuman drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.

Abstract B04: The use of whole genome methylation scanning to define genes preferentially suppressed in African American Prostate Cancer

Abstract Background: The incidence of prostate cancer (PCa) is approximately 60% higher, and the mortality rate is 2 to 3 times greater, among African American men (AA) resident in the U.S. compared with American men with a European background (EA). Men of West African ancestry from the Caribbean and South America share similar incidence and mortality to AAs, suggesting a possible genetic and/or epigenetic contribution to these outcomes. We previously carried out a sensitive expression analysis comparing the expression of both tumor and tumor-adjacent stroma of AA and EA patients by Paired T-tests for matched (age, stage, grade, tumor content, stroma content) patients (1). Remarkably, 92% of expression differences between AA and EA occurred in the stroma component and of these 97% were in the direction of lower expression in AA compared to EA patients. The downregulated genes were dominated by immune regulatory and extracellular matrix genes. This suggests the possibility that decreased immune and inflammatory processes in tumor-adjacent stroma may be responsible for the aggressive nature of prostate cancer in AA patients (1). We have begun to test this possibility by examining whether the down regulated genes are methylated. Methods: Whole Genome FFPE Methylation and Transcriptome Profiling. We used archived FFPE samples where there is a potential to examine many patients. We are in the early methods-development stage of this project. One AA and two EA prostate cancer cases were matched for age, time to relapse/survival and Gleason score. A Norgen kit was used to obtain both DNA and RNA from the same sample. Methylated regions in each case were captured by methylated DNA ChIP using the human Methyl-CpG Binding Domain-2 (hMBD2) protein MBD2 then DNA purification followed by NGS. The transcriptome of tumor stroma was obtained using the TruSeq RNA access kit which is based on capture oligonucleotides to each exon to enrich RNA of coding regions. The method works for low amounts of highly degraded RNA isolated from low quality samples. The kit includes > 425,000 probes each constructed against NCBI377 hg19 reference genome, covering 98.3% of the RefSeq exome. The probes capture >210,000 targets, spanning 21,415 genes. Results: Our preliminary data show that one AA tumor-adjacent stroma has a greater number of hypermethylated regions than in tumor-adjacent stroma of two matched EA patients. Genome-wide methylation analysis revealed 5217 sites of significant (p < 0.001) hypermethylation in AA compared to EA, and 988 other sites of hypermethylation in EA compared AA PCa cases, indicating a marked increase in methylation in this one AA PCa case compared to the two EA cases. Within coding DNA the major classes of methylated genes included immune regulatory, interferon stimulated genes, and genes of the extracellular matrix. Our studies provide the first joint methylation and transcriptome analysis of PCa patients using FFPE samples and support the possibility that epigenetic alterations might be responsible for deficiencies in tumor immunity in AA PCa. Reference: Kinseth M, A., Z. Jia, et al., 2014. Int. J. Cancer 134: 81-91. DOI: 10.1002/ijc.28326. Citation Format: Farah Rahmatpanah, Kathleen McGuire, Michael Lilly, Michael McClelland, Dan Mercola. The use of whole genome methylation scanning to define genes preferentially suppressed in African American Prostate Cancer. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B04.

Loss of Mbd2 Protects Mice Against High-Fat Diet-Induced Obesity and Insulin Resistance by Regulating the Homeostasis of Energy Storage and Expenditure.

Previous studies including ours demonstrated that methyl-CpG-binding domain 2 (MBD2) acts as a reader to decipher DNA methylome-encoded information. We thus in the current study used Mbd2-/- mice as a model to dissect the impact of high-fat diet (HFD) on DNA methylome relevant to the pathoetiology of obesity. It was interestingly noted that mice deficient in Mbd2 were protected from HFD-induced obesity and insulin resistance. Mechanistic study revealed that HFD rendered epididymal adipose tissues to undergo a DNA methylation turnover as evidenced by the changes of methylation levels and patterns. Specifically, HFD was noted with higher potency to induce DNA hypomethylation in genes relevant to energy storage than that in genes associated with energy expenditure. As a result, arrays of genes were subjected to expression changes, which led to an altered homeostasis for energy storage and expenditure in favor of obesity development. Loss of Mbd2 resulted in impaired implementation of above DNA methylation changes associated with altered energy homeostasis, which then protected mice from HFD-induced obesity and insulin resistance. Those data would provide novel insight into the understanding of the pathoetiology underlying obesity with potential for developing effective therapies against obesity in clinical settings.

MBD2 as a novel marker associated with poor survival of patients with hepatocellular carcinoma after hepatic resection.

Methyl-CpG binding domain 2 (MBD2) leads to the silencing of methylated genes in cancer cells and was implicated in the activation of prometastatic genes in hepatocellular carcinoma (HCC). The present study aimed to investigate the expression status of MBD2 in HCC and the correlation with surgical outcomes. The correlation between clinical prognostic factors and MBD2 were also evaluated. MBD2 expression was analyzed by western blotting in 20 paired HCC and paratumor liver (PTL) tissues. In addition, immunohistochemistry was performed on the 159 HCC samples following hepatic resection performed between January 2003 and October 2008. The correlation between clinicopathological factors and MBD2 expression was also evaluated by statistical analysis to determine the prognostic value of MBD2 expression in HCC. Postoperative prognostic factors were evaluated using univariate and multivariate analyses. Compared with PTL tissues, MBD2 expression was shown to be upregulated in 10 of the 20 HCC tissues (50%) by western blotting. The immunohistochemistry data indicated significant increase of the MBD2 expression level in 81 cases (50.94%) compared with the PTL tissues (0/159, 0%, P<0.001). The upregulated MBD2 expression in HCC tissues was correlated with BCLC stage B, tumor size >5 cm and microscopic vascular invasion. Multivariate analysis revealed that MBD2 was an independent prognostic factor for overall survival [HR, 2.089; P=0.001] and disease-free survival (HR, 1.601; P=0.022). In conclusion, MBD2 expression was elevated in HCC tissue, which suggesting MBD2 as a candidate prognostic marker of HCC.

Expression and targeting to methyl-CpG-binding domain 2 of microRNA-373 in hilar cholangiocarcinoma

Objective To study the expression and targeting to methyl-CpG-binding domain 2 (MBD2) of microRNA (miR)-373 in hilar cholangiocarcinoma.Methods The miR-373 expression was detected by using TaqMan miRNA assay.The mRNA and protein expression of methyl-CpG binding domain proteins (MBPs) was examined by using QuantiTect_Primer assays and Western blotting,respectively.The targeting at MBD2-3' UTR by miR-373 was evaluated by using dual-luciferase reporter gene assay.Results The miR-373 expression was decreased 2.94 times and was closely associated with poor cell differentiation (P ＜ 0.05),advanced clinical stage (P ＜ 0.05),and shorter survival (P ＜ 0.05) in hilar cholangiocarcinoma.In high miR-373 group,the MBD2 mRNA and protein expression was increased 3.75 times (P ＜0.01) and 2.34 times (P ＜ 0.05),respectively.Precursor miR-373 inhibited the luciferase activity of MBD2-3' UTR by 1.79 times.Exogenous miR-373 and anti-miR-373 suppressed MBD2 in QBC939 cells by 2.56 times (P ＜ 0.01),and enhanced MBD2 in human intrahepatic biliary epithelial cells(HIBEpic) by 2.13 times (P ＜ 0.01).Conclusion miR-373 is one negative regulator of MBD2.The increase of MBD2 induced by down-expression of miR-373 plays an important role in development and prognosis of hilar cholangiocarcinoma. Key words: MicroRNA-373; Methyl-CpG binding domain protein 2; Hilar cholangiocarcinoma

Epigenetic Control of Angiogenesis via DNA Methylation

Why some patients develop limb- and life-saving collaterals but others do not constitutes one of the great challenges that face patients with peripheral vascular and coronary artery disease. A longstanding goal in vascular disease is the establishment of therapies that enhance angiogenesis in compromised tissue beds. To achieve this goal, however, it is essential to define and understand the underlying mechanisms that control angiogenesis and may distinguish individual patient capacity for collateral formation. In this issue of Circulation , Rao and colleagues1 report that ischemic injury increases DNA methylation in several genes critical for angiogenesis. The methyl-CpG–binding domain 2 (MBD2) protein senses DNA methylation and mediates transcriptional repression of genes involved in angiogenesis and endothelial cell survival. This seminal report mechanistically links epigenetic changes in vascular cells to angiogenesis, paving the way for new therapies that could improve perfusion in patients with vascular disease. Article see p 2964 In mammals, cytosine methylation (5-methylcytosine) is a common epigenetic modification, occurring primarily in regions of DNA where a cytosine nucleotide is adjacent to a guanine nucleotide (so-called CpG sites). Up to 5% of cytosines are methylated, and nearly 60% of genes have CpG-rich islands in their 5′ regions.2 During embryonic development, DNA methylation dramatically declines by ≈30% in mice.3 Somatic cells and cancer cells also exhibit alterations in DNA methylation based on environmental stimuli. Because CG dinucleotides base pair to GC, methylation in 1 strand is mirrored by methylation in the other. During replication, methylation in the parent strand targets newly replicated DNA for methylation by recruiting DNA methyltransferases (DNMTs). In this way, stable transfer of gene methylation patterns to progeny lines is accomplished.4 DNA methylation is often present …

Global DNA methylation, DNMT1, and MBD2 in patients with systemic lupus erythematosus

To investigate the associations of DNA methylation levels and mRNA expressions of DNA cytosine-5-methyltransferase 1 (DNMT1) and methyl CpG-binding domain 2 (MBD2) with systemic lupus erythematosus (SLE), 108 patients with SLE and 97 healthy controls were enrolled in this study. DNA and total RNA were extracted from the peripheral blood mononuclear cells of the SLE patients and the controls. The global methylation levels of DNA were measured in 63 patients with SLE and 68 healthy controls by the ELISA method. DNMT1 and MBD2 mRNA were also detected in 108 SLE patients and 97 controls using the quantitative real-time polymerase chain reaction method. The global methylation level of DNA was significantly decreased in the SLE patients in comparison with that in the controls (p < 0.001, 95% CI = 0.1573-0.5052). The patients with SLE have higher expressions of DNMT1 and MBD2 mRNA than the controls (p < 0.001, 95% CI = -0.0049 - -0.0019 and p = 0.001, 95% CI = -0.0119 - -0.0029, respectively). We also found that there were no significant differences in the methylation level and the expression of DNMT1 and MBD2 mRNA between the active and the inactive SLE patients. A positive correlation was also found between DNMT1 and MBD2 mRNA expressions in the SLE patients (p < 0.001). This study demonstrated that the patients with SLE had a significantly lower level of DNA methylation than the controls. The expression of both DNMT1 and MBD2 mRNA was significantly increased in the SLE patients compared with the controls. This study also showed a positive correlation between DNMT1 and MBD2 mRNA levels in the patients with SLE.

Global DNA methylation, DNMT1, and MBD2 in patients with rheumatoid arthritis

Objectives To investigate the associations of DNA methylation levels and mRNA expressions of DNA cytosine-5-methyltransferase 1 (DNMT1) and methyl CpG-binding domain 2 (MBD2) with rheumatoid arthritis (RA). Methods The global methylation status of DNA was measured in 65 patients with RA and 64 healthy controls by the ELISA method. DNMT1 and MBD2 mRNA were also detected in 177 RA patients and 95 controls using the quantitative real-time polymerase chain reaction method. Results The global methylation of DNA was significantly decreased in the RA patients compared to the controls ( p = 0.005, 95% CI = 0.0835–0.4503). The patients with RA had higher expressions of DNMT1 and MBD2 mRNA than the controls ( p < 0.001, 95% CI = −0.0024 to −0.0053 and p < 0.001, 95% CI = −0.0079 to −0.0167, respectively). We also found that the MBD2 mRNA level was not related to the disease activity of RA. However, the expression of DNMT1 mRNA tended to be associated with the disease activity of RA ( p = 0.08). The levels of DNA methylation and DNMT1 mRNA were significantly decreased in the patients with anti-CCP antibody compared with those without ( p = 0.005, 95% CI = −0.7333 to −0.1373 and p = 0.003, 95% CI = −0.0071 to −0.0022, respectively). The differences in the methylation level and expressions of DNMT1 and MBD2 were not significant between the patients treated with and without anti-TNFα biological agents (Enbrel or Humira). Conclusion This study demonstrated that the RA patients have a significantly lower level of DNA methylation than the controls. Moreover, RA patients have higher expressions of DNMT1 and MBD2 mRNA. The anti-TNFα biological agents do not seem to affect DNA methylation and mRNA expressions of DNMT1 and MBD2 in RA patients.

Homocysteine-Mediated Expression of SAHH, DNMTs, MBD2, and DNA Hypomethylation Potential Pathogenic Mechanism in VSMCs

Homocysteine (Hcy) is a well-established risk factor for atherosclerosis and may cause dysregulation of gene expression, but the characteristics and the key links involved in its pathogenic mechanisms are still poorly understood. The aim of this study was to explore (i) the effects of Hcy on DNA methylation in vascular smooth muscle cells (VSMCs) and (ii) the underlying mechanism of Hcy-induced changes in DNA methylation patterns in relation to atherosclerosis. We examined the levels of gDNA methylation, namely, the Alu and line-1 element sequences, which can serve as a surrogate marker for gDNA methylation, and also investigated the effects of Hcy on the intracellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations as well as the expressions of SAH hydrolase (SAHH), DNA methyltransferase3a (DNMT3a), DNMT3b, and methyl-CpG-binding domain 2 (MBD2). We found that clinically relevant levels of Hcy (0-500 microM) induced elevation of SAH, declination of SAM and SAM/SAH ratio, and reduction in expression of SAHH and MBD2, but increased the activity of DNMT3a and DNMT3b compared to the control group (p < 0.05). We found also that the genome-wide hypomethylation is a common feature of gDNA in the VSMCs cultured with Hcy. In conclusion, these results suggest that Hcy-induced DNA methylation may be an important potential pathogenic mechanism in the development of atherosclerosis, and may become a therapeutic target for preventing Hcy-induced atherosclerosis.

Interaction of HTLV-1 Tax and methyl-CpG-binding domain 2 positively regulates the gene expression from the hypermethylated LTR

Epigenetic regulation of gene expression is critical in the maintenance of cellular homeostasis. Dysregulation of normal epigenetic transcription occurs in abnormal physiological conditions, such as those seen in cancer cells and cells infected with parasites, making the mechanism underlying abnormal epigenetic transcription of great interest. Gene expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by a viral transcriptional stimulator, Tax. We herein report a novel mechanism of transcription from the HTLV-1 long terminal repeat (LTR) that is regulated by Tax. In this study, we determined that Tax is able to activate transcription from the LTR, even when it was heavily methylated. In addition, the methyl-CpG-binding domain 2 (MBD2) protein played an important role in Tax-mediated transcriptional activation. We demonstrated the importance of a physical interaction between Tax and MBD2 in enhancing the transcriptional activity of Tax against the methylated LTR. Furthermore, we identified the formation of a protein complex composed of MBD2 and Tax bound to the methylated LTR. We propose a new model of epigenetic regulation by MBD2 acting in concert with a virally encoded transactivator, Tax. Our observation provides insight into the epigenetic regulation of gene expression and the diverse mechanisms of transcriptional regulation using methylated promoters.