DNA Methyltransferase 3b Expression Research Articles

Epigenetic Regulation of Macrophage Polarization by DNA Methyltransferase 3b

Adipose tissue macrophages (ATMs) undergo a phenotypic switch from alternatively activated antiinflammatory M2 macrophages in lean individuals to classically activated proinflammatory M1 macrophages in obese subjects. However, the molecular mechanism underlying this process remains unclear. In this study we aim to determine whether DNA methyltransferase 3b (DNMT3b) regulates macrophage polarization and inflammation. We found that the expression of DNMT3b was significantly induced in macrophages exposed to the saturated fatty acid stearate, was higher in ATMs isolated from obese mice, but was significantly lower in alternatively activated M2 vs classically activated M1 ATMs, suggesting a role for DNMT3b in regulation of macrophage polarization and inflammation in obesity. DNMT3b knockdown promoted macrophage polarization to alternatively activated M2 phenotype and suppressed macrophage inflammation, whereas overexpressing DNMT3b did the opposite. Importantly, in a macrophage-adipocyte coculture system, we found that DNMT3b knockdown significantly improved adipocyte insulin signaling. The promoter of peroxisome proliferator activated receptor (PPAR)γ1, a key transcriptional factor that regulates macrophage polarization, is enriched with CpG sites. Chromatin immunoprecipitation assays showed that DNMT3b bound to the methylation region at PPARγ1 promoter, which was further enhanced by stearate. Moreover, pyrosequencing analysis revealed that stearate increased DNA methylation at PPARγ1, which was prevented by DNMT3b deficiency. Therefore, our data demonstrate that DNMT3b plays an important role in regulating macrophage polarization through epigenetic mechanisms. In obesity, elevated saturated fatty acids enhance DNMT3b expression, leading to DNA methylation at the PPARγ1 promoter, which may contribute to deregulated adipose tissue macrophage polarization, inflammation, and insulin resistance.

HIV infection of human regulatory T cells downregulates Foxp3 expression by increasing DNMT3b levels and DNA methylation in the FOXP3 gene

Regulatory T cells (Tregs) play an important role in infections modulating host immune responses and avoiding overreactive immunity. The mechanisms underlying their action in HIV-infected patients have not been well established. HIV can infect Treg, but little is known about the effects of the infection on Treg phenotype and function. The objective of this study was to investigate whether in-vitro HIV infection modifies the phenotype and suppressive capacity of Treg cells. Because Treg cells are a subset of CD4 T cells, HIV infection could produce alterations in the phenotype and methylation pattern of Treg disturbing the functionality of these cells. Isolated Treg cells from healthy volunteers were cultured in the presence of HIV-1, and phenotype, methylation pattern of FOXP3 locus, cytokine secretion profile and suppressive function of infected Treg were analysed in comparison with noninfected Treg. We demonstrate that HIV-1 directly infects Treg and deregulates the function and the phenotype that define these cells. HIV infection downregulates the Foxp3 expression in Treg, which is followed by the loss of suppressive capacity and alterations in cytokine secretion pattern, with decreased production of transforming growth factor-beta (TGF-β) and an increased production of interleukin (IL)-4. Foxp3 downregulation in HIV-infected Treg was related to an increase in the expression of DNA methyltransferase3b (DNMT3b) associated with higher methylation of CpG sites in the FOXP3 locus. These findings are pivotal to our understanding of the role of Treg in HIV infection and indicate that regulatory function could be seriously impaired in HIV-infected patients contributing to the immune hyperactivation.

A prospective study of duration of smoking cessation and colorectal cancer risk by epigenetics-related tumor classification.

The effect of duration of cigarette smoking cessation on colorectal cancer risk by molecular subtypes remains unclear. Using duplication-method Cox proportional-hazards regression analyses, we examined associations between duration of smoking cessation and colorectal cancer risk according to status of CpG island methylator phenotype (CIMP), microsatellite instability, v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation, or DNA methyltransferase-3B (DNMT3B) expression. Follow-up of 134,204 individuals in 2 US nationwide prospective cohorts (Nurses' Health Study (1980-2008) and Health Professionals Follow-up Study (1986-2008)) resulted in 1,260 incident rectal and colon cancers with available molecular data. Compared with current smoking, 10-19, 20-39, and ≥40 years of smoking cessation were associated with a lower risk of CIMP-high colorectal cancer, with multivariate hazard ratios (95% confidence intervals) of 0.53 (0.29, 0.95), 0.52 (0.32, 0.85), and 0.50 (0.27, 0.94), respectively (Ptrend = 0.001), but not with the risk of CIMP-low/CIMP-negative cancer (Ptrend = 0.25) (Pheterogeneity = 0.02, between CIMP-high and CIMP-low/CIMP-negative cancer risks). Differential associations between smoking cessation and cancer risks by microsatellite instability (Pheterogeneity = 0.02), DNMT3B expression (Pheterogeneity = 0.03), and BRAF (Pheterogeneity = 0.10) status appeared to be driven by the associations of CIMP-high cancer with microsatellite instability-high, DNMT3B-positive, and BRAF-mutated cancers. These molecular pathological epidemiology data suggest a protective effect of smoking cessation on a DNA methylation-related carcinogenesis pathway leading to CIMP-high colorectal cancer.

Promoted Interaction of Nuclear Factor-κB with Demethylated Cystathionine-β-Synthetase Gene Contributes to Gastric Hypersensitivity in Diabetic Rats

Patients with long-standing diabetes frequently demonstrate gastric hypersensitivity with an unknown mechanism. The present study was designed to investigate roles for nuclear factor-κB (NF-κB) and the endogenous H2S-producing enzyme cystathionine-β-synthetase (CBS) signaling pathways by examining cbs gene methylation status in adult rats with diabetes. Intraperitoneal injection of streptozotocin (STZ) produced gastric hypersensitivity in female rats in response to gastric balloon distention. Treatment with the CBS inhibitor aminooxyacetic acid significantly attenuated STZ-induced gastric hypersensitivity in a dose-dependent fashion. Aminooxyacetic acid treatment also reversed hyperexcitability of gastric-specific dorsal root ganglion (DRG) neurons labeled by the dye DiI in diabetic rats. Conversely, the H2S donor NaHS enhanced neuronal excitability of gastric DRG neurons. Expression of CBS and p65 were markedly enhanced in gastric DRGs in diabetic rats. Blockade of NF-κB signaling using pyrrolidine dithiocarbamate reversed the upregulation of CBS expression. Interestingly, STZ treatment led to a significant demethylation of CpG islands in the cbs gene promoter region, as determined by methylation-specific PCR and bisulfite sequencing. STZ treatment also remarkably downregulated the expression of DNA methyltransferase 3a and 3b. More importantly, STZ treatment significantly enhanced the ability of cbs to bind DNA at the p65 consensus site, as shown by chromatin immunoprecipitation assays. Our findings suggest that upregulation of cbs expression is attributed to cbs promoter DNA demethylation and p65 activation and that the enhanced interaction of the cbs gene and p65 contributes to gastric hypersensitivity in diabetes. This finding may guide the development and evaluation of new treatment modalities for patients with diabetic gastric hypersensitivity.

DNA methyltransferase 3a, 3b and 3L expression in fetal germ cells and its modulation

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Immunohistochemical analysis of the DNA methyltransferase 3b expression is associated with significant improvements in the discrimination of ulcerative colitis-associated neoplastic lesions

Making a clinicopathological diagnosis of dysplasia is crucial. We herein assess the significance of the DNA methyltransferase 3b (DNMT3b) expression as a diagnostic marker of ulcerative colitis (UC)-associated neoplasia. Thirty-one patients with long-standing and extensive UC were included in this study. The expression of DNMT3b in non-neoplastic rectal epithelium (non-dysplasia in 31 patients) and colorectal neoplasia (dysplasia in 43 patients and invasive cancer in 34 patients) was determined using immunohistochemistry. The presence of immunoreactive DNMT3b was assessed in the areas with the highest density of cells with positively staining nuclei. DNMT3b was expressed as the percentage of positive cells relative to the total number of cells counted under high power magnification. The DNMT3b expression in neoplastic rectal epithelium (0.76, range 0.59-0.84) was increased compared to that observed in non-neoplastic epithelium (0.32, range 0.18-0.67, P < 0.001). A ROC curve analysis confirmed 0.68 to be the best diagnostic cut-off value for the DNMT3b expression in neoplastic epithelium (area under the curve = 0.810). The sensitivity of the diagnostic test was 66.2 %, the specificity was 86.7 %, the positive predictive value was 95.7 % and the negative predictive value was 36.1 %. The positive likelihood ratio was 4.98 and the negative likelihood ratio was 0.20. The accuracy was 69.9 %. An immunohistochemical analysis of the DNMT3b expression was associated with significant improvements in the discrimination of UC-associated neoplastic lesions.

Promoter demethylation of cystathionine-β-synthetase gene contributes to inflammatory pain in rats

Hydrogen sulfide (H2S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H2S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H2S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H2S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

DNA Methyltransferase 3B (DNMT3B) Protein Expression Predicts Survival in Patients with Acute Myeloid Leukemia (AML)

AbstractAbstract 1395▪▪This icon denotes a clinically relevant abstractAberrant hypermethylation of tumor suppressor genes plays an important role in the development of acute myeloid leukemia (AML). In order to investigate the expression pattern of DNA methyltransferases (DNMTs) in AML patients, we established a tissue microarray (TMA) on bone marrow biopsies of 127 patients diagnosed with AML, who received intensive treatment in our department between 1999 and 2008 and with available bone marrow histology specimens. Immunohistochemistry was performed on this TMA with antibodies against DNMT1, DNMT3A, DNMT3B, methylated cytosine (mC) and hydroxymethylated cytosine (hmC).127 patients having received intensive treatment in our department and with available clinical follow-up data were analyzed. Median follow-up after diagnosis was 56 (range 0–133) months. Median age at diagnosis of these patients cohort was 63 (range 21 – 85) years, 52 patients (41%) were female, 75(59%) were male. 88 patients (70%) had de novo AML, 38 (30%) had secondary AML (sAML) evolving from antecedent myelodysplastic syndrome or after previous cytotoxic treatment. For one patient the information was not available. Cytogenetic and molecular genetic risk assessment was performed according to the classification published by the European Leukemia Network (ELN). Of the 120 patients (95%) with available results, 20 patients (16.7%) were low risk, 49 (40.8%) intermediate-I, 20 (16.7%) intermediate-II and 31 (25.8%) high risk.Of 126 patients with evaluable DNMT1 expression, only 9 patients (7%) had detectable expression in ≥5% positive blasts. DNMT1 expression did not associate with any outcome parameter. DNMT3A expression analysis could not be established due to unspecific staining. By contrast, DNMT3B expression was positive (≥5%) in 80 of 116 evaluable patients (69%). Patients with absent or low (positivity in <5% of blasts) DNMT3B expression had a higher pretreatment white blood cell count (WBC) (p= 0.046), a higher peripheral blood blast percentage before treatment (p= 0.02) and a more favorable cytogenetic and molecular risk profile (p=0.002) compared to patients with high DNMT3B expression. Patients with absent or low DNMT3B expression showed a slightly, but not significantly lower positivity for mC than patients with high DNMT3B expression (mean 54 versus 50% positive blasts, p=0.56) and a trend for lower hmC positivity (mean 47 versus 56%, p=0.17). Complete remission rates were not different between low/absent or high DNMT3B expression (69 versus 62%, p=0.27). DNMT3B low/absent expression was associated with a significantly improved overall (median 42 versus 10 months, p=0.014), event-free (median 13 versus 5 months, p=0.018) and relapse-free survival (64 versus 7 months, p=0.025) compared to patients with DNMT3B positivity in ≥5% bone marrow blasts. In multivariate Cox regression analyses with the inclusion of other risk factors associated with prognosis in our cohort (age, sex, ELN risk group, de novo versus secondary AML, WBC), absent or low DNMT3B expression remained an independent favorable risc factor for overall (proportional hazard risk (HR) 0.47, 95% confidence interval (CI) 0.26 – 0.85), event-free (HR 0.61, 95% CI 0.38 – 0.97) and relapse-free survival (HR 0.48, 95% CI 0.25 – 0.92).Taken together, DNMT3B protein expression is an independent prognostic factor in patients with AML in our cohort. If validated, immunohistochemical detection of DNMT3B protein expression might become a valuable tool for prognostication in patients with AML. Disclosures:No relevant conflicts of interest to declare.

Global DNA Methylation Not Increased in Chronic Hemodialysis Patients: A Case–Control Study

Patients with chronic kidney disease have an increased risk of cardiovascular disease and mortality. Since DNA methylation is an important mechanism modulating the gene expression associated with aging, inflammation, and atherosclerosis, the objective of this study was to determine the possible effect of the uremic milieu on global DNA methylation and DNA methyltransferase (DNMT) expression in uremic status by comparing chronic hemodialysis (HD) patients with the normal population. Twenty normal subjects and twenty chronic dialysis patients with similar ages, sex, and body mass indexes (BMIs) were included. We evaluated the clinical characteristics; the levels of homocysteine, total indoxyl sulfate (IS), and total p-cresol sulfate (PCS); and the DNMT messenger RNA expression and global DNA methylation in the peripheral blood leukocytes. The chronic HD patients had significantly higher blood urea nitrogen (BUN), creatinine (Cr), uric acid, Ca, P, intact parathyroid harmone (iPTH), cholesterol, high-sensitivity C-reactive protein (hs-CRP), total indoxyl sulphate (IS) and p-cresol sulphate (PCS), and homocysteine than the normal subjects. The expression of DNMT 1 and 3a did not differ significantly between these two study groups. The chronic HD patients had significantly decreased DNMT 3b expression in the leukocytes. There were no significant correlations between the global DNA methylation and the levels of IS, PCS, and homocysteine. We concluded that chronic HD patients may have lower DNMT 3b expression than normal subjects. However, the status of global DNA methylation may not change significantly in uremic patients when compared with the normal population.

CagA mediates epigenetic regulation to attenuate let-7 expression in Helicobacter pylori-related carcinogenesis

ObjectiveMicroRNAs (miRNAs) act as tumour suppressor genes or oncogenes in the regulation of multiple carcinogenic processes. Aberrant miRNA expression is reported in Helicobacter pylori (H pylori)-related gastritis and gastric cancer....

New DNA methylation markers associated with oral cancer (OC) development (dvlpt).

5524 Background: We have reported that DNA methyltransferase-3B (DNMT3B) gene expression is a strong predictor of OC dvlpt. Using high-throughput DNA methylation profiles of oral preneoplastic lesions (OPL), we identified 86 candidate genes that were hypermethylated in patients (pts) developing OC and suggested that a CpG island methylation phenotype may occur early during OC dvlpt. Our aim was to validate some of the candidate genes and to study the value of LINE1 repetitive element methylation, a surrogate of global DNA methylation, as biomarkers of risk to develop OC. Methods: Validation cohort included 40 pts with a median follow-up of 4.2 years, including 14 pts who developed OC. None of them were used in the discovery phase. Frozen OPL were collected prospectively and subject to DNA extraction. We performed a quantitative analysis of the degree of methylation of LINE1, and AGTR1, FOXI2, PENK, and HOXA9 promoters CpG sites using pyrosequencing. Results: The degree of methylation of AGTR1 (Mann-Whitney P&lt;0.0001), FOXI2 (P=0.0002), PENK (P&lt;0.0001), but not HOXA9 (P=0.0714) was significantly higher in pts who developed OC. On the contrary, LINE1 methylation was significantly lower in pts who developed OC (P&lt;0.0001). The median percent of methylation was used to dichotomize the pts into high versus low methylation levels. Oral cancer-free survival (OCFS) was significantly worse in pts with high levels of AGTR1 (Log-rank P=0.0229), FOXI2 (P=0.0170), and PENK (P=0.0142) promoter methylation. No significant difference was found with HOXA9. A Methylation Index (MI) was developed by averaging the percent of methylation of AGTR1, FOXI2, and PENK promoters; pts with a high MI had a worse OCFS (P=0.0031). On the other hand, pts with low levels of LINE1 methylation had a significantly worse OCFS (P=0.0118). Finally, in 26 pts, a positive correlation was observed between DNMT3B gene expression levels and the MI (rho=0.65, P=0.0003); surprisingly, there was a negative correlation with LINE1 methylation (rho=-0.74, P&lt;0.0001). Conclusions: AGTR1, FOXI2 and PENK promoter methylation may be associated with increased OC risk in pts with OPL and correlate with DNMT3B expression. Global DNA hypomethylation is an early event in OC dvlpt.

Studies on Wild Yam (Dioscorea villosa) Root Extract as a Potential Chemopreventive Agent for Breast Cancer

Aberrant epigenetic alterations in the genome such as DNA methylation and chromatin remodeling are able to inactivate specific gene function which is considered as one of the mechanisms involved in breast cancer development [1]. Attempts have been made to develop demethylating epigenetic drugs (epi-drugs) that are able to reactivate the silenced gene and prevent breast cancer. The current synthetic epi-drugs are non-specific and able to induce adverse side effects. In an attempt to search new and safe gene-specific demethylating agents (epi-drugs) from natural products, this study aimed to evaluate the effects of wild yam (Dioscorea villosa) root extract in an estrogen receptor positive (ER+) breast cancer cell line (MCF-7). Two genes, DNA methyl transferase 3B (DNMT3B) and GATA3 which are reported to be over expressed in breast cancer cells were used for evaluation. Additionally, by using Methyl Primer Express V1.0 software (Applied Bioscience) we were able to identify 3 CpG rich sites in the promoter region of GATA3 and 1 site in DNMT3B. MCF-7 cells at about 70% confluency were exposed to various concentrations of wild yam root extract (0–50 ((igrave))g/ml) for 72h. After cell count, total RNA was extracted and used for GATA3 and DNMT3B mRNA analysis by quantitative real-time PCR (qPCR). Moreover, the promoter methylation pattern of a specific region of GATA3 gene was also analyzed. It was observed that wild yam root extract significantly reduced the cell proliferation in a dose-dependent manner. The mRNA expression of DNMT3B was significantly enhanced in a dose-dependent manner as observed by qPCR while GATA3 mRNA expression was unaltered. From our preliminary data, it was evident that wild yam root extract has the potential to be used as a cancer chemopreventive substance, however, to establish its efficacy as an epi-drug more studies are needed. Acknowledgements: Thanks go to the United States Department of Agriculture, Agriculture Research Service, Specific Cooperative Agreement No. 58–6408–2-0009.

DNA methyltransferase 3B expression is associated with poor outcome of stage I testicular seminoma.

AimsTo examine in testicular seminomas the expression of DNA methyltransferase 3B (DNMT3B), which is known to be associated with early embryonic development and carcinogenesis, and to obtain a predictive marker for relapse of stage I seminomas.Methods and resultsImmunohistochemical examination of DNMT3B was performed in 88 cases of seminoma, 35 (39.8%) of which showed widely scattered nuclear immunoreactivity for DNMT3B, and 53 (60.2%) of which were completely negative. The incidence of focal DNMT3B expression was higher in stage III seminomas (5/5, 100%) than in stage I (25/70, 35.7%) or stage II (5/13, 38.5%) seminomas (P = 0.011). In stage I seminomas there were no significant correlations between DNMT3B expression and tumour size, invasion of the rete testis, or lymphatic or vascular involvement. Six of 25 cases (24%) showing DNMT3B expression relapsed, whereas only 3/45 cases (6.7%) lacking such expression did so (P = 0.037). Patients with seminomas showing DNMT3B expression had a significantly lower relapse-free survival rate than patients whose tumours lacked this feature (P = 0.0464).ConclusionsPatients with seminomas showing focal DNMT3B expression are at increased risk of relapse, and should be followed up carefully.

The role of DNA methyltransferase 3b in esophageal squamous cell carcinoma

The identification of potential tumor markers can improve therapeutic planning and patient management. The objective of this study was to highlight the role of DNA methyltransferase 3b (DNMT3b) in esophageal squamous cell carcinoma (SCC). One hundred seventy-three esophageal SCC samples were analyzed using immunohistochemical staining to correlate the expression of DNMT3b with clinical outcome. Furthermore, a human esophageal SCC cell line, CE81T, was selected for cellular and animal experiments to investigate changes in tumor behavior and treatment response after the manipulation of DNMT3b expression. The incidence of nuclear DNMT3b immunoreactivity in esophageal cancer specimens was significantly higher than in nonmalignant epithelium, and this incidence was linked positively to developing distant metastasis (56% in localized disease vs 80% in distant metastasis; P = .002). Furthermore, increased expression of DNMT3b was linked significantly to lower treatment response rates (P = .002) and reduced survival rates (P = .000). Inhibition of DNMT3b expression resulted in slower cellular proliferation, increased cell death, a less invasive capacity, and less epithelial-mesenchymal-transition changes. Moreover, DNMT3b silencing vectors sensitized esophageal cancer cells to irradiation and cisplatin treatment. The current results also indicated that constitutional activation of signal transducer and activator of transcription 3 (STAT3) signaling associated with inhibited expression of suppressor of cytokine signaling 3 (SOCS3) may be the mechanism underlying more aggressive tumor growth in DNMT3b-positive esophageal cancer. DNMT3b was linked significantly to a poor prognosis for patients with esophageal cancer. Moreover, the current results indicated that targeting this enzyme may be a promising strategy for treating esophageal cancer, as evidenced by inhibited aggressive tumor behavior and treatment resistance.

Transcriptional activity of DNMT3B in pancreatic cancer cells: Effects of −149 (C → T) promoter polymorphism

Polymorphic C-to-T change in the promoter region of DNA-methyltransferase-3B (DNMT3B) gene is associated with risk of several cancers. The aim of this study was to investigate the effect of DNMT3B promoter genetic variant on its transcriptional activity and to compare activity in several pancreatic cell lines. DNMT3B promoter constructs carrying either −149C allele or −149T allele were transiently transfected into pancreatic cancer cells. In promoter assaying, carriage of −149T allele showed only a slight activity (1.1-fold) in Mia cells (p=0.462). In contrast, significant increase (3.8-fold) in activity of −149T allele was shown in SU86.86 pancreatic cancer cells (p=0.0001). These preliminary findings suggest that genetic variance may influence DNMT3B expression in pancreatic cancer. Further studies are needed.

Abstract 816: Anti-inflammatory effects of black raspberries in ulcerative colitis are associated with demethylation of genes in the Wnt signaling and protective modulation of toll-like receptor pathway

Abstract Ulcerative Colitis (UC) causes pervasive, chronic inflammation of the colon and is a potential precursor to colon cancer. Reduced Interleukin 10 (IL10), an anti-inflammatory cytokine, is associated with an increased risk of developing UC in rodents and humans. IL10 knockout (KO) mice develop UC spontaneously with subsequent development of colon tumors. Toll-like receptor and Wnt pathways are deregulated in UC patients and IL10 KO mice. Toll-like receptor pathway plays critical roles in the inflammatory response partly by mediating the Wnt pathway. We previously showed that 5-10% dietary freeze-dried black raspberries (BRBs) reduced chronic inflammation and tumor development in IL10 KO mice; aberrant promoter methylation of negative Wnt regulators, e.g., dkk2, dkk3, and sfrp1, were observed in colon from these animals. The goals of the current study were to determine if the anti-inflammatory effects of BRBs on IL10 KO mice are associated with modulation of the Wnt pathway through DNA demethylation, with alteration of enzymes regulating DNA methylation, and with modulation of toll-like receptor pathway. IL10 KO mice were fed control or 5% BRB diet for 4 wks. Cells from bone marrow and spleen as well as colon tissue were collected. Promoter methylation of dkk2, dkk3, and sfrp1 was quantified by Pyrosequencing. Protein expression of enzymes regulating DNA methylation, e.g., histone deacetylase 1, 2 (HDAC1, 2), methyl binding domain 2 (MBD2), and DNA methyltransferase 3B (DNMT3B), and β-catenin were assessed using quantitative immunohistochemistry. mRNA expression of genes associated with the Wnt and toll-like receptor pathways were quantified using real-time PCR based arrays, each of which contained 84 genes. Increased promoter methylation of dkk2, dkk3, and sfrp1 was observed in colon from KO mice when compared with colon from wild-type mice. The methylation levels of these genes in bone marrow and spleen were lower than those in colon. BRBs significantly decreased promoter methylation of dkk2 in colon and dkk3 in spleen. This was associated with decreased protein expression of HDAC1, HDAC2, MBD2, and DNMT3B. The expression of 84 genes in both pathways was different in colon of IL10 KO mice when compared to colon of wild-type mice. BRBs modulated 80% (67/84) and 95% (80/84) of differentially expressed genes toward normal levels of expression in the Wnt and toll-like receptor pathways, respectively. Protein expression of nuclear β-catenin was decreased by BRBs. In summary, berries are capable of reversing aberrant promoter methylation of genes in the Wnt pathway presumably through inhibition of proteins regulating DNA methylation. These changes could result in protective modulation of the Wnt pathway and/or its interaction with the toll-like receptor pathway which, in turn, reduce inflammation in ulcerative colitis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 816. doi:10.1158/1538-7445.AM2011-816

Genes methylated by DNA methyltransferase 3b are similar in mouse intestine and human colon cancer

Human cancer cells frequently have regions of their DNA hypermethylated, which results in transcriptional silencing of affected genes and promotion of tumor formation. However, it is still unknown whether cancer-associated aberrant DNA methylation is targeted to specific genomic regions, whether this methylation also occurs in noncancerous cells, and whether these epigenetic events are maintained in the absence of the initiating cause. Here we have addressed some of these issues by demonstrating that transgenic expression of DNA methyltransferase 3b (Dnmt3b) in the mouse colon initiates de novo DNA methylation of genes that are similar to genes that become methylated in human colon cancer. This is consistent with the notion that aberrant methylation in cancer may be attributable to targeting of specific sequences by Dnmt3b rather than to random methylation followed by clonal selection. We also showed that Dnmt3b-induced aberrant DNA methylation was maintained in regenerating tissue, even in the absence of continuous Dnmt3b expression. This supports the concept that transient stressors can cause permanent epigenetic changes in somatic stem cells and that these accumulate over the lifetime of an organism in analogy to DNA mutations.

MicroRNAs and senescence

MicroRNAs and senescence

OxLDL up‐regulates microRNA‐29b, leading to epigenetic modifications of MMP‐2/MMP‐9 genes: a novel mechanism for cardiovascular diseases

MicroRNAs (miRNAs), small noncoding RNAs, can control gene expression by binding to their target genes for degradation and/or translational repression. Epigenetic mechanisms are defined as heritable changes in gene expression that do not involve coding sequence modifications. Both mechanisms play an important role in maintaining physiological functions and are also related to disease development. However, few studies report that miRNA-mediated epigenetic regulations are involved in atherosclerosis. In the present study, oxidized low-density lipoprotein (oxLDL) significantly increased primary human aortic smooth muscle cell (HASMC) migration through MMP-2/MMP-9 up-regulation associated with decreased DNA methylation levels. Either mRNA or protein level of DNA methyltransferase 3b (DNMT3b) showed a dose-dependent down-regulation in oxLDL-mediated HASMCs. Knockdown DNMT3b expression enhanced oxLDL-induced DNA demethylation levels of MMP-2/MMP-9. The expression of miRNA-29b (miR-29b), directly targeting DNMT3b, was up-regulated by oxLDL treatment in a dose-dependent manner. OxLDL-mediated MMP-2/MMP-9 up-regulation, DNMT3b down-regulation, and DNA demethylation were all attenuated after knockdown miR-29b expression by antagomiR-29b. We find that oxLDL can up-regulate miR-29b expression, resulting in DNMT3b down-regulation in HASMCs and epigenetically regulated MMP-2/MMP-9 genes involved in cell migration. These results show that miRNA-mediated epigenetic regulation may be a novel mechanism in atherosclerosis.

112 EXPRESSION OF DNA METHYLTRANSFERASES (DNMT) IN PLACENTAL TISSUES DURING EARLY PREGNANCY IN SHEEP

Normal placental development is critical for placental function and thus for normal embryonic and fetal growth and development. Many factors, including those from the environment or from the application of assisted reproductive techniques, are known to affect embryonic development. Additionally, altered DNA methylation was reported for fetal and/or maternal placenta from compromised pregnancies, and this may contribute to high embryonic/fetal loss. DNA methylation regulated by DNA methyltransferase (DNMT) plays an important role during embryonic development. However, little is known about the expression of DNMT in placental tissues during early pregnancy in any species. To determine the mRNA expression of DNMT 3a and 3b (developmentally-regulated DNMT) in normal placenta, caruncular (CAR, maternal placenta) tissue and fetal membranes (FM, chorioallantois or fetal placenta) were collected on Days 14, 16, 18, 20, 22, 24, 26, 28, and 30 after natural mating (n = 5–8 ewes day–1) and on Day 9–11 after oestrus (n = 7; non-pregnant [NP] controls, CAR only), snap-frozen, and then used for quantitative real time RT-PCR. For each tissue, data were analysed statistically by ANOVA with the day of pregnancy as the main effect. In CAR and FM, mRNA expression of DNMT3A and 3b was affected (P &lt; 0.01–0.02) by day of pregnancy. In CAR, expression of DNMT3A was similar in NP controls and on days 14, 16, 18, and 30, was decreased (P &lt; 0.01) ∼2-fold on day 20, and then gradually increased to day 30 of pregnancy. In CAR, expression of DNMT3b was similar in NP controls and on days 14, 16, 18, 24, 26, and 28, but was greater (P &lt; 0.02) by ∼2-fold on days 22 and 30 than in NP controls or on days 24 and 26 of pregnancy. For CAR, regression analysis of DNMT3a mRNA expression demonstrated a cubic pattern (R2 = 0.253; P = 0.01) of expression during early pregnancy. In FM, DNMT3a increased (P &lt; 0.01) ∼2-fold from day 16 to 24–30, but DNMT3b gradually decreased (∼0.5–5-fold; P &lt; 0.01) from day 16 to day 30 of pregnancy. For FM, regression analysis of mRNA expression for DNMT3a demonstrated a linear increase (R2 = 0.301; P &lt; 0.01), but for DNMT3b a cubic pattern (R2 = 0.624; P &lt; 0.01) of expression during early pregnancy. These data indicate that DNMT3a and 3b mRNA are differentially expressed in CAR and FM, and the temporal pattern of expression of DNMT3a and 3b differs between maternal and fetal placental tissues during early pregnancy in sheep. Thus, significant changes in mRNA expression of DNMT3a and 3b in CAR and FM indicate that de novo methylation is present in the placenta during early pregnancy in sheep and may be regulated in part by the level of DNMT expression. These data provide a foundation for determining the basis for altered DNA methylation of placental and embryonic tissues in compromised pregnancies. Supported by USDA grant 2007-01215 to LPR and ATGB, and NIH grant HL64141 to LPR and DAR.