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Sample records for gene promoter methylation

  1. Promoter methylation of candidate genes associated with familial testicular cancer.

    PubMed

    Mirabello, Lisa; Kratz, Christian P; Savage, Sharon A; Greene, Mark H

    2012-01-01

    Recent genomic studies have identified risk SNPs in or near eight genes associated with testicular germ cell tumors (TGCT). Mouse models suggest a role for Dnd1 epigenetics in TGCT susceptibility, and we have recently reported that transgenerational inheritance of epigenetic events may be associated with familial TGCT risk. We now investigate whether aberrant promoter methylation of selected candidate genes is associated with familial TGCT risk. Pyrosequencing assays were designed to evaluate CpG methylation in the promoters of selected genes in peripheral blood DNA from 153 TGCT affecteds and 116 healthy male relatives from 101 multiple-case families. Wilcoxon rank-sum tests and logistic regression models were used to investigate associations between promoter methylation and TGCT. We also quantified gene product expression of these genes, using quantitative PCR. We observed increased PDE11A, SPRY4 and BAK1 promoter methylation, and decreased KITLG promoter methylation, in familial TGCT cases versus healthy male family controls. A significant upward risk trend was observed for PDE11A when comparing the middle and highest tertiles of methylation to the lowest [odds ratio (OR) =1.55, 95% confidence intervals (CI) 0.82-2.93, and 1.94, 95% CI 1.03-3.66], respectively; P(trend)=0.042). A significant inverse association was observed for KITLG when comparing the middle and lowest tertiles to the highest (OR=2.15, 95% CI 1.12-4.11, and 2.15, 95% CI 1.12-4.14, respectively; P(trend)=0.031). There was a weak inverse correlation between promoter methylation and KITLG expression. Our results suggest that familial TGCT susceptibility may be associated with promoter methylation of previously-identified TGCT risk-modifying genes. Larger studies are warranted. PMID:23050052

  2. Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer

    PubMed Central

    Gonzalo, Victoria; Lozano, Juan José; Muñoz, Jenifer; Balaguer, Francesc; Pellisé, Maria; de Miguel, Cristina Rodríguez; Andreu, Montserrat; Jover, Rodrigo; Llor, Xavier; Giráldez, M. Dolores; Ocaña, Teresa; Serradesanferm, Anna; Alonso-Espinaco, Virginia; Jimeno, Mireya; Cuatrecasas, Miriam; Sendino, Oriol; Castellví-Bel, Sergi; Castells, Antoni

    2010-01-01

    Background Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. Methodology/Principal Findings We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple

  3. Silencing of CHD5 Gene by Promoter Methylation in Leukemia

    PubMed Central

    Zhao, Rui; Meng, Fanyi; Wang, Nisha; Ma, Wenli; Yan, Qitao

    2014-01-01

    Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter. PMID:24454811

  4. Evolutionary Transition of Promoter and Gene Body DNA Methylation across Invertebrate–Vertebrate Boundary

    PubMed Central

    Keller, Thomas E.; Han, Priscilla; Yi, Soojin V.

    2016-01-01

    Genomes of invertebrates and vertebrates exhibit highly divergent patterns of DNA methylation. Invertebrate genomes tend to be sparsely methylated, and DNA methylation is mostly targeted to a subset of transcription units (gene bodies). In a drastic contrast, vertebrate genomes are generally globally and heavily methylated, punctuated by the limited local hypo-methylation of putative regulatory regions such as promoters. These genomic differences also translate into functional differences in DNA methylation and gene regulation. Although promoter DNA methylation is an important regulatory component of vertebrate gene expression, its role in invertebrate gene regulation has been little explored. Instead, gene body DNA methylation is associated with expression of invertebrate genes. However, the evolutionary steps leading to the differentiation of invertebrate and vertebrate genomic DNA methylation remain unresolved. Here we analyzed experimentally determined DNA methylation maps of several species across the invertebrate–vertebrate boundary, to elucidate how vertebrate gene methylation has evolved. We show that, in contrast to the prevailing idea, a substantial number of promoters in an invertebrate basal chordate Ciona intestinalis are methylated. Moreover, gene expression data indicate significant, epigenomic context-dependent associations between promoter methylation and expression in C. intestinalis. However, there is no evidence that promoter methylation in invertebrate chordate has been evolutionarily maintained across the invertebrate–vertebrate boundary. Rather, body-methylated invertebrate genes preferentially obtain hypo-methylated promoters among vertebrates. Conversely, promoter methylation is preferentially found in lineage- and tissue-specific vertebrate genes. These results provide important insights into the evolutionary origin of epigenetic regulation of vertebrate gene expression. PMID:26715626

  5. Correlation of clinical features and methylation status of MGMT gene promoter in glioblastomas.

    PubMed

    Blanc, J L; Wager, M; Guilhot, J; Kusy, S; Bataille, B; Chantereau, T; Lapierre, F; Larsen, C J; Karayan-Tapon, L

    2004-07-01

    In an effort to extend the potential relationship between the methylation status of MGMT promoter and response to CENU therapy, we examined the methylation status of MGMT promoter in 44 patients with glioblastomas. Tumor specimens were obtained during surgery before adjuvant treatment, frozen and stored at -80 degrees C until for DNA extraction process. DNA methylation patterns in the CpG island of the MGMT gene were determined in every tumor by methylation specific PCR (MSP). These results were then related to overall survival and response to alkylating agents using statistical analysis. Methylation of the MGMT promoter was detected in 68% of tumors, and 96.7% of methylated tumors exhibited also an unmethylated status. There was no relationship between the methylation status of the MGMT promoter and overall survival and response to alkylating agents. Our observations do not lead us to consider promoter methylation of MGMT gene as a prognostic factor of responsiveness to alkylating agents in glioblastomas. PMID:15332332

  6. ABERRANT PROMOTER METHYLATION OF MULTIPLE GENES IN SPUTUM FROM INDIVIDUALS EXPOSED TO SMOKY COAL EMISSIONS

    EPA Science Inventory

    Aberrant methylation in the promoter region of cancer-related genes leads to gene transcriptional inactivation and plays an integral role in lung tumorigenesis. Recent studies demonstrated that promoter methylation was detected not only in lung tumors from patients with lung canc...

  7. Concomitant promoter methylation of multiple genes in lung adenocarcinomas from current, former and never smokers

    PubMed Central

    Tessema, Mathewos; Yu, Yang Y.; Stidley, Christine A.; Machida, Emi O.; Schuebel, Kornel E.; Baylin, Stephen B.; Belinsky, Steven A.

    2009-01-01

    Aberrant promoter hypermethylation is one of the major mechanisms in carcinogenesis and some critical growth regulatory genes have shown commonality in methylation across solid tumors. Twenty-six genes, 14 identified through methylation in colon and breast cancers, were evaluated using primary lung adenocarcinomas (n = 175) from current, former and never smokers. Tumor specificity of methylation was validated through comparison of 14 lung cancer cell lines to normal human bronchial epithelial cells derived from bronchoscopy of 20 cancer-free smokers. Twenty-five genes were methylated in 11–81% of primary tumors. Prevalence for methylation of TNFRSF10C, BHLHB5 and BOLL was significantly higher in adenocarcinomas from never smokers than smokers. The relation between methylation of individual genes was examined using pairwise comparisons. A significant association was seen between 138 (42%) of the possible 325 pairwise comparisons. Most notably, methylation of MMP2, BHLHB4 or p16 was significantly associated with methylation of 16–19 other genes, thus predicting for a widespread methylation phenotype. Kaplan–Meier log-rank test and proportional hazard models identified a significant association between methylation of SULF2 (a pro-growth, -angiogenesis and -migration gene) and better patient survival (hazard ratio = 0.23). These results demonstrate a high degree of commonality for targeted silencing of genes between lung and other solid tumors and suggest that promoter hypermethylation in cancer is a highly co-ordinated event. PMID:19435948

  8. Defining the cutoff value of MGMT gene promoter methylation and its predictive capacity in glioblastoma.

    PubMed

    Brigliadori, Giovanni; Foca, Flavia; Dall'Agata, Monia; Rengucci, Claudia; Melegari, Elisabetta; Cerasoli, Serenella; Amadori, Dino; Calistri, Daniele; Faedi, Marina

    2016-06-01

    Despite advances in the treatment of glioblastoma (GBM), median survival is 12-15 months. O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status is acknowledged as a predictive marker for temozolomide (TMZ) treatment. When MGMT promoter values fall into a "methylated" range, a better response to chemotherapy is expected. However, a cutoff that discriminates between "methylated" and "unmethylated" status has yet to be defined. We aimed to identify the best cutoff value and to find out whether variability in methylation profiles influences the predictive capacity of MGMT promoter methylation. Data from 105 GBM patients treated between 2008 and 2013 were analyzed. MGMT promoter methylation status was determined by analyzing 10 CpG islands by pyrosequencing. Patients were treated with radiotherapy followed by TMZ. MGMT promoter methylation status was classified into unmethylated 0-9 %, methylated 10-29 % and methylated 30-100 %. Statistical analysis showed that an assumed methylation cutoff of 9 % led to an overestimation of responders. All patients in the 10-29 % methylation group relapsed before the 18-month evaluation. Patients with a methylation status ≥30 % showed a median overall survival of 25.2 months compared to 15.2 months in all other patients, confirming this value as the best methylation cutoff. Despite wide variability among individual profiles, single CpG island analysis did not reveal any correlation between single CpG island methylation values and relapse or death. Specific CpG island methylation status did not influence the predictive value of MGMT. The predictive role of MGMT promoter methylation was maintained only with a cutoff value ≥30 %. PMID:27029617

  9. Methylation Status of Vitamin D Receptor Gene Promoter in Benign and Malignant Adrenal Tumors

    PubMed Central

    Pilon, Catia; Rebellato, Andrea; Urbanet, Riccardo; Guzzardo, Vincenza; Cappellesso, Rocco; Sasano, Hironobu; Fassina, Ambrogio

    2015-01-01

    We previously showed a decreased expression of vitamin D receptor (VDR) mRNA/protein in a small group of adrenocortical carcinoma (ACC) tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5′ regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas) were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesis. PMID:26843863

  10. A methylation-dependent DNA-binding activity recognising the methylated promoter region of the mouse Xist gene.

    PubMed

    Huntriss, J; Lorenzi, R; Purewal, A; Monk, M

    1997-06-27

    Differential methylation of CpG sites in the promoter region of the mouse Xist gene is correlated with Xist expression and X-chromosome inactivation in the female. Using oligonucleotides encompassing the differentially methylated sites as probes in band-shift assays, we have identified a nuclear protein which binds to a specific region of the promoter (between base pairs -45 and -30 upstream from the transcription start site) only when CpG sites within the CG rich region (GCGCCGCGG, -44 to -36) are methylated. Competition experiments with methylated or unmethylated heterologous oligonucleotides demonstrate that the activity is sequence-specific as well as methylation-dependent. Analysis by Southwestern blot identifies a protein of approximately 100 kDa molecular weight and confirms strong binding to the methylated Xist promoter oligonucleotide. Using a 233bp Xist-promoter luciferase construct in which the cytosines in the three CpG sites in the -44 to -36 region are mutated to thymine, we have established that this region is required for transcription from the mouse Xist promoter. Therefore, we suggest that the binding of the 100kDa protein to the methylated sequence leads to repression of transcription from the methylated Xist allele, thus suggesting a role in the regulation of both imprinted and random Xist transcription and X-chromosome inactivation. PMID:9207230

  11. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

    Kirov, Julia V.; Adkisson, Michael; Nava, A. J.; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, K. C. Kent; de Jong, Pieter; West, David B.

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  12. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.

    PubMed

    Kirov, Julia V; Adkisson, Michael; Nava, A J; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K; Lloyd, K C Kent; de Jong, Pieter; West, David B

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  13. MGMT, GATA6, CD81, DR4, and CASP8 gene promoter methylation in glioblastoma

    PubMed Central

    2012-01-01

    Background Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome. Methods The methylation status of MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis. Results The overwhelming majority (97.3%) of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p < 0.05), while CASP8 with older (p < 0.01). MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p < 0.05), while methylation of CASP8 was more frequent in patients who survived shorter than 36 months (p < 0.05). Cox regression analysis showed patient age, treatment, MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p < 0.05). MGMT and GATA6 were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy. Conclusions High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome. PMID:22672670

  14. Aberrant promoter methylation of multiple genes in sputum from individuals exposed to smoky coal emissions

    PubMed Central

    Liu, Yang; Lan, Qing; Shen, Min; Mumford, Judy; Keohavong, Phouthone

    2010-01-01

    Summary Aberrant methylation in the promoter region of cancer-related genes leads to gene transcriptional inactivation and plays an integral role in lung tumorigenesis. Recent studies demonstrated that promoter methylation was detected not only in lung tumors from patients with lung cancer but also in sputum of smokers without the disease, suggesting the potential for aberrant gene promoter methylation in sputum as a predictive marker for lung cancer. In the present study, we investigated promoter methylation of 4 genes frequently detected in lung tumors, including p16, MGMT, RASSF1A and DAPK genes, in sputum samples obtained from 107 individuals, including 34 never-smoking females and 73 mostly smoking males, who had no evidence of lung cancer but who were exposed to smoky coal emission in Xuan Wei County, China, where lung cancer rate is more than 6 times the Chinese national average rate. Forty nine of the individuals showed evidence of chronic bronchitis while the remaining 58 individuals showed no such a symptom. Promoter methylation of p16, MGMT, RASSF1A and DAPK was detected in 51.4% (55/107), 17.8% (19/107), 29.9% (32/107), and 15.9% (17/107) of the sputum samples from these individuals, respectively. There were no differences in promoter methylation frequencies of any of these genes according to smoking status or gender of the subjects or between individuals with chronic bronchitis and those without evidence of such a symptom. Therefore, individuals exposed to smoky coal emissions in this region harbored in their sputum frequent promoter methylation of these genes that have been previously found in lung tumors and implicated in lung cancer development. PMID:18751376

  15. Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens

    PubMed Central

    Coppedè, Fabio; Migheli, Francesca; Lopomo, Angela; Failli, Alessandra; Legitimo, Annalisa; Consolini, Rita; Fontanini, Gabriella; Sensi, Elisa; Servadio, Adele; Seccia, Massimo; Zocco, Giuseppe; Chiarugi, Massimo; Spisni, Roberto; Migliore, Lucia

    2014-01-01

    We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process. PMID:24500500

  16. Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

    PubMed Central

    Ngô, V; Gourdji, D; Laverrière, J N

    1996-01-01

    The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo. PMID:8668139

  17. Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

    PubMed Central

    Brait, Mariana; Ling, Shizhang; Nagpal, Jatin K.; Chang, Xiaofei; Park, Hannah Lui; Lee, Juna; Okamura, Jun; Yamashita, Keishi; Sidransky, David; Kim, Myoung Sook

    2012-01-01

    The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer. PMID:23028699

  18. Promoter CpG methylation of multiple genes in pituitary adenomas: frequent involvement of caspase-8.

    PubMed

    Bello, M Josefa; De Campos, Jose M; Isla, Alberto; Casartelli, Cacilda; Rey, Juan A

    2006-02-01

    The epigenetic changes in pituitary adenomas were identified by evaluating the methylation status of nine genes (RB1, p14(ARF), p16(INK4a), p73, TIMP-3, MGMT, DAPK, THBS1 and caspase-8) in a series of 35 tumours using methylation-specific PCR analysis plus sequencing. The series included non-functional adenomas (n=23), prolactinomas (n=6), prolactinoma plus thyroid-stimulating hormone adenoma (n=1), growth hormone adenomas (n=4), and adrenocorticotropic adenoma (n=1). All of the tumours had methylation of at least one of these genes and 40% of samples (14 of 35) displayed concurrent methylation of at least three genes. The frequencies of aberrant methylation were: 20% for RB1, 17% for p14(ARF), 34% for p16(INK4a), 29% for p73, 11% for TIMP-3, 23% for MGMT, 6% for DAPK, 43% for THBS1 and 54% for caspase-8. No aberrant methylation was observed in two non-malignant pituitary samples from healthy controls. Although some differences in the frequency of gene methylation between functional and non-functional adenomas were detected, these differences did not reach statistical significance. Our results suggest that promoter methylation is a frequent event in pituitary adenoma tumourigenesis, a process in which inactivation of apoptosis-related genes (DAPK, caspase-8) might play a key role. PMID:16391867

  19. Promoter Methylation and mRNA Expression of Response Gene to Complement 32 in Breast Carcinoma

    PubMed Central

    Eskandari-Nasab, Ebrahim; Hashemi, Mohammad; Rafighdoost, Firoozeh

    2016-01-01

    Background. Response gene to complement 32 (RGC32), induced by activation of complements, has been characterized as a cell cycle regulator; however, its role in carcinogenesis is still controversial. In the present study we compared RGC32 promoter methylation patterns and mRNA expression in breast cancerous tissues and adjacent normal tissues. Materials and Methods. Sixty-three breast cancer tissues and 63 adjacent nonneoplastic tissues were included in our study. Design. Nested methylation-specific polymerase chain reaction (Nested-MSP) and quantitative PCR (qPCR) were used to determine RGC32 promoter methylation status and its mRNA expression levels, respectively. Results. RGC32 methylation pattern was not different between breast cancerous tissue and adjacent nonneoplastic tissue (OR = 2.30, 95% CI = 0.95–5.54). However, qPCR analysis displayed higher levels of RGC32 mRNA in breast cancerous tissues than in noncancerous tissues (1.073 versus 0.959; P = 0.001), irrespective of the promoter methylation status. The expression levels and promoter methylation of RGC32 were not correlated with any of patients' clinical characteristics (P > 0.05). Conclusion. Our findings confirmed upregulation of RGC32 in breast cancerous tumors, but it was not associated with promoter methylation patterns. PMID:27118972

  20. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    PubMed Central

    Shilpa, V.; Bhagat, Rahul; Premalata, C.S.; Pallavi, V.R.; Ramesh, G.; Krishnamoorthy, Lakshmi

    2014-01-01

    Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O6-methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O6-position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC) tissue samples, 14 low malignant potential (LMP) tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP) after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression. PMID:25579142

  1. P07.04PROMOTER METHYLATION OF THE LATS1 AND LATS2 GENES IN SCHWANNOMAS

    PubMed Central

    Ohta, T.; Oh, J.; Mittelbronn, M.; Paulus, W.; Ohgaki, H.

    2014-01-01

    Schwannoma is a benign nerve sheath tumor that is typically encapsulated and composed of well-differentiated Schwann cellswhich comprises 5-10% of all intracranial tumors in adults. Approximately 90% of schwannomas are solitary and sporadic, whereas ∼4% are considered to arise in the setting of neurofibromatosis type 2 (NF2) syndrome by NF2 germline mutations. The molecular basis of sporadic schwannomas is not fully understood, other than frequent NF2 mutations (∼60%). LATS1 and the related LATS2 are downstream molecules of NF2 and negative regulators of the YAP oncogene in the Salvador/Warts/Hippo (SWH) signaling pathway. Expression of these genes is reduced due to promoter methylation in a variety of neoplasms including gliomas. In the present study, methylation-specific PCR revealed promoter methylation of the LATS1 and LATS2 in 15 of 91 (16%) and 32 of 91 (35%) schwannomas, respectively. These alterations were significantly more frequent in spinal than in peripheral schwannomas (23% vs 3% for LATS1, P = 0.0171; 42% vs 21% for LATS2, P = 0.0386). LATS1 methylation was also detected in 3 of 4 schwannomatosis cases. Furthermore, neurofibroma / schwannoma hybrid tumors showed promoter methylation in LATS1 (3/14; 21%) and LATS2 (8/14; 57%). LATS1 and LATS2 promoter methylation were largely mutually exclusive, and there was a significant negative correlation (P = 0.003); only 10 cases had methylation in both genes. These results suggest that LATS1 and LATS2 promoter methylation may be additional molecular mechanisms resulting in an abnormal SWH pathway in schwannomas and related tumors.

  2. Association Between Promoter Methylation of Serotonin Transporter Gene and Depressive Symptoms: A Monozygotic Twin Study

    PubMed Central

    Zhao, Jinying; Goldberg, Jack; Bremner, James D.; Vaccarino, Viola

    2013-01-01

    Objective Epigenetic mechanisms have been implicated in the pathogenesis of psychiatric disorders. The serotonin transporter gene (SLC6A4) is a key candidate gene for depression. We examined the association between SLC6A4 promoter methylation variation and depressive symptoms using 84 monozygotic twin pairs. Methods DNA methylation level in the SLC6A4 promoter region was quantified by bisulfite pyrosequencing using genomic DNA isolated from peripheral blood leukocytes. The number of current depressive symptoms was assessed using the Beck Depressive Inventory II (BDI-II). The association between methylation variation and depressive symptoms was examined using matched twin-pair analyses, adjusting for body mass index, smoking, physical activity, and alcohol consumption. Multiple testing was controlled by adjusted false discovery rate (q value). Results Intrapair difference in DNA methylation variation at 10 of the 20 studied CpG sites is significantly correlated with intrapair difference in BDI scores. Linear regression using intrapair differences demonstrates that intrapair difference in BDI score was significantly associated with intrapair differences in DNA methylation variation after adjusting for potential confounders and correction for multiple testing. On average, a 10% increase in the difference in mean DNA methylation level was associated with 4.4 increase in the difference in BDI score (95% confidence interval = 0.9–7.9, p = .01). Conclusions This study provides evidence that variation in methylation level within the promoter region of the serotonin transporter gene is associated with variation in depressive symptoms in a large sample of monozygotic twin pairs. This relationship is not confounded by genetic and shared environment. The 5-HTTLPR genotype also does not modulate this association. PMID:23766378

  3. A critical re-assessment of DNA repair gene promoter methylation in non-small cell lung carcinoma

    PubMed Central

    Do, Hongdo; Wong, Nicholas C.; Murone, Carmel; John, Thomas; Solomon, Benjamin; Mitchell, Paul L.; Dobrovic, Alexander

    2014-01-01

    DNA repair genes that have been inactivated by promoter methylation offer potential therapeutic targets either by targeting the specific repair deficiency, or by synthetic lethal approaches. This study evaluated promoter methylation status for eight selected DNA repair genes (ATM, BRCA1, ERCC1, MGMT, MLH1, NEIL1, RAD23B and XPC) in 56 non-small cell lung cancer (NSCLC) tumours and 11 lung cell lines using the methylation-sensitive high resolution melting (MS-HRM) methodology. Frequent methylation in NEIL1 (42%) and infrequent methylation in ERCC1 (2%) and RAD23B (2%) are reported for the first time in NSCLC. MGMT methylation was detected in 13% of the NSCLCs. Contrary to previous studies, methylation was not detected in ATM, BRCA1, MLH1 and XPC. Data from The Cancer Genome Atlas (TCGA) was consistent with these findings. The study emphasises the importance of using appropriate methodology for accurate assessment of promoter methylation. PMID:24569633

  4. A global profile of gene promoter methylation in treatment-naïve urothelial cancer

    PubMed Central

    Ibragimova, Ilsiya; Dulaimi, Essel; Slifker, Michael J; Chen, David DY; Uzzo, Robert G; Cairns, Paul

    2014-01-01

    The epigenetic alteration of aberrant hypermethylation in the promoter CpG island of a gene is associated with repression of transcription. In neoplastic cells, aberrant hypermethylation is well described as a mechanism of allele inactivation of particular genes with a tumor suppressor function. To investigate the role of aberrant hypermethylation in the biology and progression of urothelial cancer, we examined 101 urothelial (transitional cell) carcinomas (UC), broadly representative of the disease at presentation, with no prior immunotherapy, chemotherapy or radiotherapy, by Infinium HM27 containing 14,495 genes. The genome-wide signature of aberrant promoter hypermethylation in UC consisted of 729 genes significant by a Wilcoxon test, hypermethylated in a CpG island within 1 kb of the transcriptional start site and unmethylated in normal urothelium from aged individuals. We examined differences in gene methylation between the two main groups of UC: the 75% that are superficial, which often recur but rarely progress, and the 25% with muscle invasion and poor prognosis. We further examined pairwise comparisons of the pathologic subgroups of high or low grade, invasive or non-invasive (pTa), and high grade superficial or low grade superficial UC. Pathways analysis indicated over-representation of genes involved in cell adhesion or metabolism in muscle-invasive UC. Notably, the TET2 epigenetic regulator was one of only two genes more frequently methylated in superficial tumors and the sole gene in low grade UC. Other chromatin remodeling genes, MLL3 and ACTL6B, also showed aberrant hypermethylation. The Infinium methylation value for representative genes was verified by pyrosequencing. An available mRNA expression data set indicated many of the hypermethylated genes of interest to be downregulated in UC. Unsupervised clustering of the most differentially methylated genes distinguished muscle invasive from superficial UC. After filtering, cluster analysis showed a Cp

  5. SUVH1, a Su(var)3-9 family member, promotes the expression of genes targeted by DNA methylation.

    PubMed

    Li, Shaofang; Liu, Lin; Li, Shengben; Gao, Lei; Zhao, Yuanyuan; Kim, Yun Ju; Chen, Xuemei

    2016-01-29

    Transposable elements are found throughout the genomes of all organisms. Repressive marks such as DNA methylation and histone H3 lysine 9 (H3K9) methylation silence these elements and maintain genome integrity. However, how silencing mechanisms are themselves regulated to avoid the silencing of genes remains unclear. Here, an anti-silencing factor was identified using a forward genetic screen on a reporter line that harbors a LUCIFERASE (LUC) gene driven by a promoter that undergoes DNA methylation. SUVH1, a Su(var)3-9 homolog, was identified as a factor promoting the expression of the LUC gene. Treatment with a cytosine methylation inhibitor completely suppressed the LUC expression defects of suvh1, indicating that SUVH1 is dispensable for LUC expression in the absence of DNA methylation. SUVH1 also promotes the expression of several endogenous genes with promoter DNA methylation. However, the suvh1 mutation did not alter DNA methylation levels at the LUC transgene or on a genome-wide scale; thus, SUVH1 functions downstream of DNA methylation. Histone H3 lysine 4 (H3K4) trimethylation was reduced in suvh1; in contrast, H3K9 methylation levels remained unchanged. This work has uncovered a novel, anti-silencing function for a member of the Su(var)3-9 family that has previously been associated with silencing through H3K9 methylation. PMID:26400170

  6. SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation

    PubMed Central

    Li, Shaofang; Liu, Lin; Li, Shengben; Gao, Lei; Zhao, Yuanyuan; Kim, Yun Ju; Chen, Xuemei

    2016-01-01

    Transposable elements are found throughout the genomes of all organisms. Repressive marks such as DNA methylation and histone H3 lysine 9 (H3K9) methylation silence these elements and maintain genome integrity. However, how silencing mechanisms are themselves regulated to avoid the silencing of genes remains unclear. Here, an anti-silencing factor was identified using a forward genetic screen on a reporter line that harbors a LUCIFERASE (LUC) gene driven by a promoter that undergoes DNA methylation. SUVH1, a Su(var)3–9 homolog, was identified as a factor promoting the expression of the LUC gene. Treatment with a cytosine methylation inhibitor completely suppressed the LUC expression defects of suvh1, indicating that SUVH1 is dispensable for LUC expression in the absence of DNA methylation. SUVH1 also promotes the expression of several endogenous genes with promoter DNA methylation. However, the suvh1 mutation did not alter DNA methylation levels at the LUC transgene or on a genome-wide scale; thus, SUVH1 functions downstream of DNA methylation. Histone H3 lysine 4 (H3K4) trimethylation was reduced in suvh1; in contrast, H3K9 methylation levels remained unchanged. This work has uncovered a novel, anti-silencing function for a member of the Su(var)3–9 family that has previously been associated with silencing through H3K9 methylation. PMID:26400170

  7. DNA methylation dynamics in the rat EGF gene promoter after partial hepatectomy

    PubMed Central

    Li, Deming; Fan, Jinyu; Li, Ziwei; Xu, Cunshuan

    2014-01-01

    Epidermal growth factor (EGF), a multifunctional growth factor, is a regulator in a wide variety of physiological processes. EGF plays an important role in the regulation of liver regeneration. This study was aimed at investigating the methylation level of EGF gene throughout liver regeneration. DNA of liver tissue from control rats and partial hepatectomy (PH) rats at 10 time points was extracted and a 354 bp fragment including 10 CpG sites from the transcription start was amplified after DNA was modified by sodium bisulfate. The result of sequencing suggested that methylation ratio of four CpG sites was found to be significantly changed when PH group was compared to control group, in particular two of them were extremely striking. mRNA expression of EGF was down-regulated in total during liver regeneration. We think that the rat EGF promoter region is regulated by variation in DNA methylation during liver regeneration. PMID:25071410

  8. A viral satellite DNA vector-induced transcriptional gene silencing via DNA methylation of gene promoter in Nicotiana benthamiana.

    PubMed

    Ju, Zheng; Wang, Lei; Cao, Dongyan; Zuo, Jinhua; Zhu, Hongliang; Fu, Daqi; Luo, Yunbo; Zhu, Benzhong

    2016-09-01

    Virus-induced gene silencing (VIGS) has been widely used for plant functional genomics study at the post-transcriptional level using various DNA or RNA viral vectors. However, while virus-induced transcriptional gene silencing (VITGS) via DNA methylation of gene promoter was achieved using several plant RNA viral vectors, it has not yet been done using a satellite DNA viral vector. In this study, a viral satellite DNA associated with tomato yellow leaf curl China virus (TYLCCNV), which has been modified as a VIGS vector in previous research, was developed as a VITGS vector. Firstly, the viral satellite DNA VIGS vector was further optimized to a more convenient p1.7A+2mβ vector with high silencing efficiency of the phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. Secondly, the constructed VITGS vector (TYLCCNV:35S), which carried a portion of the cauliflower mosaic virus 35S promoter, could successfully induce heritable transcriptional gene silencing (TGS) of the green fluorescent protein (GFP) gene in the 35S-GFP transgenic N. benthamiana line 16c plants. Moreover, bisulfite sequencing results revealed higher methylated cytosine residues at CG, CHG and CHH sites of the 35S promoter sequence in TYLCCNV:35S-inoculated plants than in TYLCCNV-inoculated line 16c plants (control). Overall, these results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes. PMID:27422476

  9. Promoter methylation of serotonin transporter gene is associated with obesity measures: a monozygotic twin study

    PubMed Central

    Zhao, J; Goldberg, J; Vaccarino, V

    2013-01-01

    Objective: Epigenetic mechanisms are increasingly being recognized as an important factor for obesity. The serotonin transporter gene (SLC6A4) has a critical role in regulating food intake, body weight and energy balance. This study examines the potential association between SLC6A4 promoter methylation and obesity measures in a monozygotic (MZ) twin sample. Methods: We studied 84 MZ twin pairs drawn from the Vietnam Era Twin Registry. Obesity measures include body mass index (BMI), body weight, waist circumference (WC) and waist-hip ratio (WHR). The SLC6A4 promoter methylation profile in peripheral blood leukocytes was quantified by bisulfite pyrosequencing. The association between methylation variation and obesity parameters was examined by mixed-model regression and matched pair analysis, adjusting for age, smoking, alcohol consumption, physical activity and total daily energy intake. Multiple testing was controlled using the adjusted false discovery rate (q-value). Results: Mean methylation level was positively correlated with BMI (r=0.29; P=0.0002), body weight (r=0.31; P<0.0001) and WC (r=0.20; P=0.009), but not WHR. Intra-pair differences in mean methylation were significantly correlated with intra-pair differences in BMI, body weight and WC, but not WHR. On average, a 1% increase in mean methylation was associated with 0.33 kg m−2 increase in BMI (95% CI: 0.02–0.65; P=0.03), 1.16 kg increase in body weight (95% CI, 0.16–2.16; P=0.02) and 0.78 cm increase in WC (95% CI, 0.05–1.50; P=0.03) after controlling for potential confounders. Conclusions: SLC6A4 promoter hypermethylation is significantly associated with an increased prevalence of obesity within a MZ twin study. PMID:22290534

  10. Promoter methylation of tumor suppressor genes in pre-neoplastic lesions; potential marker of disease recurrence

    PubMed Central

    2014-01-01

    Background Epigenetic alterations of specific genes have been reported to be related to colorectal cancer (CRC) transformation and would also appear to be involved in the early stages of colorectal carcinogenesis. Little data are available on the role of these alterations in determining a different risk of colorectal lesion recurrence. The aim of the present study was to verify whether epigenetic alterations present in pre-neoplastic colorectal lesions detected by colonoscopy can predict disease recurrence. Methods A retrospective series of 78 adenomas were collected and classified as low (35) or high-risk (43) for recurrence according to National Comprehensive Cancer Network guidelines. Methylation alterations were analyzed by the methylation-specific multiplex ligation probe assay (MS-MLPA) which is capable of quantifying methylation levels simultaneously in 24 different gene promoters. MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry. Results Higher levels of methylation were associated with disease recurrence. In particular, MLH1, ATM and FHIT gene promoters were found to be significantly hypermethylated in recurring adenomas. Unconditional logistic regression analysis used to evaluate the relative risk (RR) of recurrence showed that FHIT and MLH1 were independent variables with an RR of 35.30 (95% CI 4.15-300.06, P = 0.001) and 17.68 (95% CI 1.91-163.54, P = 0.011), respectively. Conclusions Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions. Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence. PMID:25091577

  11. Polymorphic tandem repeats within gene promoters act as modifiers of gene expression and DNA methylation in humans.

    PubMed

    Quilez, Javier; Guilmatre, Audrey; Garg, Paras; Highnam, Gareth; Gymrek, Melissa; Erlich, Yaniv; Joshi, Ricky S; Mittelman, David; Sharp, Andrew J

    2016-05-01

    Despite representing an important source of genetic variation, tandem repeats (TRs) remain poorly studied due to technical difficulties. We hypothesized that TRs can operate as expression (eQTLs) and methylation (mQTLs) quantitative trait loci. To test this we analyzed the effect of variation at 4849 promoter-associated TRs, genotyped in 120 individuals, on neighboring gene expression and DNA methylation. Polymorphic promoter TRs were associated with increased variance in local gene expression and DNA methylation, suggesting functional consequences related to TR variation. We identified >100 TRs associated with expression/methylation levels of adjacent genes. These potential eQTL/mQTL TRs were enriched for overlaps with transcription factor binding and DNaseI hypersensitivity sites, providing a rationale for their effects. Moreover, we showed that most TR variants are poorly tagged by nearby single nucleotide polymorphisms (SNPs) markers, indicating that many functional TR variants are not effectively assayed by SNP-based approaches. Our study assigns biological significance to TR variations in the human genome, and suggests that a significant fraction of TR variations exert functional effects via alterations of local gene expression or epigenetics. We conclude that targeted studies that focus on genotyping TR variants are required to fully ascertain functional variation in the genome. PMID:27060133

  12. Polymorphic tandem repeats within gene promoters act as modifiers of gene expression and DNA methylation in humans

    PubMed Central

    Quilez, Javier; Guilmatre, Audrey; Garg, Paras; Highnam, Gareth; Gymrek, Melissa; Erlich, Yaniv; Joshi, Ricky S.; Mittelman, David; Sharp, Andrew J.

    2016-01-01

    Despite representing an important source of genetic variation, tandem repeats (TRs) remain poorly studied due to technical difficulties. We hypothesized that TRs can operate as expression (eQTLs) and methylation (mQTLs) quantitative trait loci. To test this we analyzed the effect of variation at 4849 promoter-associated TRs, genotyped in 120 individuals, on neighboring gene expression and DNA methylation. Polymorphic promoter TRs were associated with increased variance in local gene expression and DNA methylation, suggesting functional consequences related to TR variation. We identified >100 TRs associated with expression/methylation levels of adjacent genes. These potential eQTL/mQTL TRs were enriched for overlaps with transcription factor binding and DNaseI hypersensitivity sites, providing a rationale for their effects. Moreover, we showed that most TR variants are poorly tagged by nearby single nucleotide polymorphisms (SNPs) markers, indicating that many functional TR variants are not effectively assayed by SNP-based approaches. Our study assigns biological significance to TR variations in the human genome, and suggests that a significant fraction of TR variations exert functional effects via alterations of local gene expression or epigenetics. We conclude that targeted studies that focus on genotyping TR variants are required to fully ascertain functional variation in the genome. PMID:27060133

  13. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

    PubMed Central

    2011-01-01

    Background Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Method Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Results Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Conclusions Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue. PMID:21668942

  14. Methylation pattern of ALX4 gene promoter as a potential biomarker for blood-based early detection of colorectal cancer

    PubMed Central

    Salehi, Rasoul; Atapour, Norollah; Vatandoust, Nasimeh; Farahani, Najmeh; Ahangari, Fatemeh; Salehi, Ahmad Reza

    2015-01-01

    Background: To develop a non-invasive screening method for colorectal cancer, we evaluated the methylation of ALX4 gene promoter in serum samples from patients with colorectal cancer (CRC) and equal number of healthy individuals. Materials and Methods: In serum samples from 25 patients with colorectal cancer and 25 healthy control subjects, isolated serum free-floating DNA was treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction (MSP) with primers specific for methylated or unmethylated promoter CpG island sequences of the ALX4 gene. Results: Methylation of the ALX4 gene promoter was present in the serum DNA of patients with adenoma and colorectal cancer. A sensitivity of 68% and specificity of 88% were achieved in the detection of promoter methylation in colorectal neoplasia samples. The difference in methylation status of the ALX4 promoter between the patients with colorectal neoplasia and the control group was statistically highly significant (P < 0.001). Conclusions: The results indicate that this serum free DNA test of methylation of the ALX4 gene promoter is a sensitive and specific method. Therefore in combination with other useful markers it seems ALX4 has the potential of a clinically useful test for the early detection of colorectal cancer. PMID:26918234

  15. Oxidative stress levels are correlated with P15 and P16 gene promoter methylation in myelodysplastic syndrome patients.

    PubMed

    Gonçalves, Ana Cristina; Cortesão, Emília; Oliveiros, Barbara; Alves, Vera; Espadana, Ana Isabel; Rito, Luís; Magalhães, Emília; Pereira, Sónia; Pereira, Amélia; Costa, José Manuel Nascimento; Mota-Vieira, Luisa; Sarmento-Ribeiro, Ana Bela

    2016-08-01

    Oxidative stress and abnormal DNA methylation have been implicated in some types of cancer, namely in myelodysplastic syndromes (MDS). Since both mechanisms are observed in MDS patients, we analyzed the correlation of intracellular levels of peroxides, superoxide anion, and glutathione (GSH), as well as ratios of peroxides/GSH and superoxide/GSH, with the methylation status of P15 and P16 gene promoters in bone marrow leukocytes from MDS patients. Compared to controls, these patients had lower GSH content, higher peroxide levels, peroxides/GSH and superoxide/GSH ratios, as well as higher methylation frequency of P15 and P16 gene promoters. Moreover, patients with methylated P15 gene had higher oxidative stress levels than patients without methylation (peroxides: 460 ± 42 MIF vs 229 ± 25 MIF, p = 0.001; superoxide: 383 ± 48 MIF vs 243 ± 17 MIF, p = 0.022; peroxides/GSH: 2.50 ± 0.08 vs 1.04 ± 0.34, p < 0.001; superoxide/GSH: 1.76 ± 0.21 vs 1.31 ± 0.10, p = 0.007). Patients with methylated P16 and at least one methylated gene had higher peroxide levels as well as peroxides/GSH ratio than patients without methylation. Interestingly, oxidative stress levels allow the discrimination of patients without methylation from ones with methylated P15, methylated P16, or at least one methylated (P15 or P16) promoter. Taken together, these findings support the hypothesis that oxidative stress is correlated with P15 and P16 hypermethylation. PMID:25982567

  16. Promoter methylation of APC and RAR-β genes as prognostic markers in non-small cell lung cancer (NSCLC).

    PubMed

    Feng, Hongxiang; Zhang, Zhenrong; Qing, Xin; Wang, Xiaowei; Liang, Chaoyang; Liu, Deruo

    2016-02-01

    Aberrant promoter hypermethylations of tumor suppressor genes are promising markers for lung cancer diagnosis and prognosis. The purpose of this study was to determine methylation status at APC and RAR-β promoters in primary NSCLC, and whether they have any relationship with survival. APC and RAR-β promoter methylation status were determined in 41 NSCLC patients using methylation specific PCR. APC promoter methylation was detectable in 9 (22.0%) tumor samples and 6 (14.6%) corresponding non-tumor samples (P=0.391). RAR-β promoter methylation was detectable in 13 (31.7%) tumor samples and 4 (9.8%) corresponding non-tumor samples (P=0.049) in the NSCLC patients. APC promoter methylation was found to be associated with T stage (P=0.046) and nodal status (P=0.019) in non-tumor samples, and with smoking (P=0.004) in tumor samples. RAR-β promoter methylation was found associated with age (P=0.031) in non-tumor samples and with primary tumor site in tumor samples. Patients with APC promoter methylation in tumor samples showed significantly longer survival than patients without it (Log-rank P=0.014). In a multivariate analysis of prognostic factors, APC methylation in tumor samples was an independent prognostic factor (P=0.012), as were N1 positive lymph node number (P=0.025) and N2 positive lymph node number (P=0.06). Our study shows that RAR-β methylation detected in lung tissue may be used as a predictive marker for NSCLC diagnosis and that APC methylation in tumor sample may be a useful marker for superior survival in NSCLC patients. PMID:26681652

  17. Methylation State of the EDA Gene Promoter in Chinese X-Linked Hypohidrotic Ectodermal Dysplasia Carriers

    PubMed Central

    Fan, Huali; Bian, Zhuan

    2013-01-01

    Introduction Hypodontia, hypohidrosis, sparse hair and characteristic faces are the main characters of X-linked hypohidrotic ectodermal dysplasia (XLHED) which is caused by genetic ectodysplasin A (EDA) deficiency. Heterozygous female carriers tend to have mild to moderate XLHED phenotype, even though 30% of them present no obvious symptom. Methods A large Chinese XLHED family was reported and the entire coding region and exon–intron boundaries of EDA gene were sequenced. To elucidate the mechanism for carriers’ tempered phenotype, we analyzed the methylation level on four sites of the promoter of EDA by the pyrosequencing system. Results A known frameshift mutation (c.573–574 insT) was found in this pedigree. Combined with the pedigrees we reported before, 120 samples comprised of 23 carrier females from 11 families and 97 healthy females were analyzed for the methylation state of EDA promoter. Within 95% confidence interval (CI), 18 (78.26%) carriers were hypermethylated at these 4 sites. Conclusion Chinese XLHED carriers often have a hypermethylated EDA promoter. PMID:23626789

  18. CpG Promoter Methylation Status is not a Prognostic Indicator of Gene Expression in Beryllium Challenge

    PubMed Central

    Tooker, Brian C.; Ozawa, Katie; Newman, Lee S.

    2016-01-01

    Individuals exposed to beryllium (Be) may develop Be sensitization (BeS) and progress to chronic beryllium disease (CBD). Recent studies with other metal antigens suggest epigenetic mechanisms may be involved in inflammatory disease processes, including granulomatous lung disorders and that a number of metal cations alter gene methylation. The objective of this study was to determine if Be can exert an epigenetic effect on gene expression by altering methylation in the promoter region of specific genes known to be involved in Be antigen-mediated gene expression. To investigate this objective, three macrophage tumor mouse cell lines known to differentially produce tumor necrosis factor (TNF)-α, but not interferon (IFN)-γ, in response to Be antigen were cultured with Be or controls. Following challenges, ELISA were performed to quantify induced TNFα and IFNγ expression. Bisulfate-converted DNA was evaluated by pyrosequencing to quantify CpG methylation within the promoters of TNFα and IFNγ. Be-challenged H36.12J cells expressed higher levels of TNFα compared to either H36.12E cells or P388D.1 cells. However, there were no variations in TNFα promoter CpG methylation levels between cell lines at the 6 CpG sites tested. H36.12J cell TNFα expression was shown to be metal specific by the induction of significantly more TNFα when exposed to Be than when exposed to aluminum sulfate, or nickel (II) chloride but not when exposed to cobalt (II) chloride. However, H36.12J cell methylation levels at the six CpG sites examined in the TNFα promoter did not correlate with cytokine expression differences. Nonetheless, all three cell lines had significantly more promoter methylation at the six CpG sites investigated within the IFNα promoter (a gene that is not expressed) when compared to the six CpG sites investigated in the TNFα promoter, regardless of treatment condition (p < 1.17 × 10−9). These findings suggest that in this cell system, promoter hypo-methylation

  19. CpG promoter methylation status is not a prognostic indicator of gene expression in beryllium challenge.

    PubMed

    Tooker, Brian C; Ozawa, Katherine; Newman, Lee S

    2016-05-01

    Individuals exposed to beryllium (Be) may develop Be sensitization (BeS) and progress to chronic beryllium disease (CBD). Recent studies with other metal antigens suggest epigenetic mechanisms may be involved in inflammatory disease processes, including granulomatous lung disorders and that a number of metal cations alter gene methylation. The objective of this study was to determine if Be can exert an epigenetic effect on gene expression by altering methylation in the promoter region of specific genes known to be involved in Be antigen-mediated gene expression. To investigate this objective, three macrophage tumor mouse cell lines known to differentially produce tumor necrosis factor (TNF)-α, but not interferon (IFN)-γ, in response to Be antigen were cultured with Be or controls. Following challenges, ELISA were performed to quantify induced TNFα and IFNγ expression. Bisulfate-converted DNA was evaluated by pyrosequencing to quantify CpG methylation within the promoters of TNFα and IFNγ. Be-challenged H36.12J cells expressed higher levels of TNFα compared to either H36.12E cells or P388D.1 cells. However, there were no variations in TNFα promoter CpG methylation levels between cell lines at the six CpG sites tested. H36.12J cell TNFα expression was shown to be metal-specific by the induction of significantly more TNFα when exposed to Be than when exposed to aluminum sulfate, or nickel (II) chloride, but not when exposed to cobalt (II) chloride. However, H36.12J cell methylation levels at the six CpG sites examined in the TNFα promoter did not correlate with cytokine expression differences. Nonetheless, all three cell lines had significantly more promoter methylation at the six CpG sites investigated within the IFNγ promoter (a gene that is not expressed) when compared to the six CpG sites investigated in the TNFα promoter, regardless of treatment condition (p < 1.17 × 10(-9)). These findings suggest that, in this cell system, promoter hypo-methylation

  20. Ancestral TCDD exposure promotes epigenetic transgenerational inheritance of imprinted gene Igf2: Methylation status and DNMTs.

    PubMed

    Ma, Jing; Chen, Xi; Liu, Yanan; Xie, Qunhui; Sun, Yawen; Chen, Jingshan; Leng, Ling; Yan, Huan; Zhao, Bin; Tang, Naijun

    2015-12-01

    Ancestral TCDD exposure could induce epigenetic transgenerational phenotypes, which may be mediated in part by imprinted gene inheritance. The aim of our study was to evaluate the transgenerational effects of ancestral TCDD exposure on the imprinted gene insulin-like growth factor-2 (Igf2) in rat somatic tissue. TCDD was administered daily by oral gavage to groups of F0 pregnant SD rats at dose levels of 0 (control), 200 or 800 ng/kg bw during gestation day 8-14. Animal transgenerational model of ancestral exposure to TCDD was carefully built, avoiding sibling inbreeding. Hepatic Igf2 expression of the TCDD male progeny was decreased concomitantly with hepatic damage and increased activities of serum hepatic enzymes both in the F1 and F3 generation. Imprinted Control Region (ICR) of Igf2 manifested a hypermethylated pattern, whereas methylation status in the Differentially Methylated Region 2 (DMR2) showed a hypomethylated manner in the F1 generation. These epigenetic alterations in these two regions maintained similar trends in the F3 generation. Meanwhile, the expressions of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) changed in a non-monotonic manner both in the F1 and F3 generation. This study provides evidence that ancestral TCDD exposure may promote epigenetic transgenerational alterations of imprinted gene Igf2 in adult somatic tissue. PMID:26455773

  1. The effects of omega-3 polyunsaturated fatty acids and genetic variants on methylation levels of the interleukin-6 gene promoter

    PubMed Central

    Ma, Yiyi; Smith, Caren E.; Lai, Chao-Qiang; Irvin, Marguerite R.; Parnell, Laurence D.; Lee, Yu-Chi; Pham, Lucia D.; Aslibekyan, Stella; Claas, Steven A.; Tsai, Michael Y.; Borecki, Ingrid B.; Kabagambe, Edmond K.; Ordovás, José M.; Absher, Devin M.; Arnett, Donna K.

    2016-01-01

    Scope Omega-3 PUFAs (n-3 PUFAs) reduce IL-6 gene expression, but their effects on transcription regulatory mechanisms are unknown. We aimed to conduct an integrated analysis with both population and in vitro studies to systematically explore the relationships among n-3 PUFA, DNA methylation, single nucleotide polymorphisms (SNPs), gene expression, and protein concentration of IL6. Methods and results Using data in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study and the Encyclopedia of DNA Elements (ENCODE) consortium, we found that higher methylation of IL6 promoter cg01770232 was associated with higher IL-6 plasma concentration (p = 0.03) and greater IL6 gene expression (p = 0.0005). Higher circulating total n-3 PUFA was associated with lower cg01770232 methylation (p = 0.007) and lower IL-6 concentration (p = 0.02). Moreover, an allele of IL6 rs2961298 was associated with higher cg01770232 methylation (p = 2.55 × 10−7). The association between n-3 PUFA and cg01770232 methylation was dependent on rs2961298 genotype (p = 0.02), but higher total n-3 PUFA was associated with lower cg01770232 methylation in the heterozygotes (p = 0.04) not in the homozygotes. Conclusion Higher n-3 PUFA is associated with lower methylation at IL6 promoter, which may be modified by IL6 SNPs. PMID:26518637

  2. Methylation of the Glucocorticoid Receptor Gene Promoter in Preschoolers: Links with Internalizing Behavior Problems

    ERIC Educational Resources Information Center

    Parade, Stephanie H.; Ridout, Kathryn K.; Seifer, Ronald; Armstrong, David A.; Marsit, Carmen J.; McWilliams, Melissa A.; Tyrka, Audrey R.

    2016-01-01

    Accumulating evidence suggests that early adversity is linked to methylation of the glucocorticoid receptor (GR) gene, "NR3C1," which is a key regulator of the hypothalamic-pituitary-adrenal axis. Yet no prior work has considered the contribution of methylation of "NR3C1" to emerging behavior problems and psychopathology in…

  3. Promoter methylation of fas apoptotic inhibitory molecule 2 gene is associated with obesity and dyslipidaemia in Chinese children.

    PubMed

    Wu, Lijun; Zhao, Xiaoyuan; Shen, Yue; Zhang, Mei-Xian; Yan, Yinkun; Hou, Dongqing; Meng, Linghui; Liu, Junting; Cheng, Hong; Mi, Jie

    2015-05-01

    Fas apoptotic inhibitory molecule 2 (FAIM2) is an obesity-related gene, but the mechanisms by which FAIM2 is involved in obesity are not understood. Epigenetic alterations are important factors in the development of obesity. The purpose of this study was to investigate the potential associations of FAIM2 promoter methylation with obesity and components of dyslipidaemia in Chinese children. We studied FAIM2 promoter methylation in 59 obese and 39 lean children using the Sequenom MassARRAY platform. The methylation levels at 8 CpG sites in the FAIM2 promoter were significantly different between the obese and lean subjects, especially the methylation level at CpG site 500 (p = 0.01). The methylation levels at several of the examined CpG sites were significantly associated with dyslipidaemia and its components after adjusting for age, gender and body mass index (BMI). The methylation levels at two CpG sites (sites -362 and -360 and site -164) were highly significantly associated with high level of triglycerides (p = 0.00002 and 0.0009, respectively). This study provides the first evidence that the methylation levels of the FAIM2 promoter are significantly associated with obesity and are independently associated with dyslipidaemia and its components in Chinese children. PMID:25696115

  4. Cigarette Smoking, BPDE-DNA Adducts, and Aberrant Promoter Methylations of Tumor Suppressor Genes (TSGs) in NSCLC from Chinese Population.

    PubMed

    Jin, Yongtang; Xu, Peiwei; Liu, Xinneng; Zhang, Chunye; Tan, Cong; Chen, Chunmei; Sun, Xiaoyu; Xu, Yingchun

    2016-01-01

    Non-small cell lung cancer (NSCLC) is related to the genetic and epigenetic factors. The goal of this study was to determine association of cigarette smoking and BPDE-DNA adducts with promoter methylations of several genes in NSCLC. Methylation of the promoters of p16, RARβ, DAPK, MGMT, and TIMP-3 genes of tumor tissues from 199 lung cancer patients was analyzed with methylation-specific PCR (MSP), and BPDE-DNA adduct level in lung cancer tissue was obtained by ELISA. Level of BPDE-DNA adduct increased significantly in males, aged people (over 60 years), and smokers; however, no significant difference was found while comparing the BPDE-DNA adduct levels among different tumor types, locations, and stages. Cigarette smoking was also associated with increased BPDE-DNA adducts level (OR = 2.43, p > .05) and increased methylation level in at least 1 gene (OR = 5.22, p < .01), both in dose-response manner. Similarly, cigarette smoking also significantly increase the risk of p16 or DAPK methylation (OR = 3.02, p < .05 for p16, and 3.66, p < .05 for DAPK). The highest risk of BPDE-DNA adducts was detected among individuals with cigarette smoking for more than 40 pack-years (OR = 4.21, p < .01). Furthermore, the present study did not show that BPDE-DNA adducts are significantly associated with abnormal TSGs methylations in NSCLC, including SCC and AdO, respectively. Conclusively, cigarette smoking is significantly associated with the increase of BPDE-DNA adduct level, promoter hypermethylation of p16 and DAPK genes, while BPDE-DNA adduct was not significantly related to abnormal promoter hypermethylation in TSGs, suggesting that BPDE-DNA adducts and TSGs methylations play independent roles in NSCLC. PMID:27042875

  5. Aberrant Methylation of the E-Cadherin Gene Promoter Region in the Endometrium of Women With Uterine Fibroids.

    PubMed

    Li, Yan; Ran, Ran; Guan, Yingxia; Zhu, Xiaoxiong; Kang, Shan

    2016-08-01

    A uterine fibroid is a leiomyoma that originates from the smooth muscle layer of the uterus. A variety of endometrial abnormalities are associated with uterine fibroids. This study aims to investigate the methylation status of the E-cadherin gene (CDH1) promoter region in the endometrium of patients with uterine fibroids. The methylation of CDH1 was studied using methylation-specific polymerase chain reaction in the endometrial tissue of 102 patients with uterine fibroids and 50 control patients. The E-cadherin expression was examined by flow cytometry. The methylation rate of CDH1 promoter region was 33.3% in the endometrium of patients with uterine fibroids and 8% in the endometrium of women without fibroids. The frequency of CDH1 promoter methylation in the endometrium of patients with fibroids was significantly higher than that in the endometrium of women without fibroids (P = .001). Furthermore, the E-cadherin expression level in methylation-positive tissues was significantly lower than that in methylation-negative tissues (P = .017). These results suggest that epigenetic aberration of CDH1 may occur in the endometrium of patients with fibroids, which may be associated with E-cadherin protein expression in endometrial tissue. PMID:26880767

  6. Large sex differences in chicken behavior and brain gene expression coincide with few differences in promoter DNA-methylation.

    PubMed

    Nätt, Daniel; Agnvall, Beatrix; Jensen, Per

    2014-01-01

    While behavioral sex differences have repeatedly been reported across taxa, the underlying epigenetic mechanisms in the brain are mostly lacking. Birds have previously shown to have only limited dosage compensation, leading to high sex bias of Z-chromosome gene expression. In chickens, a male hyper-methylated region (MHM) on the Z-chromosome has been associated with a local type of dosage compensation, but a more detailed characterization of the avian methylome is limiting our interpretations. Here we report an analysis of genome wide sex differences in promoter DNA-methylation and gene expression in the brain of three weeks old chickens, and associated sex differences in behavior of Red Junglefowl (ancestor of domestic chickens). Combining DNA-methylation tiling arrays with gene expression microarrays we show that a specific locus of the MHM region, together with the promoter for the zinc finger RNA binding protein (ZFR) gene on chromosome 1, is strongly associated with sex dimorphism in gene expression. Except for this, we found few differences in promoter DNA-methylation, even though hundreds of genes were robustly differentially expressed across distantly related breeds. Several of the differentially expressed genes are known to affect behavior, and as suggested from their functional annotation, we found that female Red Junglefowl are more explorative and fearful in a range of tests performed throughout their lives. This paper identifies new sites and, with increased resolution, confirms known sites where DNA-methylation seems to affect sexually dimorphic gene expression, but the general lack of this association is noticeable and strengthens the view that birds do not have dosage compensation. PMID:24782041

  7. Large Sex Differences in Chicken Behavior and Brain Gene Expression Coincide with Few Differences in Promoter DNA-Methylation

    PubMed Central

    Nätt, Daniel; Agnvall, Beatrix; Jensen, Per

    2014-01-01

    While behavioral sex differences have repeatedly been reported across taxa, the underlying epigenetic mechanisms in the brain are mostly lacking. Birds have previously shown to have only limited dosage compensation, leading to high sex bias of Z-chromosome gene expression. In chickens, a male hyper-methylated region (MHM) on the Z-chromosome has been associated with a local type of dosage compensation, but a more detailed characterization of the avian methylome is limiting our interpretations. Here we report an analysis of genome wide sex differences in promoter DNA-methylation and gene expression in the brain of three weeks old chickens, and associated sex differences in behavior of Red Junglefowl (ancestor of domestic chickens). Combining DNA-methylation tiling arrays with gene expression microarrays we show that a specific locus of the MHM region, together with the promoter for the zinc finger RNA binding protein (ZFR) gene on chromosome 1, is strongly associated with sex dimorphism in gene expression. Except for this, we found few differences in promoter DNA-methylation, even though hundreds of genes were robustly differentially expressed across distantly related breeds. Several of the differentially expressed genes are known to affect behavior, and as suggested from their functional annotation, we found that female Red Junglefowl are more explorative and fearful in a range of tests performed throughout their lives. This paper identifies new sites and, with increased resolution, confirms known sites where DNA-methylation seems to affect sexually dimorphic gene expression, but the general lack of this association is noticeable and strengthens the view that birds do not have dosage compensation. PMID:24782041

  8. Methylation of the Glucocorticoid Receptor Gene Promoter in Preschoolers: Links With Internalizing Behavior Problems.

    PubMed

    Parade, Stephanie H; Ridout, Kathryn K; Seifer, Ronald; Armstrong, David A; Marsit, Carmen J; McWilliams, Melissa A; Tyrka, Audrey R

    2016-01-01

    Accumulating evidence suggests that early adversity is linked to methylation of the glucocorticoid receptor (GR) gene, NR3C1, which is a key regulator of the hypothalamic-pituitary-adrenal axis. Yet no prior work has considered the contribution of methylation of NR3C1 to emerging behavior problems and psychopathology in childhood. This study examined the links between methylation of NR3C1 and behavior problems in preschoolers. Data were drawn from a sample of preschoolers with early adversity (n = 171). Children ranged in age from 3 to 5 years, were racially and ethnically diverse, and nearly all qualified for public assistance. Seventy-one children had child welfare documentation of moderate to severe maltreatment in the past 6 months. Structured record review and interviews in the home were used to assess early adversity. Parents reported on child internalizing and externalizing behavior problems. Methylation of NR3C1 at exons 1D , 1F , and 1H were measured via sodium bisulfite pyrosequencing from saliva DNA. Methylation of NR3C1 at exons 1D and 1F was positively associated with internalizing (r = .21, p < .01 and r = .23, p < .01, respectively), but not externalizing, behavior problems. Furthermore, NR3C1 methylation mediated effects of early adversity on internalizing behavior problems. These results suggest that methylation of NR3C1 contributes to psychopathology in young children, and NR3C1 methylation from saliva DNA is salient to behavioral outcomes. PMID:26822445

  9. RARβ Promoter Methylation as an Epigenetic Mechanism of Gene Silencing in Non-small Cell Lung Cancer.

    PubMed

    Dutkowska, A; Antczak, A; Pastuszak-Lewandoska, D; Migdalska-Sek, M; Czarnecka, K H; Górski, P; Kordiak, J; Nawrot, E; Brzeziańska-Lasota, E

    2016-01-01

    The retinoid acid receptor-p (RARβ) gene is one of the tumor suppressor genes (TSGs), which is frequently deleted or epigenetically silenced at an early stage of tumor progression. In this study we investigated the promoter methylation and expression status of the RARβ gene in 60 surgically resected non-small cell lung cancer (NSCLC) tissue samples and 60 corresponding unchanged lung tissue samples, using methylation-specific PCR and real-time-polymerase chain reaction (qPCR) techniques. We correlated the results with the pathological features of tumors and clinical characteristics of patients. qPCR analysis detected a significantly lower RARβ expression in the patients with adenocarcinoma (AC) and large cell carcinoma (LCC) than in those with squamous cell carcinoma (SCC) (AC vs. SCC, p = 0.032; AC and LCC vs. SCC, p = 0.0 13). Additionally, significantly lower expression of the RARβ gene was revealed in the patients with non-squamous cell cancer with a history of smoking assessed as pack-years (PY < 40 vs. PY ≥ 40, p = 0.045). Regarding RARβ promoter methylation, we found significant differences in the methylation index in the SCC group when considering pTNM staging; with higher index values in T1a + T1b compared with T2a + T2b and T3 + T4 groups (p = 0.024). There was no correlation between the methylation status and expression level of the RARβ gene, which suggests that other molecular mechanisms influence the RARβ expression in NSCLC patients. In conclusion, different expression of the RARβ gene in SCC and NSCC makes the RARβ gene a valuable diagnostic marker for differentiating the NSCLC subtypes. PMID:26453065

  10. Racial Differences in DNA-Methylation of CpG Sites Within Preterm-Promoting Genes and Gene Variants.

    PubMed

    Salihu, H M; Das, R; Morton, L; Huang, H; Paothong, A; Wilson, R E; Aliyu, M H; Salemi, J L; Marty, P J

    2016-08-01

    Objective To evaluate the role DNA methylation may play in genes associated with preterm birth for higher rates of preterm births in African-American women. Methods Fetal cord blood samples from births collected at delivery and maternal demographic and medical information were used in a cross-sectional study to examine fetal DNA methylation of genes implicated in preterm birth among black and non-black infants. Allele-specific DNA methylation analysis was performed using a methylation bead array. Targeted maximum likelihood estimation was applied to examine the relationship between race and fetal DNA methylation of candidate preterm birth genes. Receiver-operating characteristic analyses were then conducted to validate the CpG site methylation marker within the two racial groups. Bootstrapping, a method of validation and replication, was employed. Results 42 CpG sites were screened within 20 candidate gene variants reported consistently in the literature as being associated with preterm birth. Of these, three CpG sites on TNFAIP8 and PON1 genes (corresponding to: cg23917399; cg07086380; and cg07404485, respectively) were significantly differentially methylated between black and non-black individuals. The three CpG sites showed lower methylation status among infants of black women. Bootstrapping validated and replicated results. Conclusion for Practice Our study identified significant differences in levels of methylation on specific genes between black and non-black individuals. Understanding the genetic/epigenetic mechanisms that lead to preterm birth may lead to enhanced prevention strategies to reduce morbidity and mortality by eventually providing a means to identify individuals with a genetic predisposition to preterm labor. PMID:27000849

  11. Correlations of Promoter Methylation in WIF-1, RASSF1A, and CDH13 Genes with the Risk and Prognosis of Esophageal Cancer

    PubMed Central

    Guo, Qiang; Wang, Hai-Bo; Li, Yong-Hui; Li, He-Fei; Li, Ting-Ting; Zhang, Wen-Xue; Xiang, Sha-Sha; Sun, Zhen-Qing

    2016-01-01

    Background This study was designed to explore the correlations of promoter methylation in Wnt inhibitory factor-1 (WIF-1), ras-association domain family member 1A (RASSF1A), and Cadherin 13 (CDH13) genes with the risk and prognosis of esophageal cancer (EC). Material/Methods A total of 71 EC tissues from resection and 35 adjacent normal tissues were collected. Methylation status in the promoter region was detected by methylation- and non-methylation-specific primers. Corresponding mRNA levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Correlations between the methylations of these 3 genes and clinicopathologic characteristics were analyzed. Kaplan-Meier method and Cox regression model were used to investigate the relationships between WIF-1, RASSF1A, and CDH13 promoter methylations and the prognosis of EC. Results Compared with adjacent normal tissues, the methylation frequencies of WIF-1, RASSF1A, and CDH13 genes were significantly higher but the mRNA levels of these 3 genes were significantly lower in EC tissues (all P<0.05). WIF-1 and CDH13 promoter methylations were associated with the degree of tumor differentiation and WIF-1 and RASSF1A promoter methylations were associated with age (all P<0.05). The survival rates of patients with WIF-1, RASSF1A, and CDH13 methylations were significantly lower than those of patients without methylation (all P<0.05). WIF-1, RASSF1A, and CDH13 promoter methylations were independent risk factors affecting the prognosis of EC (all P<0.05). Conclusions WIF-1, RASSF1A, and CDH13 promoter methylations are associated with EC. The methylation levels are negatively related with the prognosis in EC. PMID:27506957

  12. Aberrant Promoter Methylation of p16 and MGMT Genes in Lung Tumors from Smoking and Never-Smoking Lung Cancer Patients1

    PubMed Central

    Liu, Yang; Lan, Qing; Siegfried, Jill M; Luketich, James D; Keohavong, Phouthone

    2006-01-01

    Abstract Aberrant methylation in gene promoter regions leads to transcriptional inactivation of cancer-related genes and plays an integral role in tumorigenesis. This alteration has been investigated in lung tumors primarily from smokers, whereas only a few studies involved never-smokers. Here, we applied methylation-specific polymerase chain reaction to compare the frequencies of the methylated promoter of p16 and O6-methylguanine-DNA methyltransferase (MGMT) genes in lung tumors from 122 patients with non-small cell lung cancer, including 81 smokers and 41 never-smokers. Overall, promoter methylation was detected in 52.5% (64 of 122) and 30.3% (37 of 122) of the p16 and MGMT genes, respectively. Furthermore, the frequency of promoter methylation was significantly higher among smokers, compared with never-smokers, for both the p16 [odds ratio (OR) = 3.28; 95% confidence interval (CI) = 1.28-8.39; P = .013] and MGMT (OR = 3.93; 95% CI = 1.27-12.21; P = .018) genes. The trend for a higher promoter methylation frequency of these genes was also observed among female smokers compared with female never-smokers. Our results suggest an association between tobacco smoking and an increased incidence of aberrant promoter methylation of the p16 and MGMT genes in non-small cell lung cancer. PMID:16533425

  13. Developmental genes significantly afflicted by aberrant promoter methylation and somatic mutation predict overall survival of late-stage colorectal cancer

    PubMed Central

    An, Ning; Yang, Xue; Cheng, Shujun; Wang, Guiqi; Zhang, Kaitai

    2015-01-01

    Carcinogenesis is an exceedingly complicated process, which involves multi-level dysregulations, including genomics (majorly caused by somatic mutation and copy number variation), DNA methylomics, and transcriptomics. Therefore, only looking into one molecular level of cancer is not sufficient to uncover the intricate underlying mechanisms. With the abundant resources of public available data in the Cancer Genome Atlas (TCGA) database, an integrative strategy was conducted to systematically analyze the aberrant patterns of colorectal cancer on the basis of DNA copy number, promoter methylation, somatic mutation and gene expression. In this study, paired samples in each genomic level were retrieved to identify differentially expressed genes with corresponding genetic or epigenetic dysregulations. Notably, the result of gene ontology enrichment analysis indicated that the differentially expressed genes with corresponding aberrant promoter methylation or somatic mutation were both functionally concentrated upon developmental process, suggesting the intimate association between development and carcinogenesis. Thus, by means of random walk with restart, 37 significant development-related genes were retrieved from a priori-knowledge based biological network. In five independent microarray datasets, Kaplan–Meier survival and Cox regression analyses both confirmed that the expression of these genes was significantly associated with overall survival of Stage III/IV colorectal cancer patients. PMID:26691761

  14. DNA methylation analysis of human myoblasts during in vitro myogenic differentiation: de novo methylation of promoters of muscle-related genes and its involvement in transcriptional down-regulation

    PubMed Central

    Miyata, Kohei; Miyata, Tomoko; Nakabayashi, Kazuhiko; Okamura, Kohji; Naito, Masashi; Kawai, Tomoko; Takada, Shuji; Kato, Kiyoko; Miyamoto, Shingo; Hata, Kenichiro; Asahara, Hiroshi

    2015-01-01

    Although DNA methylation is considered to play an important role during myogenic differentiation, chronological alterations in DNA methylation and gene expression patterns in this process have been poorly understood. Using the Infinium HumanMethylation450 BeadChip array, we obtained a chronological profile of the genome-wide DNA methylation status in a human myoblast differentiation model, where myoblasts were cultured in low-serum medium to stimulate myogenic differentiation. As the differentiation of the myoblasts proceeded, their global DNA methylation level increased and their methylation patterns became more distinct from those of mesenchymal stem cells. Gene ontology analysis revealed that genes whose promoter region was hypermethylated upon myoblast differentiation were highly significantly enriched with muscle-related terms such as ‘muscle contraction’ and ‘muscle system process’. Sequence motif analysis identified 8-bp motifs somewhat similar to the binding motifs of ID4 and ZNF238 to be most significantly enriched in hypermethylated promoter regions. ID4 and ZNF238 have been shown to be critical transcriptional regulators of muscle-related genes during myogenic differentiation. An integrated analysis of DNA methylation and gene expression profiles revealed that de novo DNA methylation of non-CpG island (CGI) promoters was more often associated with transcriptional down-regulation than that of CGI promoters. These results strongly suggest the existence of an epigenetic mechanism in which DNA methylation modulates the functions of key transcriptional factors to coordinately regulate muscle-related genes during myogenic differentiation. PMID:25190712

  15. Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing.

    PubMed

    Kon, Tatsuya; Yoshikawa, Nobuyuki

    2014-01-01

    Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification. PMID:25426109

  16. Promoter Methylation Status of Breast Cancer Susceptibility Gene 1 and 17 Beta Hydroxysteroid Dehydrogenase Type 1 Gene in Sporadic Breast Cancer Patients

    PubMed Central

    Hosny, Marwa M.; Sabek, Nagwan A.; El-Abaseri, Taghrid B.; Hassan, Fathalla M.; Farrag, Sherif H.

    2016-01-01

    Epigenetic modifications are involved in breast carcinogenesis. Identifying genes that are epigenetically silenced via methylation could select target patients for diagnostic as well as therapeutic potential. We assessed promoter methylation of breast cancer susceptibility gene 1 (BRCA1) and 17 Beta Hydroxysteroid Dehydrogenase Type 1 (17βHSD-1) in normal and cancer breast tissues of forty sporadic breast cancer (BC) cases using restriction enzyme based methylation-specific PCR (REMS-PCR). In cancerous tissues, BRCA1 and 17βHSD-1 were methylated in 42.5% and 97.5%, respectively, while normal tissues had 35% and 95% methylation, respectively. BRCA1 methylation in normal tissues was 12.2-fold more likely to associate with methylation in cancer tissues (p < 0.001). It correlated significantly with increased age at menopause, mitosis, the negative status of Her2, and the molecular subtype “luminal A” (p = 0.048, p = 0.042, p = 0.007, and p = 0.049, resp.). Methylation of BRCA1 and 17βHSD-1 related to luminal A subtype of breast cancer. Since a small proportion of normal breast epithelial cells had BRCA1 methylation, our preliminary findings suggest that methylation of BRCA1 may be involved in breast tumors initiation and progression; therefore, it could be used as a biomarker for the early detection of sporadic breast cancer. Methylation of 17βHSD-1 in normal and cancer tissue could save patients the long term use of adjuvant antiestrogen therapies. PMID:27413552

  17. Gene-specific promoter methylation is associated with micronuclei frequency in urothelial cells from individuals exposed to organic solvents and paints.

    PubMed

    Hoyos-Giraldo, L S; Escobar-Hoyos, L F; Saavedra-Trujillo, D; Reyes-Carvajal, I; Muñoz, A; Londoño-Velasco, E; Tello, A; Cajas-Salazar, N; Ruíz, M; Carvajal, S; Santella, R M

    2016-05-01

    Sufficient epidemiologic evidence has established an etiologic link between bladder cancer risk and occupational exposure as a painter to organic solvents. Currently, it remains to be established whether gene-specific promoter methylation contributes to bladder cancer development, including by enhancing chromosome breakage or loss. We investigated the effect of chronic exposure to organic solvents and paints on DNA methylation profiles in the promoter regions of four genes (GSTP1, p16(INK4a), APC and CDH1) and micronucleus (MN) frequency in exfoliated urothelial cells from voided urine from Colombian male non-smoking car painters and age-matched unexposed individuals. The exposed group had a higher percentage of individuals with >2 MNs/2000 cells compared with the unexposed group (P=0.04). Gene-specific analysis showed a significantly higher percentage of individuals with methylated GSTP1, p16(INK4a) and APC in the exposed group. Poisson regression analysis indicated that exposed individuals with methylated GSTP1 and p16(INK4a) promoters were more than twofold more likely to have an increase in MN frequency as compared with the reference. Finally, among exposed individuals with GSTP1 and p16(INK4a) methylated promoters, those with a greater age had a higher RR of increased MN frequency compared with younger exposed individuals with methylated promoters. These results support the conclusion that gene-specific promoter methylation may increase MN frequency in a dependent or independent interaction with occupational exposure to organic solvents. PMID:25993025

  18. Splice variants and promoter methylation status of the Bovine Vasa Homology (Bvh) gene may be involved in bull spermatogenesis

    PubMed Central

    2013-01-01

    Background Vasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation. Results Here we report the molecular characterization, alternative splicing pattern, expression and promoter methylation status of Bvh. The full-length coding region of Bvh was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. Bvh is expressed only in the ovary and testis of adult cattle. Two splice variants were identified and termed Bvh-V4 (2112 bp and 703 aa) and Bvh-V45 (2040 bp and 679 aa). In male cattle, full-length Bvh (Bvh-FL), Bvh-V4 and Bvh-V45 are exclusively expressed in the testes in the ratio of 2.2:1.6:1, respectively. Real-time PCR revealed significantly reduced mRNA expression of Bvh-FL, Bvh-V4 and Bvh-V45 in testes of cattle-yak hybrids, with meiotic arrest compared with cattle and yaks with normal spermatogenesis (P < 0.01). The promoter methylation level of Bvh in the testes of cattle-yak hybrids was significantly greater than in cattle and yaks (P < 0.01). Conclusion In the present study, Bvh was isolated and characterized. These data suggest that Bvh functions in bovine spermatogenesis, and that transcription of the gene in testes were regulated by alternative splice and promoter methylation. PMID:23815438

  19. Expression and promoter methylation status of hMLH1, MGMT, APC, and CDH1 genes in patients with colon adenocarcinoma.

    PubMed

    Michailidi, Christina; Theocharis, Stamatios; Tsourouflis, Gerasimos; Pletsa, Vasiliki; Kouraklis, Gregorios; Patsouris, Efstratios; Papavassiliou, Athanasios G; Troungos, Constantinos

    2015-12-01

    Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor's formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient's age and tumor's clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis. PMID:25908636

  20. Simultaneous Analysis of SEPT9 Promoter Methylation Status, Micronuclei Frequency, and Folate-Related Gene Polymorphisms: The Potential for a Novel Blood-Based Colorectal Cancer Biomarker

    PubMed Central

    Ravegnini, Gloria; Zolezzi Moraga, Juan Manuel; Maffei, Francesca; Musti, Muriel; Zenesini, Corrado; Simeon, Vittorio; Sammarini, Giulia; Festi, Davide; Hrelia, Patrizia; Angelini, Sabrina

    2015-01-01

    One challenge in colorectal cancer (CRC) is identifying novel biomarkers to be introduced in screening programs. The present study investigated the promoter methylation status of the SEPT9 gene in peripheral blood samples of subjects’ positive fecal occult blood test (FOBT). In order to add new insights, we investigated the association between SEPT9 promoter methylation and micronuclei frequency, and polymorphisms in the folate-related pathway genes. SEPT9 promoter methylation, micronuclei frequency, and genotypes were evaluated on 74 individuals’ FOBT positive. Individuals were subjected to a colonoscopy that provided written informed consent for study participation. SEPT9 promoter methylation status was significantly lower in the CRC group than controls (p = 0.0006). In contrast, the CaCo2 cell-line, analyzed as a tissue specific model of colon adenocarcinoma, showed a significantly higher percentage of SEPT9 promoter methylation compared to the CRC group (p < 0.0001). Linear regression analysis showed an inverse correlation between micronuclei frequency and the decrease in the methylation levels of SEPT9 promoter region among CRC patients (β = −0.926, p = 0.0001). With regard to genotype analysis, we showed the involvement of the DHFR polymorphism (rs70991108) in SEPT9 promoter methylation level in CRC patients only. In particular, the presence of at least one 19 bp del allele significantly correlates with decreased SEPT9 promoter methylation, compared to the 19 bp ins/ins genotype (p = 0.007). While remaining aware of the strengths and limitations of the study, this represents the first evidence of a novel approach for the early detection of CRC, using SEPT9 promoter methylation, micronuclei frequency and genotypes, with the potential to improve CRC risk assessment. PMID:26633373

  1. Simultaneous Analysis of SEPT9 Promoter Methylation Status, Micronuclei Frequency, and Folate-Related Gene Polymorphisms: The Potential for a Novel Blood-Based Colorectal Cancer Biomarker.

    PubMed

    Ravegnini, Gloria; Zolezzi Moraga, Juan Manuel; Maffei, Francesca; Musti, Muriel; Zenesini, Corrado; Simeon, Vittorio; Sammarini, Giulia; Festi, Davide; Hrelia, Patrizia; Angelini, Sabrina

    2015-01-01

    One challenge in colorectal cancer (CRC) is identifying novel biomarkers to be introduced in screening programs. The present study investigated the promoter methylation status of the SEPT9 gene in peripheral blood samples of subjects' positive fecal occult blood test (FOBT). In order to add new insights, we investigated the association between SEPT9 promoter methylation and micronuclei frequency, and polymorphisms in the folate-related pathway genes. SEPT9 promoter methylation, micronuclei frequency, and genotypes were evaluated on 74 individuals' FOBT positive. Individuals were subjected to a colonoscopy that provided written informed consent for study participation. SEPT9 promoter methylation status was significantly lower in the CRC group than controls (p = 0.0006). In contrast, the CaCo2 cell-line, analyzed as a tissue specific model of colon adenocarcinoma, showed a significantly higher percentage of SEPT9 promoter methylation compared to the CRC group (p < 0.0001). Linear regression analysis showed an inverse correlation between micronuclei frequency and the decrease in the methylation levels of SEPT9 promoter region among CRC patients (β = -0.926, p = 0.0001). With regard to genotype analysis, we showed the involvement of the DHFR polymorphism (rs70991108) in SEPT9 promoter methylation level in CRC patients only. In particular, the presence of at least one 19 bp del allele significantly correlates with decreased SEPT9 promoter methylation, compared to the 19 bp ins/ins genotype (p = 0.007). While remaining aware of the strengths and limitations of the study, this represents the first evidence of a novel approach for the early detection of CRC, using SEPT9 promoter methylation, micronuclei frequency and genotypes, with the potential to improve CRC risk assessment. PMID:26633373

  2. Promoter methylation status of tumor suppressor genes and inhibition of expression of DNA methyltransferase 1 in non-small cell lung cancer.

    PubMed

    Liu, Bangqing; Song, Jianfei; Luan, Jiaqiang; Sun, Xiaolin; Bai, Jian; Wang, Haiyong; Li, Angui; Zhang, Lifei; Feng, Xiaoyan; Du, Zhenzong

    2016-08-01

    DNA methylation is an epigenetic DNA modification catalyzed by DNA methyltransferase 1 (DNMT1). The purpose of this study was to investigate DNMT1 gene and protein expression and the effects of methylation status on tumor suppressor genes in human non-small cell lung cancer (NSCLC) cell lines grown in vitro and in vivo Human lung adenocarcinoma cell lines, A549 and H838, were grown in vitro and inoculated subcutaneously into nude mice to form tumors and were then treated with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine, with and without treatment with the benzamide histone deacetylase inhibitor, entinostat (MS-275). DNMT1 protein expression was quantified by Western blot. Promoter methylation status of tumor suppressor genes (RASSF1A, ASC, APC, MGMT, CDH13, DAPK, ECAD, P16, and GATA4) was evaluated by methylation-specific polymerase chain reaction. Methylation status of the tumor suppressor genes was regulated by the DNMT1 gene, with the decrease of DNMT1 expression following DNA methylation treatment. Demethylation of tumor suppressor genes (APC, ASC, and RASSF1A) restored tumor growth in nude mice. The results of this study support a role for methylation of DNA as a potential epigenetic clinical biomarker of prognosis or response to therapy and for DNMT1 as a potential therapeutic target in NSCLC. PMID:27190263

  3. Developmental changes in DNA methylation and covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin gene promoters in Papio anubis.

    PubMed

    Lavelle, Donald; Vaitkus, Kestis; Hankewych, Maria; Singh, Mahipal; DeSimone, Joseph

    2006-01-01

    The baboon is a suitable and relevant animal model to study the mechanism of human globin gene switching. This investigation addresses the role of DNA methylation and histone coding in globin gene switching in the baboon, Papio anubis. Bisulfite sequencing and chromatin immunoprecipitation studies were performed in erythroid cells purified from fetuses of varying gestational ages and from adult bone marrow to analyze the manner that changes in DNA methylation of the epsilon-, gamma-, and beta-globin promoters and association of ac-H3, ac-H4, H3-dimeK4, H3-dimeK36, and H3-dimeK79 with the epsilon-, gamma-, and beta-globin promoters occur during development. Changes in DNA methylation of the epsilon- and gamma-globin gene promoters during transitional stages of globin gene switching were consistent with the stochastic model of methylation and a role of DNA methylation in gene silencing. Enrichment of ac-H3, ac-H4, and pol II at the promoters of developmentally active genes was observed, while the pattern of distribution of H3-dimeK4 and H3-dimeK79 suggests that these modifications are found near both currently and formerly active promoters. Enrichment of H3-dimeK36 at the silenced epsilon-globin gene promoter was observed. These studies demonstrate that coordinated epigenetic modifications in the chromatin structure of the beta-like globin gene promoters accompany the highly regulated changes in expression patterns of these genes during development. PMID:16527500

  4. PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells

    SciTech Connect

    Gonen, Nitzan; Bram, Eran E.; Assaraf, Yehuda G.

    2008-11-28

    The proton-coupled folate transporter (PCFT/SLC46A1) displays optimal and prominent folate and antifolate transport activity at acidic pH in human carcinoma cells but poor activity in leukemia cells. Consistently herein, human leukemia cell lines expressed poor PCFT transcript levels, whereas various carcinoma cell lines showed substantial PCFT gene expression. We identified a CpG island with high density at nucleotides -200 through +100 and explored its role in PCFT promoter silencing. Leukemia cells with barely detectable PCFT transcripts consistently harbored 85-100% methylation of this CpG island, whereas no methylation was found in carcinoma cells. Treatment with 5-Aza-2'-deoxycytidine which induced demethylation but not with the histone deacetylase inhibitor trichostatin A, restored 50-fold PCFT expression only in leukemia cells. These findings constitute the first demonstration of the dominant epigenetic silencing of the PCFT gene in leukemia cells. The potential translational implications of the restoration of PCFT expression in chemotherapy of leukemia are discussed.

  5. Aging and chronic alcohol consumption are determinants of p16 gene expression, genomic DNA methylation and p16 promoter methylation in the mouse colon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elder age and chronic alcohol consumption are important risk factors for the development of colon cancer. Each factor can alter genomic and gene-specific DNA methylation. This study examined the effects of aging and chronic alcohol consumption on genomic and p16-specific methylation, and p16 express...

  6. Methylation of the leukocyte glucocorticoid receptor gene promoter in adults: associations with early adversity and depressive, anxiety and substance-use disorders.

    PubMed

    Tyrka, A R; Parade, S H; Welch, E S; Ridout, K K; Price, L H; Marsit, C; Philip, N S; Carpenter, L L

    2016-01-01

    Early adversity increases risk for developing psychopathology. Epigenetic modification of stress reactivity genes is a likely mechanism contributing to this risk. The glucocorticoid receptor (GR) gene is of particular interest because of the regulatory role of the GR in hypothalamic-pituitary-adrenal (HPA) axis function. Mounting evidence suggests that early adversity is associated with GR promoter methylation and gene expression. Few studies have examined links between GR promoter methylation and psychopathology, and findings to date have been mixed. Healthy adult participants (N=340) who were free of psychotropic medications reported on their childhood experiences of maltreatment and parental death and desertion. Lifetime depressive and anxiety disorders and past substance-use disorders were assessed using the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Methylation of exon 1F of the GR gene (NR3C1) was examined in leukocyte DNA via pyrosequencing. On a separate day, a subset of the participants (n=231) completed the dexamethasone/corticotropin-releasing hormone (Dex/CRH) test. Childhood adversity and a history of past substance-use disorder and current or past depressive or anxiety disorders were associated with lower levels of NR3C1 promoter methylation across the region as a whole and at individual CpG sites (P<0.05). The number of adversities was negatively associated with NR3C1 methylation in participants with no lifetime disorder (P=0.018), but not in those with a lifetime disorder. GR promoter methylation was linked to altered cortisol responses to the Dex/CRH test (P<0.05). This study presents evidence of reduced methylation of NR3C1 in association with childhood maltreatment and depressive, anxiety and substance-use disorders in adults. This finding stands in contrast to our prior work, but is consistent with emerging findings, suggesting complexity in the regulation of this gene. PMID

  7. Methylation of the leukocyte glucocorticoid receptor gene promoter in adults: associations with early adversity and depressive, anxiety and substance-use disorders

    PubMed Central

    Tyrka, A R; Parade, S H; Welch, E S; Ridout, K K; Price, L H; Marsit, C; Philip, N S; Carpenter, L L

    2016-01-01

    Early adversity increases risk for developing psychopathology. Epigenetic modification of stress reactivity genes is a likely mechanism contributing to this risk. The glucocorticoid receptor (GR) gene is of particular interest because of the regulatory role of the GR in hypothalamic–pituitary–adrenal (HPA) axis function. Mounting evidence suggests that early adversity is associated with GR promoter methylation and gene expression. Few studies have examined links between GR promoter methylation and psychopathology, and findings to date have been mixed. Healthy adult participants (N=340) who were free of psychotropic medications reported on their childhood experiences of maltreatment and parental death and desertion. Lifetime depressive and anxiety disorders and past substance-use disorders were assessed using the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Methylation of exon 1F of the GR gene (NR3C1) was examined in leukocyte DNA via pyrosequencing. On a separate day, a subset of the participants (n=231) completed the dexamethasone/corticotropin-releasing hormone (Dex/CRH) test. Childhood adversity and a history of past substance-use disorder and current or past depressive or anxiety disorders were associated with lower levels of NR3C1 promoter methylation across the region as a whole and at individual CpG sites (P<0.05). The number of adversities was negatively associated with NR3C1 methylation in participants with no lifetime disorder (P=0.018), but not in those with a lifetime disorder. GR promoter methylation was linked to altered cortisol responses to the Dex/CRH test (P<0.05). This study presents evidence of reduced methylation of NR3C1 in association with childhood maltreatment and depressive, anxiety and substance-use disorders in adults. This finding stands in contrast to our prior work, but is consistent with emerging findings, suggesting complexity in the regulation of this gene. PMID

  8. Identification of aberrant gene expression associated with aberrant promoter methylation in primordial germ cells between E13 and E16 rat F3 generation vinclozolin lineage

    PubMed Central

    2015-01-01

    Background Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then re-established during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. Methods This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. Results The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Conclusions Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study. PMID:26677731

  9. Differential vitamin D 24-hydroxylase/CYP24A1 gene promoter methylation in endothelium from benign and malignant human prostate

    PubMed Central

    Karpf, Adam R; Omilian, Angela R; Bshara, Wiam; Tian, Lili; Tangrea, Michael A; Morrison, Carl D; Johnson, Candace S

    2011-01-01

    Epigenetic alterations occur in tumor-associated vessels in the tumor microenvironment. Methylation of the CYP24A1 gene promoter differs in endothelial cells isolated from tumors and non-tumor microenvironments in mice. The epigenetic makeup of endothelial cells of human tumor-associated vasculature is unknown due to difficulty of isolating endothelial cells populations from a heterogeneous tissue microenvironment. To ascertain CYP24A1 promoter methylation in tumor-associated endothelium, we utilized laser microdissection guided by CD31 immunohistochemistry to procure endothelial cells from human prostate tumor specimens. Prostate tissues were obtained following robotic radical prostatectomy from men with clinically localized prostate cancer. Adjacent histologically benign prostate tissues were used to compare endothelium from benign versus tumor microenvironments. Sodium bisulfite sequencing of CYP24A1 promoter region showed that the average CYP24A1 promoter methylation in the endothelium was 20% from the tumor microenvironment compared with 8.2% in the benign microenvironment (p < 0.05). A 2-fold to 17-fold increase in CYP24A1 promoter methylation was observed in the prostate tumor endothelium compared with the matched benign prostate endothelium in four patient samples, while CYP24A1 promoter methylation remained unchanged in two patient samples. In addition, there is no correlation of the level of CYP24A1 promoter methylation in prostate tumor-associated endothelium with that of epithelium/stroma. This study demonstrates that the CYP24A1 promoter is methylated in tumor-associated endothelium, indicating that epigenetic alterations in CYP24A1 may play a role in determining the phenotype of tumor-associated vasculature in the prostate tumor microenvironment. PMID:21725204

  10. Gene methylation in gastric cancer.

    PubMed

    Qu, Yiping; Dang, Siwen; Hou, Peng

    2013-09-23

    Gastric cancer is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide. Over 70% of new cases and deaths occur in developing countries. In the early years of the molecular biology revolution, cancer research mainly focuses on genetic alterations, including gastric cancer. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer, including DNA methylation, histone modifications, nucleosome positioning, noncoding RNAs, and microRNAs. Aberrant DNA methylation in the promoter regions of gene, which leads to inactivation of tumor suppressor and other cancer-related genes in cancer cells, is the most well-defined epigenetic hallmark in gastric cancer. The advantages of gene methylation as a target for detection and diagnosis of cancer in biopsy specimens and non-invasive body fluids such as serum and gastric washes have led to many studies of application in gastric cancer. This review focuses on the most common and important phenomenon of epigenetics, DNA methylation, in gastric cancer and illustrates the impact epigenetics has had on this field. PMID:23669186

  11. Ubiquinol affects the expression of genes involved in PPARα signalling and lipid metabolism without changes in methylation of CpG promoter islands in the liver of mice

    PubMed Central

    Schmelzer, Constance; Kitano, Mitsuaki; Hosoe, Kazunori; Döring, Frank

    2012-01-01

    Coenzyme Q10 is an essential cofactor in the respiratory chain and serves as a potent antioxidant in biological membranes. Recent studies in vitro and in vivo provide evidence that Coenzyme Q10 is involved in inflammatory processes and lipid metabolism via gene expression. To study these effects at the epigenomic level, C57BL6J mice were supplemented for one week with reduced Coenzyme Q10 (ubiquinol). Afterwards, gene expression signatures and DNA promoter methylation patterns of selected genes were analysed. Genome-wide transcript profiling in the liver identified 1112 up-regulated and 571 down-regulated transcripts as differentially regulated between ubiquinol-treated and control animals. Text mining and GeneOntology analysis revealed that the ”top 20” ubiquinol-regulated genes play a role in lipid metabolism and are functionally connected by the PPARα signalling pathway. With regard to the ubiquinol-induced changes in gene expression of about +3.14-fold (p≤0.05), +2.18-fold (p≤0.01), and −2.13-fold (p≤0.05) for ABCA1, ACYP1, and ACSL1 genes, respectively, hepatic DNA methylation analysis of 282 (sense orientation) and 271 (antisense) CpG units in the respective promoter islands revealed no significant effect of ubiquinol. In conclusion, ubiquinol affects the expression of genes involved in PPARα signalling and lipid metabolism without changing the promoter DNA methylation status in the liver of mice. PMID:22448092

  12. Short Hairpin RNA Causes the Methylation of Transforming Growth Factor-β Receptor II Promoter and Silencing of the Target Gene in Rat Hepatic Stellate Cells

    PubMed Central

    Kim, Jin-Wook; Zhang, Yan-Hong; Zern, Mark A; Rossi, John J.; Wu, Jian

    2008-01-01

    Small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in plant and animal cells. RNA dependent DNA methylation (RdDM) accounts for TGS in plants, but it is unclear whether siRNA induces RdDM in mammalian cells. To determine whether stable expression of short hairpin siRNA (shRNA) induces DNA methylation in mammalian cells, we transduced rat hepatic stellate SBC10 cells with lentiviral vectors which encode an U6 promoter-driven shRNA expression cassette homologous to the transforming growth factor-β receptor (TGFβRII) promoter region. Sequencing analysis of bisulfite-modified genomic DNA showed the methylation of cytosine residues both in CpG dinucleotides and non-CpG sites around the target region of the TGFβRII promoter in SBC10 cells transduced with the promoter-targeting lentiviral vector. In these cells, real-time RT-PCR showed a decrease in TGFβRII mRNA levels which were reversed by treatment with 5-aza-2-deoxycytidine. Our results demonstrate that recombinant lentivirus-mediated shRNA delivery resulted in the methylation of the homologous promoter area in mammalian cells, and this approach may be used as a tool for transcriptional gene silencing by epigenetic modification of mammalian cell promoters. PMID:17533113

  13. Aberrant Promoter Methylation at CpG Cytosines Induce the Upregulation of the E2F5 Gene in Breast Cancer

    PubMed Central

    Ali, Arshad; Ullah, Farman; Ali, Irum Sabir; Faraz, Ahmad; Khan, Mumtaz; Shah, Syed Tahir Ali; Ali, Nawab

    2016-01-01

    Purpose The promoter methylation status of cell cycle regulatory genes plays a crucial role in the regulation of the eukaryotic cell cycle. CpG cytosines are actively subjected to methylation during tumorigenesis, resulting in gain/loss of function. E2F5 gene has growth repressive activities; various studies suggest its involvement in tumorigenesis. This study aims to investigate the epigenetic regulation of E2F5 in breast cancer to better understand tumor biology. Methods The promoter methylation status of 50 breast tumor tissues and adjacent normal control tissues was analyzed. mRNA expression was determined using SYBR® green quantitative polymerase chain reaction (PCR), and methylation-specific PCR was performed for bisulfite-modified genomic DNA using E2F5-specific primers to assess promoter methylation. Data was statistically analyzed. Results Significant (p<0.001) upregulation was observed in E2F5 expression among tumor tissues, relative to the control group. These samples were hypo-methylated at the E2F5 promoter region in the tumor tissues, compared to the control. Change in the methylation status (Δmeth) was significantly lower (p=0.022) in the tumor samples, indicating possible involvement in tumorigenesis. Patients at the postmenopausal stage showed higher methylation (75%) than those at the premenopausal stage (23.1%). Interestingly, methylation levels gradually increased from the early to the advanced stages of the disease (p<0.001), which suggests a putative role of E2F5 methylation in disease progression that can significantly modulate tumor biology at more advanced stage and at postmenopausal age (Pearson's r=0.99 and 0.86, respectively). Among tissues with different histological status, methylation frequency was higher in invasive lobular carcinoma (80.0%), followed by invasive ductal carcinoma (46.7%) and ductal carcinoma in situ (20.0%). Conclusion Methylation is an important epigenetic factor that might be involved in the upregulation of E2F5

  14. Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

    NASA Astrophysics Data System (ADS)

    Huang, Yong-Zhen; Zhang, Liang-Zhi; Lai, Xin-Sheng; Li, Ming-Xun; Sun, Yu-Jia; Li, Cong-Jun; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-04-01

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

  15. Down-regulation of promoter methylation level of CD4 gene after MDV infection in MD-susceptible chicken line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) is an oncovirus that induces lymphoid tumors in susceptible chickens, and may affect the epigenetic stability of the CD4 gene. The purpose of this study was to find how the effect of MDV infection on DNA methylation status of the CD4 gene differed between MD-resistant (L6...

  16. Gene promoter methylation signature predicts survival of head and neck squamous cell carcinoma patients.

    PubMed

    Kostareli, Efterpi; Hielscher, Thomas; Zucknick, Manuela; Baboci, Lorena; Wichmann, Gunnar; Holzinger, Dana; Mücke, Oliver; Pawlita, Michael; Del Mistro, Annarosa; Boscolo-Rizzo, Paolo; Da Mosto, Maria Cristina; Tirelli, Giancarlo; Plinkert, Peter; Dietz, Andreas; Plass, Christoph; Weichenhan, Dieter; Hess, Jochen

    2016-01-01

    Infection with high-risk types of human papilloma virus (HPV) is currently the best-established prognostic marker for head and neck squamous cell carcinoma (HNSCC), one of the most common and lethal human malignancies worldwide. Clinical trials have been launched to address the concept of treatment de-escalation for HPV-positive HNSCC with the final aim to reduce treatment related toxicity and debilitating long-term impacts on the quality of life. However, HPV-related tumors are mainly restricted to oropharyngeal SCC (OPSCC) and there is an urgent need to establish reliable biomarkers for all patients at high risk for treatment failure. A patient cohort (n = 295) with mainly non-OPSCC (72.9%) and a low prevalence of HPV16-related tumors (8.8%) was analyzed by MassARRAY to determine a previously established prognostic methylation score (MS). Kaplan-Meier revealed a highly significant correlation between a high MS and a favorable survival for OPSCC (P = 0.0004) and for non-OPSCC (P<0.0001), which was confirmed for all HNSCC by multivariate Cox regression models (HR: 9.67, 95% CI [4.61-20.30], P<0.0001). Next, we established a minimal methylation signature score (MMSS), which consists of ten most informative of the originally 62 CpG units used for the MS. The prognostic value of the MMSS was confirmed by Kaplan-Meier analysis for all HNSCC (P<0.0001) and non-OPSCC (P = 0.0002), and was supported by multivariate Cox regression models for all HNSCC (HR: 2.15, 95% CI [1.36-3.41], P = 0.001). In summary, the MS and the MMSS exhibit an excellent performance as prognosticators for survival, which is not limited by the anatomical site, and both could be implemented in future clinical trials. PMID:26786582

  17. Promoter CpG Island Methylation of Genes in Key Cancer Pathways Associates with Clinical Outcome in High Grade Serous Ovarian Cancer

    PubMed Central

    Masrour, Nahal; Siddiqui, Nadeem; Paul, James; Brown, Robert

    2013-01-01

    Purpose We aimed to identify DNA methylation biomarkers of progression free survival (PFS) to platinum-based chemotherapy in high grade serous ovarian cancer (HGSOC) within biologically relevant ovarian cancer associated pathways. Experimental Design Association with PFS of CpG island (CGI) promoter DNA methylation at genes in the pathways Akt/mTOR, p53, redox and homologous recombination DNA repair was sought with PFS as the primary objective in a prospectively collected ovarian cancer cohort (n=150). Significant loci were validated for associations between PFS, methylation and gene expression in an independent TCGA data set of HGSOC (n=311). Results DNA methylation at 29 CGI loci linked to 28 genes was significantly associated with PFS, independent from conventional clinical prognostic factors (adjusted p<0.05). Of 17 out of the 28 genes represented in the TCGA data set, methylation of VEGFB, VEGFA, HDAC11, FANCA, E2F1, GPX4, PRDX2, RAD54L and RECQL4 was prognostic in this independent patient cohort (one-sided p<0.05, FDR<10%). A multivariate Cox model was constructed, with clinical parameters (age, stage, grade and histological type) and significant loci. The final model included NKD1, VEGFB and PRDX2 as the three best predictors of PFS (p=6.62x10-6, permutation test p<0.05). Focussing only on known VEGFs in the TCGA cohort showed that methylation at promoters of VEGFA, VEGFB and VEGFC was significantly associated with PFS. Conclusions A three loci model of DNA methylation could identify two distinct prognostic groups of ovarian cancer patients (PFS: HR=2.29, p=3.34×10-5; Overall Survival: HR= 1.87, p=0.007) and patients more likely to have poor response to chemotherapy (OR=3.45, p=0.012). PMID:23965899

  18. Alterations in gene promoter methylation and transcript expression induced by cisplatin in comparison to 5-Azacytidine in HeLa and SiHa cervical cancer cell lines.

    PubMed

    Sood, Swati; Srinivasan, Radhika

    2015-06-01

    Despite recent advances in treatment, cervical cancer still remains one of the leading causes of cancer related mortality among women worldwide including India. Chemoradiation treatment is the standard-of-care which involves administration of cisplatin, a radiosensitizer along with radiation. The epigenetic changes induced by cisplatin are not known and so we designed this in vitro experimental study. We evaluated the changes induced by cisplatin administration in gene promoter methylation and the transcript levels of set of 7 genes and compared it to the changes induced by 5-Azacytidine, a known demethylating agent in two cervical cancer cell lines: HeLa (adenocarcinoma derived) and SiHa (squamous cell carcinoma derived) cell lines. Overall, there was a pronounced cytotoxic and growth inhibitory effect of both the drugs alone and in combination for both the cell lines which was dose and time dependent. Cisplatin as well as 5-Azacytidine treatment affected gene promoter methylation status resulting in demethylation and re-expression of the genes under investigation which was more pronounced in case of SiHa cells as compared to HeLa cells. Further, both the drugs acted in synergism as evident from their combination treatment. Therefore, at the cellular level, cisplatin and 5-Azacytidine can induce epigenetic changes in gene promoter methylation with altered expression which can have implications for treatment of cervical cancer. PMID:25772483

  19. Gene Expression Status and Methylation Pattern in Promoter of P15INK4b and P16INK4a in Cord Blood CD34+ Stem Cells

    PubMed Central

    Azad, Mehdi; Kaviani, Saeid; Noruzinia, Mehrdad; Mortazavi, Yousef; Mobarra, Naser; Alizadeh, Shaban; Shahjahani, Mohammad; Skandari, Fatemeh; Ahmadi, Mohammad Hosein; Atashi, Amir; Abroun, Saeid; Zonoubi, Zahra

    2013-01-01

    Objective(s) : Stem cell differentiation into different cell lineages depends upon several factors, cell cycle control elements and intracellular signaling elements, including P15INK4b and P16INK4a genes. Epigenetics may be regarded as a control mechanism which is affected by these factors with respect to their promoter structure. Materials and Methods : The CD34 + cord blood stem cells were purified, isolated and then expanded. The undifferentiated day genome was isolated from part of the cultured cells, and the seventh day differentiated genome was isolated from the other part after differentiation to erythroid lineage. The procedure was followed by a separate Real-Time PCR for the two genes using the obtained cDNA. The processed DNA of the former stages was used for MSP (Methylation Specific PCR) reaction. Finally, pre- and post differentiation results were compared.  Results : After performing MSP for each gene, it became clear that P15INK4b gene has undergone methylation and expression in predifferentiation stage. In addition, its status has not been changed after differentiation. P15INK4b gene expression was reduced after the differentiation. The other gene, P16INK4a, showed no predifferentiation methylation. Itwas completely expressed methylated and underwent reduced expression after differentiation. Conclusion : Specific predifferentiation expression of P15INK4b and P16INK4a genes along with reduction in their expression after erythroid differentiation indicated animportant role for these two genes in biology of CD34+ cells in primary stages and before differentiation. In addition, both genes are capable of epigenetic modifications due to the structure of their promoters. PMID:23997911

  20. Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene.

    PubMed

    Niculescu, Mihai D; Yamamuro, Yutaka; Zeisel, Steven H

    2004-06-01

    Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline-deficient IMR-32 neuroblastoma cells, the promoter of the cyclin-dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase-associated phosphatase (KAP), which inhibits the G(1)/S transition of the cell cycle by dephosphorylating cyclin-dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline-deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline-deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline-deficient rat and mouse fetuses. PMID:15147518

  1. Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene

    PubMed Central

    Niculescu, Mihai D.; Yamamuro, Yutaka; Zeisel, Steven H.

    2006-01-01

    Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline-deficient IMR-32 neuroblastoma cells, the promoter of the cyclin-dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase-associated phosphatase (KAP), which inhibits the G1/S transition of the cell cycle by dephosphorylating cyclin-dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline-deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline-deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline-deficient rat and mouse fetuses. PMID:15147518

  2. ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

    PubMed Central

    2014-01-01

    Background Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. Methods Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). Results Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. Conclusion Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment. PMID:24495356

  3. Differences in Expression of DPP4 in Steatotic Rat Liver Are Not Related to Differences in the Methylation of its Gene Promoter.

    PubMed

    Tarantola, Eleonora; Gobbato, Sara; Ferrigno, Andrea; Bertone, Vittorio; Capelli, Enrica

    2015-01-01

    The aim of the present study was to investigate the methylation status in the promoter region of Dipeptidyl peptidase-IV (Dpp4) gene, in livers from obese Zucker rats with different patterns of immunohistochemical positivity. Molecular analysis was carried-out on DNA obtained from livers of obese and lean Zucker rats and of control Wistar rats using the bisulfite conversion method and DNA sequencing. Our study focused on the genomic region of 1,000 bp, which includes the final part of 680 bp of the Dpp4 gene promoter and a small stretch of 320 bp at the beginning of the gene. The results indicate that the different immunohistochemical pattern of DPP4 observed in obese (fa/fa) and lean (fa/-) Zucker rats is not correlated to DNA methylation of its promoter. This is in agreement with the results of other studies carried-out on visceral and subcutaneous adipose tissue with varying levels of enzyme expression, in which differences in the methylation pattern of the Dpp4 promoter region were not observed. PMID:26359413

  4. DNA Promoter Methylation-dependent Transcription of the Double C2-like Domain β (DOC2B) Gene Regulates Tumor Growth in Human Cervical Cancer*

    PubMed Central

    Kabekkodu, Shama Prasada; Bhat, Samatha; Radhakrishnan, Raghu; Aithal, Abhijit; Mascarenhas, Roshan; Pandey, Deeksha; Rai, Lavanya; Kushtagi, Pralhad; Mundyat, Gopinath Puthiya; Satyamoorthy, Kapaettu

    2014-01-01

    Double C2-like domain β (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region −630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth. PMID:24570007

  5. Size and positional effects of promoter RNA segments on virus-induced RNA-directed DNA methylation and transcriptional gene silencing

    PubMed Central

    Otagaki, Shungo; Kawai, Miou; Masuta, Chikara; Kanazawa, Akira

    2011-01-01

    DNA methylation at a gene promoter can be triggered by double-stranded RNAs (dsRNAs) through the RNA-directed DNA methylation (RdDM) pathway and induces transcriptional gene silencing (TGS). Although genes involved in the RdDM pathway have been identified, whether dsRNAs of different promoter regions have different extent of effects on RdDM and/or TGS is unknown. Here, we addressed this question by targeting the CaMV 35S promoter in the plant genome using a recombinant Cucumber mosaic virus that contained various portions of the promoter. The efficiency of the induction of TGS depended on the length of the promoter segment triggering the RdDM; the lower size limit for TGS induction was 81-91 nt. TGS was induced when 70-nt fragments were connected in tandem, none of which solely induced TGS. TGS induction did not simply depend on the production of small interfering RNAs corresponding to the promoter. Along with the induction of RdDM, spreading of DNA methylation from the originally targeted site toward the adjacent regions was detected. The maintenance of TGS in the progeny that lacks an RNA trigger depended on the promoter segments triggering the RdDM in the former generation and was correlated with the number of cytosines at symmetrical sites in the targeted region. These results indicate that both the length of dsRNA above the threshold and the frequency of cytosines at symmetric sites in the region targeted by dsRNA are the major factors that allow induction of heritable TGS via RdDM. PMID:21610318

  6. p53 inhibits the expression of p125 and the methylation of POLD1 gene promoter by downregulating the Sp1-induced DNMT1 activities in breast cancer

    PubMed Central

    Zhang, Liang; Yang, Weiping; Zhu, Xiao; Wei, Changyuan

    2016-01-01

    p125 is one of four subunits of human DNA polymerases – DNA Pol δ as well as one of p53 target protein encoded by POLD1. However, the function and significance of p125 and the role that p53 plays in regulating p125 expression are not fully understood in breast cancer. Tissue sections of human breast cancer obtained from 70 patients whose median age was 47.6 years (range: 38–69 years) with stage II–III breast cancer were studied with normal breast tissue from the same patients and two human breast cell lines (MCF-7 and MCF-10A). p53 expression levels were reduced, while p125 protein expression was increased in human breast cancer tissues and cell line detected by Western blot and quantitative reverse transcriptase-polymerase chain reaction. The methylation level of the POLD1 gene promoter was greater in breast cancer tissues and cells when compared with normal tissues and cells. In MCF-7 cell model, p53 overexpression caused a decrease in the level of p125 protein, while the methylation level of the p125 gene promoter was also inhibited by p53 overexpression. To further investigate the regulating mechanism of p53 on p125 expression, our study focused on DNA methyltransferase 1 (DNMT1) and transcription factor Sp1. Both DNMT1 and Sp1 protein expression were reduced when p53 was overexpressed in MCF-7 cells. The Sp1 binding site appears to be important for DNMT1 gene transcription; Sp1 and p53 can bind together, which means that DNMT1 gene expression may be downregulated by p53 through binding to Sp1. Because DNMT1 methylation level of the p125 gene promoter can affect p125 gene transcription, we propose that p53 may indirectly regulate p125 gene promoter expression through the control of DNMT1 gene transcription. In conclusion, the data from this preliminary study have shown that p53 inhibits the methylation of p125 gene promoter by downregulating the activities of Sp1 and DNMT1 in breast cancer. PMID:27022290

  7. Methylation of Exons 1D, 1F, and 1H of the Glucocorticoid Receptor Gene Promoter and Exposure to Adversity in Pre-School Aged Children

    PubMed Central

    Tyrka, Audrey R.; Parade, Stephanie H.; Eslinger, Nicole M.; Marsit, Carmen J.; Lesseur, Corina; Armstrong, David A.; Philip, Noah S.; Josefson, Brittney; Seifer, Ronald

    2016-01-01

    Epigenetic modifications to the genome are a key mechanism involved in the biological encoding of experience. Animal studies and a growing body of literature in humans have shown that early adversity is linked to methylation of the gene for the glucocorticoid receptor (GR) which is a key regulator of the hypothalamic-pituitary-adrenal (HPA) axis as well as a broad range of physiological systems including metabolic and immune function. One hundred eighty-four families participated, including n=74 with child welfare documentation of moderate-severe maltreatment in the past six months. Children ranged in age from 3 to 5 years, and were racially and ethnically diverse. Structured record review and interviews in the home were used to assess a history of maltreatment, other traumas, and contextual life stressors, and a composite variable assessed the number exposures to these adversities. Methylation of regions 1D, 1F, and 1H of the GR gene was measured via sodium bisulfite pyrosequencing. The composite measure of adversity was positively correlated with methylation at exons 1D and 1F in the promoter of NR3C1. Individual stress measures were significantly associated with a several CpG sites in these regions. GR gene methylation may be a mechanism of the bio-behavioral effects of adverse exposures in young children. PMID:25997773

  8. GENE METHYLATION CHANGES IN TUMOR SUPPRESSOR GENES INDUCED BY ARSENIC

    EPA Science Inventory

    The choice of a dose-response model used for extrapolation can be influenced by knowledge of mechanism of action. We have already showed that arsenic affects methylation of the human p53 gene promoter. Evidence that genes other than the p53 tumor suppressor gene are affected woul...

  9. DNA Methylation of BDNF Gene in Schizophrenia

    PubMed Central

    Çöpoğlu, Ümit Sertan; İğci, Mehri; Bozgeyik, Esra; Kokaçya, M. Hanifi; İğci, Yusuf Ziya; Dokuyucu, Recep; Arı, Mustafa; Savaş, Haluk A.

    2016-01-01

    Background Although genetic factors are risk factors for schizophrenia, some environmental factors are thought to be required for the manifestation of disease. Epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of DNA. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. It is established that methylation status of the BDNF gene is associated with fear learning, memory, and stressful social interactions. In this study, we aimed to investigate the DNA methylation status of BDNF gene in patients with schizophrenia. Material/Methods The study included 49 patients (33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (46 male and 19 female). Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA results in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA. Results There was no significant difference in methylated or un-methylated status for BDNF promoters between schizophrenia patients and controls. The mean duration of illness was significantly lower in the hemi-methylated group compared to the non-methylated group for BDNF gene CpG island-1 in schizophrenia patients. Conclusions Although there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls, there was an association between duration of illness and DNA methylation. PMID:26851233

  10. Nicotine induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production

    SciTech Connect

    Wang, Tingting; Chen, Man; Liu, Lian; Cheng, Huaiyan; Yan, You-E; Feng, Ying-Hong; Wang, Hui

    2011-12-15

    Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming. -- Highlights: Black-Right-Pointing-Pointer Nicotine-induced StAR inhibition in two human adrenal cell models. Black-Right-Pointing-Pointer Nicotine-induced single CpG site methylation in StAR promoter. Black-Right-Pointing-Pointer Persistent StAR inhibition and single CpG methylation after nicotine termination

  11. Aberrant methylation of the CDKN2a/p16INK4a gene promoter region in preinvasive bronchial lesions: a prospective study in high-risk patients without invasive cancer.

    PubMed

    Lamy, Aude; Sesboüé, Richard; Bourguignon, Jeannette; Dautréaux, Brigitte; Métayer, Josette; Frébourg, Thierry; Thiberville, Luc

    2002-07-10

    Among the identified factors involved in malignant transformation, abnormal methylation of the CDKN2A/p16(INK4a) gene promoter has been described as an early event, particularly in bronchial cell cancerization. Precancerous bronchial lesions (n = 70) prospectively sampled during fluorescence endoscopy in a series of 37 patients at high risk for lung cancer were studied with respect to the methylation status of the CDKN2A gene. Methylation-specific polymerase chain reaction was performed on DNA extracted from pure bronchial cell populations derived from biopsies and detection of p16 protein was studied by immunohistochemistry on contiguous parallel biopsies. Aberrant methylation of the CDKN2A gene promoter was found in 19% of preinvasive lesions and its frequency increased with the histologic grade of the lesions. Methylation in at least 1 bronchial site was significantly more frequent in patients with cancer history, although there was no difference in the outcome of patients with or without methylation in bronchial epithelium. The other risk factors studied (tobacco and asbestos exposure) did not influence the methylation status. There was no relationship between CDKN2A methylation and the evolutionary character of the lesions. Our results confirm that abnormal methylation of the CDKN2A gene promoter is an early event in bronchial cell cancerization, which can persist for several years after carcinogen exposure cessation, and show that this epigenetic alteration cannot predict the evolution of precancerous lesions within a 2-year follow-up. PMID:12115568

  12. The bacterial cell cycle regulator GcrA is a σ70 cofactor that drives gene expression from a subset of methylated promoters.

    PubMed

    Haakonsen, Diane L; Yuan, Andy H; Laub, Michael T

    2015-11-01

    Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ(70)-dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ(70) interact with domain 4, GcrA interfaces with domain 2, the region that binds the -10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼ 200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively. PMID:26545812

  13. The bacterial cell cycle regulator GcrA is a σ70 cofactor that drives gene expression from a subset of methylated promoters

    PubMed Central

    Haakonsen, Diane L.; Yuan, Andy H.; Laub, Michael T.

    2015-01-01

    Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ70-dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ70 interact with domain 4, GcrA interfaces with domain 2, the region that binds the −10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively. PMID:26545812

  14. ZCT1 and ZCT2 transcription factors repress the activity of a gene promoter from the methyl erythritol phosphate pathway in Madagascar periwinkle cells.

    PubMed

    Chebbi, Mouadh; Ginis, Olivia; Courdavault, Vincent; Glévarec, Gaëlle; Lanoue, Arnaud; Clastre, Marc; Papon, Nicolas; Gaillard, Cécile; Atanassova, Rossitza; St-Pierre, Benoit; Giglioli-Guivarc'h, Nathalie; Courtois, Martine; Oudin, Audrey

    2014-10-15

    In Catharanthus roseus, accumulating data highlighted the existence of a coordinated transcriptional regulation of structural genes that takes place within the secoiridoid biosynthetic branch, including the methyl erythritol phosphate (MEP) pathway and the following steps leading to secologanin. To identify transcription factors acting in these pathways, we performed a yeast one-hybrid screening using as bait a promoter region of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene involved in the responsiveness of C. roseus cells to hormonal signals inducing monoterpene indole alkaloid (MIA) production. We identified that ZCT2, one of the three members of the zinc finger Catharanthus protein (ZCT) family, can bind to a HDS promoter region involved in hormonal responsiveness. By trans-activation assays, we demonstrated that ZCT1 and ZCT2 but not ZCT3 repress the HDS promoter activity. Gene expression analyses in C. roseus cells exposed to methyljasmonate revealed a persistence of induction of ZCT2 gene expression suggesting the existence of feed-back regulatory events acting on HDS gene expression in correlation with the MIA production. PMID:25108262

  15. Dynamic Changes in the Follicular Transcriptome and Promoter DNA Methylation Pattern of Steroidogenic Genes in Chicken Follicles throughout the Ovulation Cycle

    PubMed Central

    Zhu, Guiyu; Mao, Yong; Zhou, Wendi; Jiang, Yunliang

    2015-01-01

    The molecular mechanisms associated with follicle maturation and ovulation are not well defined in avian species. In this study, we used RNA-seq to study the gene expression profiles of the chicken follicles from different developmental stages (pre-hierarchical, pre-ovulatory and post-ovulatory). Transcriptomic analysis revealed a total of 1,277 and 2,310 genes were differentially expressed when follicles progressed through the pre-hierarchical to hierarchical and pre-ovulatory to post-ovulatory transitions, respectively. The differentially expressed genes (DEG) were involved in signaling pathways such as adherens junction, apoptosis and steroid biosynthesis. We further investigated the transcriptional regulation of follicular steroidogenesis by examining the follicle-specific methylation profiles of Star (steroidogenic acute regulatory protein), Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1) and Hsd3b (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1), genes encoding the key enzymes for progesterone synthesis. The varied patterns of DNA methylation in proximal promoters of Star and Cyp11a1but not Hsd3b in different follicles could play a major role in controlling gene expression as well as follicular steroidogenic activity. Finally, the promoter-reporter analysis suggests that TGF-β could be involved in the regulation of Hsd3b expression during ovulation. Together, current data not only provide novel insights into the molecular mechanisms of follicular physiology in chicken follicles, but also present the first evidence of epigenetic regulation of ovarian steroidogenesis in avian species. PMID:26716441

  16. Promoter methylation of p16 and RASSF1A genes may contribute to the risk of papillary thyroid cancer: A meta-analysis

    PubMed Central

    JIANG, JIA-LI; TIAN, GUI-LAN; CHEN, SHU-JIAO; XU, LI; WANG, HUI-QIN

    2015-01-01

    The aim of the present meta-analysis was to investigate the correlation of promoter methylation of the p16 and Ras association domain family 1 isoform A (RASSF1A) genes with the risk of the development of papillary thyroid cancer (PTC). A number of electronic databases were searched without language restrictions as follows: Medline (1966–2013), the Cochrane Library database (Issue 12, 2013), Embase (1980–2013), CINAHL (1982–2013), Web of Science (1945–2013) and the Chinese Biomedical Database (CBM; 1982–2013). A meta-analysis was performed with the use of Stata statistical software. The odds ratios (ORs), ratio differences (RDs) and 95% confidence intervals (95% CIs) were calculated. In the present meta-analysis, eleven clinical cohort studies with a total of 734 patients with PTC were included. The results of the current meta-analysis indicated that the frequency of promoter methylation of p16 in cancer tissues was significantly higher compared with that in normal, adjacent and benign tissues (cancer tissues vs. normal tissues: OR=7.14; 95% CI, 3.30–15.47; P<0.001; cancer tissues vs. adjacent tissues: OR=11.90; 95% CI, 5.55–25.52; P<0.001; cancer tissues vs. benign tissues: OR=2.25; 95% CI, 1.67–3.03; P<0.001, respectively). The results also suggest that RASSF1A promoter methylation may be implicated in the pathogenesis of PTC (cancer tissues vs. normal tissues: RD=0.53; 95% CI, 0.42–0.64; P<0.001; cancer tissues vs. adjacent tissues: RD=0.39; 95% CI, 0.31–0.48; P<0.001; cancer tissues vs. benign tissues: RD=0.39; 95% CI, 0.31–0.47; P<0.001; respectively). Thus, the present meta-analysis indicates that aberrant promoter methylation of p16 and RASSF1A genes may play a crucial role in the pathogenesis of PTC. PMID:26622524

  17. Molecular detection of noninvasive and invasive bladder tumor tissues and exfoliated cells by aberrant promoter methylation of laminin-5 encoding genes.

    PubMed

    Sathyanarayana, Ubaradka G; Maruyama, Riichiroh; Padar, Asha; Suzuki, Makoto; Bondaruk, Jolanta; Sagalowsky, Arthur; Minna, John D; Frenkel, Eugene P; Grossman, H Barton; Czerniak, Bogdan; Gazdar, Adi F

    2004-02-15

    Laminin-5 (LN5) anchors epithelial cells to the underlying basement membrane, and it is encoded by three distinct genes: LAMA3, LAMB3, and LAMC2. To metastasize and grow, cancer cells must invade and destroy the basement membrane. Our previous work has shown that epigenetic inactivation is a major mechanism of silencing LN5 genes in lung cancers. We extended our methylation studies to resected bladder tumors (n = 128) and exfoliated cell samples (bladder washes and voided urine; n = 71) and correlated the data with clinicopathologic findings. Nonmalignant urothelium had uniform expression of LN5 genes and lacked methylation. The methylation frequencies for LN5 genes in tumors were 21-45%, and there was excellent concordance between methylation in tumors and corresponding exfoliated cells. Methylation of LAMA3 and LAMB3 and the methylation index were correlated significantly with several parameters of poor prognosis (tumor grade, growth pattern, muscle invasion, tumor stage, and ploidy pattern), whereas methylation of LAMC2 and methylation index were associated with shortened patient survival. Of particular interest, methylation frequencies of LAMA3 helped to distinguish invasive (72%) from noninvasive (12%) tumors. These results suggest that methylation of LN5 genes has potential clinical applications in bladder cancers. PMID:14973053

  18. GSH2 promoter methylation in pancreatic cancer analyzed by quantitative methylation-specific polymerase chain reaction

    PubMed Central

    GAO, FEI; HUANG, HAO-JIE; GAO, JUN; LI, ZHAO-SHEN; MA, SHU-REN

    2015-01-01

    Tumor suppressor gene silencing via promoter hypermethylation is an important event in pancreatic cancer pathogenesis. Aberrant DNA hypermethylation events are highly tumor specific, and may provide a diagnostic tool for pancreatic cancer patients. The objective of the current study was to identify novel methylation-related genes that may potentially be used to establish novel therapeutic and diagnostic strategies against pancreatic cancer. The methylation status of the GS homeobox 2 (GSH2) gene was analyzed using the sodium bisulfite sequencing method. The GSH2 methylation ratio was examined in primary carcinomas and corresponding normal tissues derived from 47 patients with pancreatic cancer, using quantitative methylation-specific polymerase chain reaction. Methylation ratios were found to be associated with the patient's clinicopathological features. GSH2 gene methylation was detected in 26 (55.3%) of the 47 pancreatic cancer patients, indicating that it occurs frequently in pancreatic cancer. A significant association with methylation was observed for tumor-node-metastasis stage (P=0.031). GSH2 may be a novel methylation-sensitive tumor suppressor gene in pancreatic cancer and may be a tumor-specific biomarker of the disease. PMID:26171036

  19. Promoter methylation of E-cadherin, p16, and RAR-beta(2) genes in breast tumors and dietary intake of nutrients important in one-carbon metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aberrant DNA methylation plays a critical role in carcinogenesis, and the availability of dietary factors involved in 1-carbon metabolism may contribute to aberrant DNA methylation. We investigated the association of intake of folate, vitamins B(2), B(6), B(12), and methionine with promoter methylat...

  20. MicroRNA-mediated regulation of target genes in several brain regions is correlated to both microRNA-targeting-specific promoter methylation and differential microRNA expression

    PubMed Central

    2013-01-01

    Background Public domain databases nowadays provide multiple layers of genome-wide data e.g., promoter methylation, mRNA expression, and miRNA expression and should enable integrative modeling of the mechanisms of regulation of gene expression. However, researches along this line were not frequently executed. Results Here, the public domain dataset of mRNA expression, microRNA (miRNA) expression and promoter methylation patterns in four regions, the frontal cortex, temporal cortex, pons and cerebellum, of human brain were sourced from the National Center for Biotechnology Informations gene expression omnibus, and reanalyzed computationally. A large number of miRNA-mediated regulation of target genes and miRNA-targeting-specific promoter methylation were identified in the six pairwise comparisons among the four brain regions. The miRNA-mediated regulation of target genes was found to be highly correlated with one or both of miRNA-targeting-specific promoter methylation and differential miRNA expression. Genes enriched for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were related to brain function and/or development were found among the target genes of miRNAs whose differential expression patterns were highly correlated with the miRNA-mediated regulation of their target genes. Conclusions The combinatorial analysis of miRNA-mediated regulation of target genes, miRNA-targeting-specific promoter methylation and differential miRNA expression can help reveal the brain region-specific contributions of miRNAs to brain function and development. PMID:23725297

  1. Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

    PubMed Central

    2012-01-01

    Background Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers. Results To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2'-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival. Conclusions This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma. PMID:23034519

  2. Cigarette smoke induces methylation of the tumor suppressor gene NISCH

    PubMed Central

    Ostrow, Kimberly Laskie; Michalidi, Christina; Guerrero-Preston, Rafael; Hoque, Mohammad O.; Greenberg, Alissa; Rom, William; Sidransky, David

    2013-01-01

    We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (< 20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients’ plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer. PMID:23503203

  3. The evolution of invertebrate gene body methylation.

    PubMed

    Sarda, Shrutii; Zeng, Jia; Hunt, Brendan G; Yi, Soojin V

    2012-08-01

    DNA methylation of transcription units (gene bodies) occurs in the genomes of many animal and plant species. Phylogenetic persistence of gene body methylation implies biological significance; yet, the functional roles of gene body methylation remain elusive. In this study, we analyzed methylation levels of orthologs from four distantly related invertebrate species, including the honeybee, silkworm, sea squirt, and sea anemone. We demonstrate that in all four species, gene bodies distinctively cluster to two groups, which correspond to high and low methylation levels. This pattern resembles that of sequence composition arising from the mutagenetic effect of DNA methylation. In spite of this effect, our results show that protein sequences of genes targeted by high levels of methylation are conserved relative to genes lacking methylation. Our investigation identified many genes that either gained or lost methylation during the course of invertebrate evolution. Most of these genes appear to have lost methylation in the insect lineages we investigated, particularly in the honeybee. We found that genes that are methylated in all four invertebrate taxa are enriched for housekeeping functions related to transcription and translation, whereas the loss of DNA methylation occurred in genes whose functions include cellular signaling and reproductive processes. Overall, our study helps to illuminate the functional significance of gene body methylation and its impacts on genome evolution in diverse invertebrate taxa. PMID:22328716

  4. Importance of Tumour Suppressor Gene Methylation in Sinonasal Carcinomas.

    PubMed

    Chmelařová, M; Sirák, I; Mžik, M; Sieglová, K; Vošmiková, H; Dundr, P; Němejcová, K; Michálek, J; Vošmik, M; Palička, V; Laco, J

    2016-01-01

    Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in samples of sinonasal carcinoma by comparison with normal sinonasal tissue. To search for epigenetic events we used methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to compare the methylation status of 64 tissue samples of sinonasal carcinomas with 19 control samples. We also compared the human papilloma virus (HPV) status with DNA methylation. Using a 20% cut-off for methylation, we observed significantly higher methylation in RASSF1, CDH13, ESR1 and TP73 genes in the sinonasal cancer group compared with the control group. HPV positivity was found in 15/64 (23.4 %) of all samples in the carcinoma group and in no sample in the control group. No correlation was found between DNA methylation and HPV status. In conclusion, our study showed that there are significant differences in promoter methylation in the RASSF1, ESR 1, TP73 and CDH13 genes between sinonasal carcinoma and normal sinonasal tissue, suggesting the importance of epigenetic changes in these genes in carcinogenesis of the sinonasal area. These findings could be used as prognostic factors and may have implications for future individualised therapies based on epigenetic changes. PMID:27516190

  5. MGMT Gene Promoter Methylation as a Potent Prognostic Factor in Glioblastoma Treated With Temozolomide-Based Chemoradiotherapy: A Single-Institution Study

    SciTech Connect

    Kim, Young Suk; Kim, Se Hoon; Cho, Jaeho; Kim, Jun Won; Chang, Jong Hee; Kim, Dong Suk; Lee, Kyu Sung; Suh, Chang-Ok

    2012-11-01

    Purpose: Recently, cells deficient in O{sup 6}-methylguanine-DNA methyltransferase (MGMT) were found to show increased sensitivity to temozolomide (TMZ). We evaluated whether hypermethylation of MGMT was associated with survival in patients with glioblastoma multiforme (GBM). Methods and Materials: We retrospectively analyzed 93 patients with histologically confirmed GBM who received involved-field radiotherapy with TMZ from 2001 to 2008. The median age was 58 years (range, 24-78 years). Surgical resection was total in 39 patients (42%), subtotal in 30 patients (32%), and partial in 17 patients (18%); only a biopsy was performed in 7 patients (8%). Postoperative radiotherapy began within 3 weeks of surgery in 87% of the patients. Radiotherapy doses ranged from 50 to 74 Gy (median, 70 Gy). MGMT gene methylation was determined in 78 patients; MGMT was unmethylated in 43 patients (55%) and methylated in 35 patients (45%). The median follow-up period was 22 months (range, 3-88 months) for all patients. Results: The median overall survival (OS) was 22 months, and progression-free survival (PFS) was 11 months. MGMT gene methylation was an independently significant prognostic factor for both OS (p = 0.002) and PFS (p = 0.008) in multivariate analysis. The median OS was 29 months for the methylated group and 20 months for the unmethylated group. In 35 patients with methylated MGMT genes, the 2-year and 5-year OS rates were 54% and 31%, respectively. Six patients with combined prognostic factors of methylated MGMT genes, age {<=}50 years, and total/subtotal resections are all alive 38 to 77 months after operation, whereas the median OS in 8 patients with unmethylated MGMT genes, age >50 years, and less than subtotal resection was 13.2 months. Conclusion: We confirmed that MGMT gene methylation is a potent prognostic factor in patients with GBM. Our results suggest that early postoperative radiotherapy and a high total/subtotal resection rate might further improve the

  6. Gene Body Methylation can alter Gene Expression and is a Therapeutic Target in Cancer

    PubMed Central

    Yang, Xiaojing; Han, Han; De Carvalho, Daniel D.; Lay, Fides D.; Jones, Peter A.; Liang, Gangning

    2014-01-01

    SUMMARY DNA methylation in promoters is well known to silence genes and is the presumed therapeutic target of methylation inhibitors. Gene body methylation is positively correlated with expression yet its function is unknown. We show that 5-aza-2'-deoxycytidine treatment not only reactivates genes but decreases the over-expression of genes, many of which are involved in metabolic processes regulated by c-MYC. Down-regulation is caused by DNA demethylation of the gene bodies and restoration of high levels of expression requires remethylation by DNMT3B. Gene body methylation may therefore be an unexpected therapeutic target for DNA methylation inhibitors, resulting in the normalization of gene over-expression induced during carcinogenesis. Our results provide direct evidence for a causal relationship between gene body methylation and transcription. PMID:25263941

  7. CpG methylation has differential effects on the binding of YY1 and ETS proteins to the bi-directional promoter of the Surf-1 and Surf-2 genes.

    PubMed Central

    Gaston, K; Fried, M

    1995-01-01

    The divergently transcribed Surf-1 and Surf-2 housekeeping genes are separated by a bi-directional, TATA-less promoter which lies within a CpG-rich island. Here we show that CpG methylation severely reduces transcription in the direction of both Surf-1 and Surf-2. Previous work has identified three promoter elements (Su1, Su2 and Su3) which are conserved between the human and mouse Surf-1/Surf-2 promoters. These elements bind transcription factors present in human and mouse cell nuclear extracts in vitro and mutations which prevent factor binding also reduce promoter activity in vivo. Transcription initiation factor YY1 binds to the Su1 site and stimulates transcription in the direction of Surf-1 and, to a lesser extent, Surf-2. Here we show that members of the ETS family of transcription factors bind to the Su2 site. Although the Su1 factor binding site contains three CpG dinucleotides, the binding of YY1 is not affected by CpG methylation. In contrast, CpG methylation abolishes the binding of ETS proteins to the Su2 site; methylation of a single cytosine, at position 3 of the consensus ETS site, is sufficient to prevent factor binding. This direct effect on the binding of ETS proteins is, however, not in itself sufficient to explain the repression of this promoter by CpG methylation. A mutation of the Su2 site which removes the sequence CpG, but which does not prevent ETS factor binding, fails to relieve this promoter from repression by CpG methylation. Images PMID:7731802

  8. Analysis of aberrant methylation on promoter sequences of tumor suppressor genes and total DNA in sputum samples: a promising tool for early detection of COPD and lung cancer in smokers

    PubMed Central

    2012-01-01

    Background Chronic obstructive pulmonary disease (COPD) is a disorder associated to cigarette smoke and lung cancer (LC). Since epigenetic changes in oncogenes and tumor suppressor genes (TSGs) are clearly important in the development of LC. In this study, we hypothesize that tobacco smokers are susceptible for methylation in the promoter region of TSGs in airway epithelial cells when compared with non-smoker subjects. The purpose of this study was to investigate the usefulness of detection of genes promoter methylation in sputum specimens, as a complementary tool to identify LC biomarkers among smokers with early COPD. Methods We determined the amount of DNA in induced sputum from patients with COPD (n = 23), LC (n = 26), as well as in healthy subjects (CTR) (n = 33), using a commercial kit for DNA purification, followed by absorbance measurement at 260 nm. The frequency of CDKN2A, CDH1 and MGMT promoter methylation in the same groups was determined by methylation-specific polymerase chain reaction (MSP). The Fisher’s exact test was employed to compare frequency of results between different groups. Results DNA concentration was 7.4 and 5.8 times higher in LC and COPD compared to the (CTR) (p < 0.0001), respectively. Methylation status of CDKN2A and MGMT was significantly higher in COPD and LC patients compared with CTR group (p < 0.0001). Frequency of CDH1 methylation only showed a statistically significant difference between LC patients and CTR group (p < 0.05). Conclusions We provide evidence that aberrant methylation of TSGs in samples of induced sputum is a useful tool for early diagnostic of lung diseases (LC and COPD) in smoker subjects. Virtual slides The abstract MUST finish with the following text: Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1127865005664160 PMID:22818553

  9. Elevated DRD4 promoter methylation increases the risk of Alzheimer's disease in males.

    PubMed

    Ji, Huihui; Wang, Yunliang; Jiang, Danjie; Liu, Guili; Xu, Xuting; Dai, Dongjun; Zhou, Xiaohui; Cui, Wei; Li, Jinfeng; Chen, Zhongming; Li, Ying; Zhou, Dongsheng; Zha, Qin; Zhuo, Renjie; Jiang, Liting; Liu, Yu; Shen, Lili; Zhang, Beibei; Xu, Lei; Hu, Haochang; Zhang, Yuzheng; Yin, Honglei; Duan, Shiwei; Wang, Qinwen

    2016-09-01

    Aberrant promoter methylation of multiple genes is associated with various diseases, including Alzheimer's disease (AD). The goal of the present study was to determine whether dopamine receptor D4 (DRD4) promoter methylation is associated with AD. In the current study, the methylation levels of the DRD4 promoter were measured in 46 AD patients and 61 controls using bisulfite pyrosequencing technology. The results of the present study demonstrated that DRD4 promoter methylation was significantly higher in AD patients than in controls. A further breakdown analysis by gender revealed that there was a significant association of DRD4 promoter methylation with AD in males (23 patients and 45 controls). In conclusion, the results of the present study demonstrated that elevated DRD4 promoter methylation was associated with AD risk in males. PMID:27485706

  10. Aberrant methylation of candidate tumor suppressor genes in neuroblastoma.

    PubMed

    Hoebeeck, Jasmien; Michels, Evi; Pattyn, Filip; Combaret, Valérie; Vermeulen, Joëlle; Yigit, Nurten; Hoyoux, Claire; Laureys, Geneviève; De Paepe, Anne; Speleman, Frank; Vandesompele, Jo

    2009-01-18

    CpG island hypermethylation has been recognized as an alternative mechanism for tumor suppressor gene inactivation. In this study, we performed methylation-specific PCR (MSP) to investigate the methylation status of 10 selected tumor suppressor genes in neuroblastoma. Seven of the investigated genes (CD44, RASSF1A, CASP8, PTEN, ZMYND10, CDH1, PRDM2) showed high frequencies (> or =30%) of methylation in 33 neuroblastoma cell lines. In 42 primary neuroblastoma tumors, the frequencies of methylation were 69%, CD44; 71%, RASSF1A; 56%, CASP8; 25%, PTEN; 15%, ZMYND10; 8%, CDH1; and 0%, PRDM2. Furthermore, CASP8 and CDH1 hypermethylation was significantly associated with poor event-free survival. Meta-analysis of 115 neuroblastoma tumors demonstrated a significant correlation between CASP8 methylation and MYCN amplification. In addition, there was a correlation between ZMYND10 methylation and MYCN amplification. The MSP data, together with optimized mRNA re-expression experiments (in terms of concentration and time of treatment and use of proper reference genes) further strengthen the notion that epigenetic alterations could play a significant role in NB oncogenesis. This study thus warrants the need for a global profiling of gene promoter hypermethylation to identify genome-wide aberrantly methylated genes in order to further understand neuroblastoma pathogenesis and to identify prognostic methylation markers. PMID:18819746

  11. Developmental- and differentiation-specific patterns of human gamma- and beta-globin promoter DNA methylation.

    PubMed

    Mabaera, Rodwell; Richardson, Christine A; Johnson, Kristin; Hsu, Mei; Fiering, Steven; Lowrey, Christopher H

    2007-08-15

    The mechanisms underlying the human fetal-to-adult beta-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human gamma- and beta-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at -162 of the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and FL, respectively. We also studied gamma-globin promoter methylation during in vitro differentiation of erythroid cells. The gamma promoters are initially hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient gamma-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human gamma- and beta-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human beta-globin locus gene switching. PMID:17456718

  12. Regulation of Tissue LC-PUFA Contents, Δ6 Fatty Acyl Desaturase (FADS2) Gene Expression and the Methylation of the Putative FADS2 Gene Promoter by Different Dietary Fatty Acid Profiles in Japanese Seabass (Lateolabrax japonicus)

    PubMed Central

    Ai, Qinghui; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Zuo, Rantao

    2014-01-01

    The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53±0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter. PMID:24498178

  13. A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies

    PubMed Central

    Shames, David S; Girard, Luc; Gao, Boning; Sato, Mitsuo; Lewis, Cheryl M; Shivapurkar, Narayan; Jiang, Aixiang; Perou, Charles M; Kim, Young H; Pollack, Jonathan R; Fong, Kwun M; Lam, Chi-Leung; Wong, Maria; Shyr, Yu; Nanda, Rita; Olopade, Olufunmilayo I; Gerald, William; Euhus, David M; Shay, Jerry W; Gazdar, Adi F; Minna, John D

    2006-01-01

    Background Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets. Methods and Findings In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. Conclusions By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation

  14. Genome-wide Mapping Reveals Conservation of Promoter DNA Methylation Following Chicken Domestication

    PubMed Central

    Li, Qinghe; Wang, Yuanyuan; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Ning

    2015-01-01

    It is well-known that environment influences DNA methylation, however, the extent of heritable DNA methylation variation following animal domestication remains largely unknown. Using meDIP-chip we mapped the promoter methylomes for 23,316 genes in muscle tissues of ancestral and domestic chickens. We systematically examined the variation of promoter DNA methylation in terms of different breeds, differentially expressed genes, SNPs and genes undergo genetic selection sweeps. While considerable changes in DNA sequence and gene expression programs were prevalent, we found that the inter-strain DNA methylation patterns were highly conserved in promoter region between the wild and domestic chicken breeds. Our data suggests a global preservation of DNA methylation between the wild and domestic chicken breeds in either a genome-wide or locus-specific scale in chick muscle tissues. PMID:25735894

  15. DAPK1, MGMT and RARB promoter methylation as biomarkers for high-grade cervical lesions.

    PubMed

    Sun, Yin; Li, Shu; Shen, Keng; Ye, Shuang; Cao, Dongyan; Yang, Jiaxin

    2015-01-01

    Gene promoter methylation may be used a potential biomarker for detecting solid tumor including cervical cancer. Here, we used methylation sensitive-high resolution melting (MS-HRM) analysis to detecting promoter methylation ratios of DAPK1, MGMT and RARB gene in patients with different cervical disease grade. The detection of gene promoter methylation was conducted in two hundred fifty patients' samples including normal cytology (n=48), cervical intraepithelial neoplasia grade 1 (CIN1, n=54), cervical intraepithelial neoplasia grade 2 (CIN2, n=47), cervical intraepithelial neoplasia grade 3 (CIN3, n=56) and cervical squamous cell carcinomas (SCS, n=45). We found there were a significant positive correlation between the promoter methylation status of DAPK1 and cervical disease grade (P=0.022). In addition, the methylated promoters of DAPK1 combined with MGMT, MGMT combined with RARB, DAPK1 combined with RARB were positive correlated with cervical disease grade (P < 0.05). All three genes promoters methylated were positive correlated with cervical disease grade (P < 0.001). Receiver operating characteristic (ROC) curves was conducted to evaluate whether the three genes methylation could be used to be a potential marker for diagnosing high grade cervical disease (HSIL and SCC). The cutoff values for the methylation rates of all these genes were 0-5%. Regrettably, only the methylation of MGMT combined with DAPK1 gave 43.4% sensitivity and 68.6% specificity. The current results indicated that MS-HRM-based testing for DNA methylations of MGMT plus DAPK1 genes holds some promise for high grade cervical disease screening. PMID:26823825

  16. DAPK1, MGMT and RARB promoter methylation as biomarkers for high-grade cervical lesions

    PubMed Central

    Sun, Yin; Li, Shu; Shen, Keng; Ye, Shuang; Cao, Dongyan; Yang, Jiaxin

    2015-01-01

    Gene promoter methylation may be used a potential biomarker for detecting solid tumor including cervical cancer. Here, we used methylation sensitive-high resolution melting (MS-HRM) analysis to detecting promoter methylation ratios of DAPK1, MGMT and RARB gene in patients with different cervical disease grade. The detection of gene promoter methylation was conducted in two hundred fifty patients’ samples including normal cytology (n=48), cervical intraepithelial neoplasia grade 1 (CIN1, n=54), cervical intraepithelial neoplasia grade 2 (CIN2, n=47), cervical intraepithelial neoplasia grade 3 (CIN3, n=56) and cervical squamous cell carcinomas (SCS, n=45). We found there were a significant positive correlation between the promoter methylation status of DAPK1 and cervical disease grade (P=0.022). In addition, the methylated promoters of DAPK1 combined with MGMT, MGMT combined with RARB, DAPK1 combined with RARB were positive correlated with cervical disease grade (P < 0.05). All three genes promoters methylated were positive correlated with cervical disease grade (P < 0.001). Receiver operating characteristic (ROC) curves was conducted to evaluate whether the three genes methylation could be used to be a potential marker for diagnosing high grade cervical disease (HSIL and SCC). The cutoff values for the methylation rates of all these genes were 0-5%. Regrettably, only the methylation of MGMT combined with DAPK1 gave 43.4% sensitivity and 68.6% specificity. The current results indicated that MS-HRM-based testing for DNA methylations of MGMT plus DAPK1 genes holds some promise for high grade cervical disease screening. PMID:26823825

  17. Increased MTHFR promoter methylation in mothers of Down syndrome individuals.

    PubMed

    Coppedè, Fabio; Denaro, Maria; Tannorella, Pierpaola; Migliore, Lucia

    2016-05-01

    Despite that advanced maternal age at conception represents the major risk factor for the birth of a child with Down syndrome (DS), most of DS babies are born from women aging less than 35 years. Studies performed in peripheral lymphocytes of those women revealed several markers of global genome instability, including an increased frequency of micronuclei, shorter telomeres and impaired global DNA methylation. Furthermore, young mothers of DS individuals (MDS) are at increased risk to develop dementia later in life, suggesting that they might be "biologically older" than mothers of euploid babies of similar age. Mutations in folate pathway genes, and particularly in the methylenetetrahydrofolate reductase (MTHFR) one, have been often associated with maternal risk for a DS birth as well as with risk of dementia in the elderly. Recent studies pointed out that also changes in MTHFR methylation levels can contribute to human disease, but nothing is known about MTHFR methylation in MDS tissues. We investigated MTHFR promoter methylation in DNA extracted from perypheral lymphocytes of 40 MDS and 44 matched control women that coinceived their children before 35 years of age, observing a significantly increased MTHFR promoter methylation in the first group (33.3 ± 8.1% vs. 28.3 ± 5.8%; p=0.001). In addition, the frequency of micronucleated lymphocytes was available from the women included in the study, was higher in MDS than control mothers (16.1 ± 8.6‰ vs. 10.5 ± 4.3‰; p=0.0004), and correlated with MTHFR promoter methylation levels (r=0.33; p=0.006). Present data suggest that MTHFR epimutations are likely to contribute to the increased genomic instability observed in cells from MDS, and could play a role in the risk of birth of a child with DS as well as in the onset of age related diseases in those women. PMID:26926955

  18. Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis.

    PubMed Central

    Issa, J P; Vertino, P M; Boehm, C D; Newsham, I F; Baylin, S B

    1996-01-01

    We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8876210

  19. Methylation of the Sox9 and Oct4 promoters and its correlation with gene expression during testicular development in the laboratory mouse

    PubMed Central

    Pamnani, Mamta; Sinha, Puja; Singh, Alka; Nara, Seema; Sachan, Manisha

    2016-01-01

    Abstract Sox9 and Oct4 are two important regulatory factors involved in mammalian development. Sox9, a member of the group E Sox transcription factor family, has a crucial role in the development of the genitourinary system, while Oct4, commonly known as octamer binding transcription factor 4, belongs to class V of the transcription family. The expression of these two proteins exhibits a dynamic pattern with regard to their expression sites and levels. The aim of this study was to investigate the role of de novo methylation in the regulation of the tissue- and site-specific expression of these proteins. The dynamics of the de novo methylation of 15 CpGs and six CpGs in Sox9 and Oct4 respectively, was studied with sodium bisulfite genomic DNA sequencing in mouse testis at different developmental stages. Consistent methylation of three CpGs was observed in adult ovary in which the expression of Sox9 was feeble, while the level of methylation in somatic tissue was greater in Oct4 compared to germinal tissue. The promoter-chromatin status of Sox9 was also studied with a chromatin immune-precipitation assay. PMID:27560488

  20. Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation

    PubMed Central

    Chalertpet, Kanwalat; Pakdeechaidan, Watcharapong; Patel, Vyomesh; Mutirangura, Apiwat; Yanatatsaneejit, Pattamawadee

    2015-01-01

    Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7–Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation. PMID:26250467

  1. DNA methylation of distal regulatory sites characterizes dysregulation of cancer genes

    PubMed Central

    2013-01-01

    Background Abnormal epigenetic marking is well documented in gene promoters of cancer cells, but the study of distal regulatory siteshas lagged behind.We performed a systematic analysis of DNA methylation sites connected with gene expression profilesacross normal and cancerous human genomes. Results Utilizing methylation and expression data in 58 cell types, we developed a model for methylation-expression relationships in gene promoters and extrapolated it to the genome. We mapped numerous sites at which DNA methylation was associated with expression of distal genes. These sites bind transcription factors in a methylation-dependent manner, and carry the chromatin marks of a particular class of transcriptional enhancers. In contrast to the traditional model of one enhancer site per cell type, we found that single enhancer sites may define gradients of expression levels across many different cell types. Strikingly, the identified sites were drastically altered in cancers: hypomethylated enhancer sites associated with upregulation of cancer-related genes and hypermethylated sites with downregulation. Moreover, the association between enhancer methylation and gene deregulation in cancerwas significantly stronger than the association of promoter methylationwith gene deregulation. Conclusions Methylation of distal regulatory sites is closely related to gene expression levels across the genome. Single enhancers may modulate ranges of cell-specific transcription levels, from constantlyopen promoters. In contrast to the remote relationships between promoter methylation and gene dysregulation in cancer, altered methylation of enhancer sites is closely related to gene expression profiles of transformed cells. PMID:23497655

  2. Methylation Alterations at Imprinted Genes Detected Among Long Term Shiftworkers

    PubMed Central

    Jacobs, Daniel I.; Hansen, Johnni; Fu, Alan; Stevens, Richard G.; Tjonneland, Anne; Vogel, Ulla B.; Zheng, Tongzhang; Zhu, Yong

    2016-01-01

    Exposure to light at night through shiftwork has been linked to alterations in DNA methylation and increased risk of cancer development. Using an Illumina Infinium Methylation Assay, we analyzed methylation levels of 397 CpG sites in the promoter regions of 56 normally imprinted genes to investigate whether shiftwork is associated with alteration of methylation patterns. Methylation was significantly higher at 20 CpG sites and significantly lower at 30 CpG sites (P < 0.05) in 10 female long-term shiftworkers as compared to 10 female age- and folate intake-matched day workers. The strongest evidence for altered methylation patterns in shiftworkers was observed for DLX5, IGF2AS, and TP73 based on the magnitude of methylation change and consistency in the direction of change across multiple CpG sites, and consistent results were observed using quantitative DNA methylation analysis. We conclude that long-term shiftwork may alter methylation patterns at imprinted genes, which may be an important mechanism by which shiftwork has carcinogenic potential and warrants further investigation. PMID:23193016

  3. DNA methylation in cystathionine-γ-lyase (CSE) gene promoter induced by ox-LDL in macrophages and in apoE knockout mice.

    PubMed

    Du, Hua-Ping; Li, Jiaojiao; You, Shou-Jiang; Wang, Ya-Li; Wang, Fen; Cao, Yong-Jun; Hu, Li-Fang; Liu, Chun-Feng

    2016-01-15

    Recent studies suggest that epigenetic alterations such as DNA methylation control many aspects of monocytes/macrophages and participate in the pathogenesis of atherosclerosis, a lipid-driven inflammatory disorder. Our and other groups demonstrated that dysregulation of cystathionine γ-lyase (CSE) -hydrogen sulfide (H2S) pathway was involved in monocyte/macrophages-mediated inflammation and atherosclerosis. However, it remains unknown whether altered cse methylation in macrophages may play a role in linking CSE-H2S dysregulation and atherosclerosis. In the present study, we showed that plasma H2S and H2S production in the peritoneal macrophages of apolipoprotein knockout (apoE(-/-)) mice gradually decreased with ages, and were also lower than that in control mice at 12 weeks older. Moreover, CSE mRNA expressions decreased while DNA methyltransferase (DNMT) expressions increased in the peritoneal macrophages isolated from apoE(-/-) mice, compared to age-matched wildtype mice. Similar observations were obtained in an in vitro study. In oxidized low-density lipoprotein (ox-LDL)-treated raw264.7 macrophages, cse transcription was down-regulated while the expression and activity of DNMT was up-regulated, associated with enhanced DNA methylation in cse promoter. Suppression of DNMT with its inhibitor or siRNA reversed the decrease of CSE mRNA. Therefore, our data suggest that DNA hypermethylation of CpG rich region in cse promoter might contribute to the decrease of cse transcription and H2S production in macrophages, and thus contribute to atherosclerosis development. PMID:26692478

  4. Quantitative assessment of the diagnostic role of CDH13 promoter methylation in lung cancer.

    PubMed

    Zhong, Yun-Hua; Peng, Hao; Cheng, Hong-Zhong; Wang, Ping

    2015-01-01

    In order to explore the association between cadherin 13 (CDH13) gene promoter methylation and lung carcinoma (LC) risk, we carried out a meta-analysis with searching of PubMed, Web of Science. Ultimately, 17 articles were identified and analysised by STATA 12.0 software. Overall, we found a significant relationship between CDH13 promoter methylation and LC risk (odds ratio=6.98, 95% confidence interval: 4.21-11.56, p<0.001). Subgroup analyses further revealed that LC risk was increased for individuals carrying the methylated CDH13 compared with those with unmethylated CDH13. Hence, our study identified a strong association between CDH13 gene promoter methylation and LC and highlighted a promising potential for CDH13 methylation in LC risk prediction. PMID:25735345

  5. Personalized treatment strategies in glioblastoma: MGMT promoter methylation status

    PubMed Central

    Thon, Niklas; Kreth, Simone; Kreth, Friedrich-Wilhelm

    2013-01-01

    The identification of molecular genetic biomarkers considerably increased our current understanding of glioma genesis, prognostic evaluation, and treatment planning. In glioblastoma, the most malignant intrinsic brain tumor entity in adults, the promoter methylation status of the gene encoding for the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) indicates increased efficacy of current standard of care, which is concomitant and adjuvant chemoradiotherapy with the alkylating agent temozolomide. In the elderly, MGMT promoter methylation status has recently been introduced to be a predictive biomarker that can be used for stratification of treatment regimes. This review gives a short summery of epidemiological, clinical, diagnostic, and treatment aspects of patients who are currently diagnosed with glioblastoma. The most important molecular genetic markers and epigenetic alterations in glioblastoma are summarized. Special focus is given to the physiological function of DNA methylation–in particular, of the MGMT gene promoter, its clinical relevance, technical aspects of status assessment, its correlation with MGMT mRNA and protein expressions, and its place within the management cascade of glioblastoma patients. PMID:24109190

  6. Methylation of tumor suppressor genes is related with copy number aberrations in breast cancer

    PubMed Central

    Murria, Rosa; Palanca, Sarai; de Juan, Inmaculada; Egoavil, Cecilia; Alenda, Cristina; García-Casado, Zaida; Juan, María J; Sánchez, Ana B; Santaballa, Ana; Chirivella, Isabel; Segura, Ángel; Hervás, David; Llop, Marta; Barragán, Eva; Bolufer, Pascual

    2015-01-01

    This study investigates the relationship of promoter methylation in tumor suppressor genes with copy-number aberrations (CNA) and with tumor markers in breast cancer (BCs). The study includes 98 formalin fixed paraffin-embedded BCs in which promoter methylation of 24 tumour suppressor genes were assessed by Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA), CNA of 20 BC related genes by MLPA and ER, PR, HER2, CK5/6, CK18, EGFR, Cadherin-E, P53, Ki-67 and PARP expression by immunohistochemistry (IHC). Cluster analysis classed BCs in two groups according to promoter methylation percentage: the highly-methylated group (16 BCs), containing mostly hyper-methylated genes, and the sparsely-methylated group (82 BCs) with hypo-methylated genes. ATM, CDKN2A, VHL, CHFR and CDKN2B showed the greatest differences in the mean methylation percentage between these groups. We found no relationship of the IHC parameters or pathological features with methylation status, except for Catherin-E (p = 0.008). However the highly methylated BCs showed higher CNA proportion than the sparsely methylated BCs (p < 0.001, OR = 1.62; IC 95% [1.26, 2.07]). CDC6, MAPT, MED1, PRMD14 and AURKA showed the major differences in the CNA percentage between the two groups, exceeding the 22%. Methylation in RASSF1, CASP8, DAPK1 and GSTP1 conferred the highest probability of harboring CNA. Our results show a new link between promoter methylation and CNA giving support to the importance of methylation events to establish new BCs subtypes. Our findings may be also of relevance in personalized therapy assessment, which could benefit the hyper methylated BC patients group. PMID:25628946

  7. CG Methylation Covaries with Differential Gene Expression between Leaf and Floral Bud Tissues of Brachypodium distachyon

    PubMed Central

    Roessler, Kyria; Takuno, Shohei; Gaut, Brandon S.

    2016-01-01

    DNA methylation has the potential to influence plant growth and development through its influence on gene expression. To date, however, the evidence from plant systems is mixed as to whether patterns of DNA methylation vary significantly among tissues and, if so, whether these differences affect tissue-specific gene expression. To address these questions, we analyzed both bisulfite sequence (BSseq) and transcriptomic sequence data from three biological replicates of two tissues (leaf and floral bud) from the model grass species Brachypodium distachyon. Our first goal was to determine whether tissues were more differentiated in DNA methylation than explained by variation among biological replicates. Tissues were more differentiated than biological replicates, but the analysis of replicated data revealed high (>50%) false positive rates for the inference of differentially methylated sites (DMSs) and differentially methylated regions (DMRs). Comparing methylation to gene expression, we found that differential CG methylation consistently covaried negatively with gene expression, regardless as to whether methylation was within genes, within their promoters or even within their closest transposable element. The relationship between gene expression and either CHG or CHH methylation was less consistent. In total, CG methylation in promoters explained 9% of the variation in tissue-specific expression across genes, suggesting that CG methylation is a minor but appreciable factor in tissue differentiation. PMID:26950546

  8. Global and gene-specific DNA methylation pattern discriminates cholecystitis from gallbladder cancer patients in Chile

    PubMed Central

    Kagohara, Luciane Tsukamoto; Schussel, Juliana L; Subbannayya, Tejaswini; Sahasrabuddhe, Nandini; Lebron, Cynthia; Brait, Mariana; Maldonado, Leonel; Valle, Blanca L; Pirini, Francesca; Jahuira, Martha; Lopez, Jaime; Letelier, Pablo; Brebi-Mieville, Priscilla; Ili, Carmen; Pandey, Akhilesh; Chatterjee, Aditi; Sidransky, David; Guerrero-Preston, Rafael

    2015-01-01

    Aim The aim of the study was to evaluate the use of global and gene-specific DNA methylation changes as potential biomarkers for gallbladder cancer (GBC) in a cohort from Chile. Material & methods DNA methylation was analyzed through an ELISA-based technique and quantitative methylation-specific PCR. Results Global DNA Methylation Index (p = 0.02) and promoter methylation of SSBP2 (p = 0.01) and ESR1 (p = 0.05) were significantly different in GBC when compared with cholecystitis. Receiver curve operator analysis revealed promoter methylation of APC, CDKN2A, ESR1, PGP9.5 and SSBP2, together with the Global DNA Methylation Index, had 71% sensitivity, 95% specificity, a 0.97 area under the curve and a positive predictive value of 90%. Conclusion Global and gene-specific DNA methylation may be useful biomarkers for GBC clinical assessment. PMID:25066711

  9. Methylization analysis of the FMR1 gene in carrier females

    SciTech Connect

    Meyers, S.; Cappon, S.; Khalifa, M.M.

    1994-09-01

    The fragile X syndrome mutation is associated with an expansion of a CGG repeat sequence and methylation of the CpG island in the promoter of the FMR1 gene. Methylation of the CpG island silences the FMR1 gene, thereby generating the disease phenotypes. Previous studies suggest that the normal FMR1 gene has the properties of an X-linked housekeeping gene that is subject to X inactivation, i.e., its CpG island is unmethylated on the active X chromosome and methylated on the inactive X. Because methylation of the mutant FMR1 gene occurs in both males and females with the full mutation, inactivating the FMR1 gene in these females might be a localized event independent from X inactivation. To test this hypothesis we compared the methylation pattern of two housekeeping genes, PGK1 and androgen receptor (AR) with that of the FMR1 in 46 female carriers of the fragile X syndrome. Twenty eight females were in the premutation range (63-193 repeats) and 16 were carriers of the full mutation (263-996 repeats). The data revealed complete correlation between the methylation pattern of PGK1 and AR. There was also a close correlation between X inactivation pattern detected by PGK1 and/or AR and that detected by FMR1 in female carriers of the premutation. In all female carriers of the full mutation there was complete methylation of the BssHII site in the expanded FMR1 allele. The X chromosome inactivation pattern in these females as detected by PGK1 and/or AR was as follows: in 10 cases the X inactivation was skewed in favor of the mutant FMR1, i.e. the mutant allele was on the inactive X chromosome, in 3 the inactivation was random and in 3 the inactivation was skewed in favor of the normal allele. These data suggest that the methylation of the FMR1 gene in females with the full mutation is a localized event and methylation of the FMR1 gene in these females cannot be used as a predictor of X inactivation.

  10. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates.

    PubMed

    Rivière, Guillaume

    2014-01-01

    DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing, and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position, and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster's developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5'-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment. PMID:24778620

  11. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates

    PubMed Central

    Rivière, Guillaume

    2014-01-01

    DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing, and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position, and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster's developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5′-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment. PMID:24778620

  12. Methylation of CTNNA1 promoter: frequent but not an adverse prognostic factor in acute myeloid leukemia.

    PubMed

    Chen, Xing-xing; Lin, Jiang; Qian, Jun; Qian, Wei; Yang, Jing; Ma, Ji-chun; Deng, Zhao-qun; An, Cui; Tang, Chun-yan; Qian, Zhen; Liu, Qing

    2014-05-01

    The reduced expression of CTNNA1 gene, a putative tumor suppressor gene, has been found in several cancers including acute myeloid leukemia (AML). CTNNA1 expression is regulated by methylation and histone deacetylation. However, the clinical significance of CTNNA1 methylation in AML is rarely known. The present study was aimed to investigate the methylation status of CTNNA1 promoter region using methylation-specific PCR (MSP) and its clinical relevance in Chinese AML patients. Patients with CTNNA1 hypermethylation had significantly lower level of CTNNA1 transcript than those without CTNNA1 hypermethylation (P=0.031). The relationship of CTNNA1 methylation with clinical parameters was evaluated. Aberrant hypermethylation of CTNNA1 gene was found in 23.9% (37/155) AML cases. The status of CTNNA1 methylation was not correlated with the mutations of seven genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, N/K-RAS and C/EBPA). There was no significant difference in the rates of complete remission (CR) between patients with and without CTNNA1 methylation. Although the overall survival (OS) time of the CTNNA1-methylated AML was shorter than that of CTNNA1-unmethylated group (6 months vs 9 months), the difference was not statistically significant (P=0.681). Our data suggest that CTNNA1 methylation is a recurrent event but has no influence on prognosis in AML. PMID:24685333

  13. Potential clinical significance of ERβ ON promoter methylation in sporadic breast cancer.

    PubMed

    Božović, Ana; Markićević, Milan; Dimitrijević, Bogomir; Jovanović Ćupić, Snežana; Krajnović, Milena; Lukić, Silvana; Mandušić, Vesna

    2013-01-01

    The aim of the study was to assess how hypermethylation of the ON promoter of the estrogen receptor beta (ERβ) gene affects its expression (at the mRNA and protein level) and to correlate these with some clinical and histopathological parameters. A total of 131 samples of frozen breast cancer tissue was analyzed. A custom-designed, two-step PCR method was used to measure the methylation index of the ERβ gene ON promoter region. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to quantify mRNA of the ERβ1 isoform, while ERβ1 protein was determined using the Western blot method. There was a significant difference in the methylation index of the ERβ gene ON promoter between the groups of patients with negative and positive axillary lymph node status (P = 0.03). In addition, the methylation index of the ON promoter was positively correlated with estrogen receptor alfa (ERα) protein levels (ρ = 0.31, P = 0.02). There was a significant difference in the methylation index of the ON promoter between the progesterone receptor (PR)-negative and PR-positive groups of patients (P = 0.01). ERβ1 protein levels were negatively correlated with ERα protein (ρ = -0.27, P < 0.01). The methylation index of the ON promoter could be a more reliable additional parameter for prediction and/or prognosis in breast cancer than ERβ1-mRNA and/or protein levels. PMID:23794253

  14. COMPARISON OF THE METHYL REDUCTASE GENES AND GENE PRODUCTS

    EPA Science Inventory

    The DNA sequences encoding component C of methyl coenzyme M reductase (mcr genes) in Methanothermus fervidus, Methanobacterium thermoautotrophicum, Methanococcus vannielii, and Methanosarcina barkeri have been published. omparisons of transcription initiation and termination site...

  15. Methylation of the adenomatous polyposis coli (APC) gene in human placenta and hypermethylation in choriocarcinoma cells.

    PubMed

    Wong, N C; Novakovic, B; Weinrich, B; Dewi, C; Andronikos, R; Sibson, M; Macrae, F; Morley, R; Pertile, M D; Craig, J M; Saffery, R

    2008-09-01

    Methylation of the human APC gene promoter is associated with several different types of cancers and has also been documented in some pre-cancerous tissues. We have examined the methylation of APC gene promoters in human placenta and choriocarcinoma cells. This revealed a general hypomethylation of the APC-1b promoter and a pattern with monoallelic methylation of the APC-1a promoter in full term placental tissue. However, there was no evidence of a parent-of-origin effect, suggesting random post zygotic origin of methylation. Increased methylation of this promoter was observed in all choriocarcinoma-derived trophoblast cell lines, suggesting a trophoblastic origin of placental APC methylation and implicating APC hypermethylation in the development of this group of gestational tumours. Our demonstration of placental methylation of the APC-1a promoter represents the first observation of monoallelic methylation of this gene in early development, and provides further support for a role of canonical Wnt signalling in placental trophoblast invasiveness. This also implicates tumour suppressor gene silencing as an integral part of normal human placental development. PMID:18485586

  16. Arabidopsis EDM2 promotes IBM1 distal polyadenylation and regulates genome DNA methylation patterns

    PubMed Central

    Lei, Mingguang; La, Honggui; Lu, Kun; Wang, Pengcheng; Miki, Daisuke; Ren, Zhizhong; Duan, Cheng-Guo; Wang, Xingang; Tang, Kai; Zeng, Liang; Yang, Lan; Zhang, Heng; Nie, Wenfeng; Liu, Pan; Zhou, Jianping; Liu, Renyi; Zhong, Yingli; Liu, Dong; Zhu, Jian-Kang

    2014-01-01

    DNA methylation is important for the silencing of transposons and other repetitive elements in many higher eukaryotes. However, plant and mammalian genomes have evolved to contain repetitive elements near or inside their genes. How these genes are kept from being silenced by DNA methylation is not well understood. A forward genetics screen led to the identification of the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) as a cellular antisilencing factor and regulator of genome DNA methylation patterns. EDM2 contains a composite Plant Homeo Domain that recognizes both active and repressive histone methylation marks at the intronic repeat elements in genes such as the Histone 3 lysine 9 demethylase gene Increase in BONSAI Methylation 1 (IBM1) and is necessary for maintaining the expression of these genes by promoting mRNA distal polyadenylation. Because of its role in maintaining IBM1 expression, EDM2 is required for preventing CHG methylation in the bodies of thousands of genes. Our results thus increase the understanding of antisilencing, genome methylation patterns, and regulation of alternative RNA processing by intronic heterochromatin. PMID:24248388

  17. Brain-derived neurotrophic factor promoter methylation and cortical thickness in recurrent major depressive disorder

    PubMed Central

    Na, Kyoung-Sae; Won, Eunsoo; Kang, June; Chang, Hun Soo; Yoon, Ho-Kyoung; Tae, Woo Suk; Kim, Yong-Ku; Lee, Min-Soo; Joe, Sook-Haeng; Kim, Hyun; Ham, Byung-Joo

    2016-01-01

    Recent studies have reported that methylation of the brain-derived neurotrophic factor (BDNF) gene promoter is associated with major depressive disorder (MDD). This study aimed to investigate the association between cortical thickness and methylation of BDNF promoters as well as serum BDNF levels in MDD. The participants consisted of 65 patients with recurrent MDD and 65 age- and gender-matched healthy controls. Methylation of BDNF promoters and cortical thickness were compared between the groups. The right medial orbitofrontal, right lingual, right lateral occipital, left lateral orbitofrontal, left pars triangularis, and left lingual cortices were thinner in patients with MDD than in healthy controls. Among the MDD group, right pericalcarine, right medical orbitofrontal, right rostral middle frontal, right postcentral, right inferior temporal, right cuneus, right precuneus, left frontal pole, left superior frontal, left superior temporal, left rostral middle frontal and left lingual cortices had inverse correlations with methylation of BDNF promoters. Higher levels of BDNF promoter methylation may be closely associated with the reduced cortical thickness among patients with MDD. Serum BDNF levels were significantly lower in MDD, and showed an inverse relationship with BDNF methylation only in healthy controls. Particularly the prefrontal and occipital cortices seem to indicate key regions in which BDNF methylation has a significant effect on structure. PMID:26876488

  18. A CPG ISLAND AT THE PROMOTER OF THE PDE8B GENE IS METHYLATED IN PLACENTA AND HYDATIDIFORM MOLES, BUT NOT IN CONTROL DNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: We used a genome-wide CpG methylation screen, restriction landmark genome scanning (RLGS) to identify CpG islands that have altered methylation in complete hydatidiform moles (CHM), compared to control genomic DNA. Because CHM are diploid, but of uniparental parental inheritance and uniq...

  19. Detection of MGMT promoter methylation in glioblastoma using pyrosequencing

    PubMed Central

    Xie, Hao; Tubbs, Raymond; Yang, Bin

    2015-01-01

    Recent clinical trials on patients with glioblastoma revealed that O6-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient’s response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation. PMID:25755756

  20. Detection of MGMT promoter methylation in glioblastoma using pyrosequencing

    PubMed Central

    Xie, Hao; Tubbs, Raymond; Yang, Bin

    2015-01-01

    Recent clinical trials on patients with glioblastoma revealed that O6-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient’s response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation. PMID:25973069

  1. Gene Body Methylation Patterns in Daphnia Are Associated with Gene Family Size

    PubMed Central

    Asselman, Jana; De Coninck, Dieter I. M.; Pfrender, Michael E.; De Schamphelaere, Karel A. C.

    2016-01-01

    The relation between gene body methylation and gene function remains elusive. Yet, our understanding of this relationship can contribute significant knowledge on how and why organisms target specific gene bodies for methylation. Here, we studied gene body methylation patterns in two Daphnia species. We observed both highly methylated genes and genes devoid of methylation in a background of low global methylation levels. A small but highly significant number of genes was highly methylated in both species. Remarkably, functional analyses indicate that variation in methylation within and between Daphnia species is primarily targeted to small gene families whereas large gene families tend to lack variation. The degree of sequence similarity could not explain the observed pattern. Furthermore, a significant negative correlation between gene family size and the degree of methylation suggests that gene body methylation may help regulate gene family expansion and functional diversification of gene families leading to phenotypic variation. PMID:27017526

  2. Association of BRCA1 promoter methylation with sporadic breast cancers: Evidence from 40 studies.

    PubMed

    Zhang, Li; Long, Xinghua

    2015-01-01

    Breast cancer susceptibility gene 1 (BRCA1) located at chromosome 17q12-21 is a classic tumor suppressor gene, and has been considered as a significant role in hereditary breast cancers. Moreover, numerous studies demonstrated the methylation status of CpG islands in the promoter regions of BRCA1 gene was aberrant in patients with sporadic breast tumors compared with healthy females or patients with benign diseases. However, these conclusions were not always consistent. Hence, a meta-analysis was performed to get a more precise estimate for these associations. Crude odds ratio with 95% confidence interval were used to assess the association of BRCA1 promoter methylation and the risk or clinicopathologic characteristics of breast cancers under fixed or random effect model. A total of 40 studies were eligible for this present study. We observed the frequency of BRCA1 promoter methylation was statistically significant higher in breast cancers than non-cancer controls. Furthermore, BRCA1 methylation was statistically associated with lymph node metastasis, histological grade 3, ER(-), PR(-), triple-negative phenotype, and decreased or lack levels of BRCA1 protein expression. In conclusion, this study indicated that BRCA1 promoter methylation appeared to be a useful predictive or prognostic biomarker for breast cancers in clinical assessment. PMID:26643130

  3. Association of BRCA1 promoter methylation with sporadic breast cancers: Evidence from 40 studies

    PubMed Central

    Zhang, Li; Long, Xinghua

    2015-01-01

    Breast cancer susceptibility gene 1 (BRCA1) located at chromosome 17q12-21 is a classic tumor suppressor gene, and has been considered as a significant role in hereditary breast cancers. Moreover, numerous studies demonstrated the methylation status of CpG islands in the promoter regions of BRCA1 gene was aberrant in patients with sporadic breast tumors compared with healthy females or patients with benign diseases. However, these conclusions were not always consistent. Hence, a meta-analysis was performed to get a more precise estimate for these associations. Crude odds ratio with 95% confidence interval were used to assess the association of BRCA1 promoter methylation and the risk or clinicopathologic characteristics of breast cancers under fixed or random effect model. A total of 40 studies were eligible for this present study. We observed the frequency of BRCA1 promoter methylation was statistically significant higher in breast cancers than non-cancer controls. Furthermore, BRCA1 methylation was statistically associated with lymph node metastasis, histological grade 3, ER(-), PR(-), triple-negative phenotype, and decreased or lack levels of BRCA1 protein expression. In conclusion, this study indicated that BRCA1 promoter methylation appeared to be a useful predictive or prognostic biomarker for breast cancers in clinical assessment. PMID:26643130

  4. Interleukin-6 Promotes Tumorigenesis by Altering DNA Methylation in Oral Cancer Cells

    PubMed Central

    Gasche, Jacqueline A.; Hoffmann, Jürgen; Boland, C. Richard; Goel, Ajay

    2011-01-01

    Worldwide oral squamous cell carcinoma (OSCC) accounts for more than 100,000 deaths each year. Chronic inflammation constitutes one of the key risk factors for OSCC. Accumulating evidence suggests that aberrant DNA methylation may contribute to OSCC tumorigenesis. This study investigated whether chronic inflammation alters DNA methylation and expression of cancer-associated genes in OSCC. We established an in-vitro model of interleukin (IL)-6 mediating chronic inflammation in OSCC cell lines. Thereafter, we measured the ability of IL-6 to induce global hypomethylation of LINE-1 sequences, as well as CpG methylation changes using multiple methodologies including quantitative pyrosequencing, methylation-specific multiplex ligation-dependent probe amplification, and sensitive melting analysis after real-time methylation specific PCR. Gene expression was investigated by quantitative Reverse Transcriptase-PCR. IL-6 induced significant global LINE-1 hypomethylation (p=0.016) in our in-vitro model of inflammatory stress in OSCC cell lines. Simultaneously, IL-6 induced CpG promoter methylation changes in several important putative tumor suppressor genes including CHFR, GATA5, and PAX6. Methylation changes correlated inversely with the changes in the expression of corresponding genes. Our results indicate that IL-6-induced inflammation promotes tumorigenesis in the oral cavity by altering global LINE-1 hypomethylation. In addition, concurrent hypermethylation of multiple tumor suppressor genes by IL-6 suggests that epigenetic gene silencing may be an important consequence of chronic inflammation in the oral cavity. These findings have clinical relevance, as both methylation and inflammation are suitable targets for developing novel preventive and therapeutic measures. PMID:21710491

  5. A significant association between BDNF promoter methylation and the risk of drug addiction.

    PubMed

    Xu, Xuting; Ji, Huihui; Liu, Guili; Wang, Qinwen; Liu, Huifen; Shen, Wenwen; Li, Longhui; Xie, Xiaohu; Zhou, Wenhua; Duan, Shiwei

    2016-06-10

    As a member of the neurotrophic factor family, brain derived neurotrophic factor (BDNF) plays an important role in the survival and differentiation of neurons. The aim of our work was to evaluate the role of BDNF promoter methylation in drug addiction. A total of 60 drug abusers (30 heroin and 30 methylamphetamine addicts) and 52 healthy age- and gender-matched controls were recruited for the current case control study. Bisulfite pyrosequencing technology was used to determine the methylation levels of five CpGs (CpG1-5) on the BDNF promoter. Among the five CpGs, CpG5 methylation was significantly lower in drug abusers than controls. Moreover, significant associations were found between CpG5 methylation and addictive phenotypes including tension-anxiety, anger-hostility, fatigue-inertia, and depression-dejection. In addition, luciferase assay showed that the DNA fragment of BDNF promoter played a key role in the regulation of gene expression. Our results suggest that BDNF promoter methylation is associated with drug addiction, although further studies are needed to understand the mechanisms by which BDNF promoter methylation contributes to the pathophysiology of drug addiction. PMID:26976342

  6. Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients

    PubMed Central

    Nguyen, Albert; Mamarbachi, Maya; Turcot, Valérie; Lessard, Samuel; Yu, Carol; Luo, Xiaoyan; Lalongé, Julie; Hayami, Doug; Gayda, Mathieu; Juneau, Martin; Thorin-Trescases, Nathalie; Lettre, Guillaume; Nigam, Anil; Thorin, Eric

    2016-01-01

    DNA methylation is believed to regulate gene expression during adulthood in response to the constant changes in environment. The methylome is therefore proposed to be a biomarker of health through age. ANGPTL2 is a circulating pro-inflammatory protein that increases with age and prematurely in patients with coronary artery diseases; integrating the methylation pattern of the promoter may help differentiate age- vs. disease-related change in its expression. We believe that in a pro-inflammatory environment, ANGPTL2 is differentially methylated, regulating ANGPTL2 expression. To test this hypothesis we investigated the changes in promoter methylation of ANGPTL2 gene in leukocytes from patients suffering from post-acute coronary syndrome (ACS). DNA was extracted from circulating leukocytes of post-ACS patients with cardiovascular risk factors and from healthy young and age-matched controls. Methylation sites (CpGs) found in the ANGPTL2 gene were targeted for specific DNA methylation quantification. The functionality of ANGPTL2 methylation was assessed by an in vitro luciferase assay. In post-ACS patients, C-reactive protein and ANGPTL2 circulating levels increased significantly when compared to healthy controls. Decreased methylation of specific CpGs were found in the promoter of ANGPTL2 and allowed to discriminate age vs. disease associated methylation. In vitro DNA methylation of specific CpG lead to inhibition of ANGPTL2 promoter activity. Reduced leukocyte DNA methylation in the promoter region of ANGPTL2 is associated with the pro-inflammatory environment that characterizes patients with post-ACS differently from age-matched healthy controls. Methylation of different CpGs in ANGPTL2 gene may prove to be a reliable biomarker of coronary disease. PMID:27101308

  7. Ammonium Inhibits Chromomethylase 3-Mediated Methylation of the Arabidopsis Nitrate Reductase Gene NIA2

    PubMed Central

    Kim, Joo Yong; Kwon, Ye Jin; Kim, Sung-Il; Kim, Do Youn; Song, Jong Tae; Seo, Hak Soo

    2016-01-01

    Gene methylation is an important mechanism regulating gene expression and genome stability. Our previous work showed that methylation of the nitrate reductase (NR) gene NIA2 was dependent on chromomethylase 3 (CMT3). Here, we show that CMT3-mediated NIA2 methylation is regulated by ammonium in Arabidopsis thaliana. CHG sequences (where H can be A, T, or C) were methylated in NIA2 but not in NIA1, and ammonium [(NH4)2SO4] treatment completely blocked CHG methylation in NIA2. By contrast, ammonium had no effect on CMT3 methylation, indicating that ammonium negatively regulates CMT3-mediated NIA2 methylation without affecting CMT3 methylation. Ammonium upregulated NIA2 mRNA expression, which was consistent with the repression of NIA2 methylation by ammonium. Ammonium treatment also reduced the overall genome methylation level of wild-type Arabidopsis. Moreover, CMT3 bound to specific promoter and intragenic regions of NIA2. These combined results indicate that ammonium inhibits CMT3-mediated methylation of NIA2 and that of other target genes, and CMT3 selectively binds to target DNA sequences for methylation. PMID:26834755

  8. Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients

    PubMed Central

    Kimura, Naoki; Nagasaka, Takeshi; Murakami, Jun; Sasamoto, Hiromi; Murakami, Masahiro; Tanaka, Noriaki; Matsubara, Nagahide

    2005-01-01

    Aberrant methylation of DNA has been shown to play an important role in a variety of human cancers, developmental disorders and aging. Hence, aberrant methylation patterns in genes can be a molecular marker for such conditions. Therefore, a reliable but uncomplicated method to detect DNA methylation is preferred, not merely for research purposes but for daily clinical practice. To achieve these aims, we have established a precise system to identify DNA methylation patterns based on an oligonucleotide microarray technology. Our microarray method has an advantage over conventional methods and is unique because it allows the precise measurement of the methylation patterns within a target region. Our simple signal detection system depends on using an avidin–biotinylated peroxidase complex and does not require an expensive laser scanner or hazardous radioisotope. In this study, we applied our technique to detect promoter methylation status of O6-methylguanine-DNA methyltransferase (MGMT) gene. Our easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylation analyses for clinical purposes. PMID:15760842

  9. MGMT promoter methylation in non-neoplastic brain.

    PubMed

    Hsu, Chih-Yi; Ho, Hsiang-Ling; Chang-Chien, Yi-Chun; Chang, Yi-Wen; Ho, Donald Ming-Tak

    2015-02-01

    O(6)-methylguanine-DNA-methyltransferase (MGMT) is mainly regulated by cytosine-guanine island promoter methylation that is believed to occur only in neoplastic tissue. The present study was undertaken to investigate whether methylation occurs also in non-neoplastic brains by collecting 45 non-neoplastic brains from autopsies and 56 lobectomy specimens from epileptic surgeries. The promoter methylation status of MGMT was studied by methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), while protein expression was studied by immunohistochemical stain (IHC). The methylation rates, as determined by MSP and PSQ, were 3.0 % (3/101) and 2.9 % (2/69), respectively. Of note, no case had positive result concomitantly from both MSP and PSQ (3 were MSP+/PSQ- and 2 were MSP-/PSQ+), and all the positive samples were further confirmed by cloning and Sanger sequencing. All the methylated cases, except for those having indeterminate IHC results from autopsy specimens, revealed no loss of MGMT protein expression and similar staining pattern to that of the unmethylated cases. In conclusion, the current study demonstrated that MGMT promoter methylation could occur in a low percentage of non-neoplastic brains but did not affect the status of protein expression, which could be regarded as a normal variation in non-neoplastic brains. PMID:25391970

  10. Silencing of TESTIN by dense biallelic promoter methylation is the most common molecular event in childhood acute lymphoblastic leukaemia

    PubMed Central

    2010-01-01

    Background Aberrant promoter DNA methylation has been reported in childhood acute lymphoblastic leukaemia (ALL) and has the potential to contribute to its onset and outcome. However, few reports demonstrate consistent, prevalent and dense promoter methylation, associated with tumour-specific gene silencing. By screening candidate genes, we have detected frequent and dense methylation of the TESTIN (TES) promoter. Results Bisulfite sequencing showed that 100% of the ALL samples (n = 20) were methylated at the TES promoter, whereas the matched remission (n = 5), normal bone marrow (n = 6) and normal PBL (n = 5) samples were unmethylated. Expression of TES in hyperdiploid, TEL-AML+, BCR-ABL+, and E2A-PBX+ subtypes of B lineage ALL was markedly reduced compared to that in normal bone marrow progenitor cells and in B cells. In addition TES methylation and silencing was demonstrated in nine out of ten independent B ALL propagated as xenografts in NOD/SCID mice. Conclusion In total, 93% of B ALL samples (93 of 100) demonstrated methylation with silencing or reduced expression of the TES gene. Thus, TES is the most frequently methylated and silenced gene yet reported in ALL. TES, a LIM domain-containing tumour suppressor gene and component of the focal adhesion complex, is involved in adhesion, motility, cell-to-cell interactions and cell signalling. Our data implicate TES methylation in ALL and provide additional evidence for the involvement of LIM domain proteins in leukaemogenesis. PMID:20573277

  11. Gene Expression and Methylation Pattern in HRK Apoptotic Gene in Myelodysplastic Syndrome

    PubMed Central

    Zaker, Farhad; Amirizadeh, Naser; Nasiri, Nahid; Razavi, Seyed Mohsen; Teimoori-Toolabi, Ladan; Yaghmaie, Marjan; Mehrasa, Roya

    2016-01-01

    Myelodysplastic syndromes (MDSs) are a clonal bone marrow (BM) disease characterized by ineffective hematopoiesis, dysplastic maturation and progression to acute myeloid leukemia (AML). Methylation silencing of HRK has been found in several human malignancies. In this study, we explored the association of HRK methylation status with its expression, clinical parameters and MDS subtypes in MDS patients. To study the methylation status of HRK gene, we applied Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) in MDS patients, as well as healthy controls and EpiTect®PCR Control DNA. Real time RT-PCR was used for gene expression analysis. Methylation frequency in promoter region of HRK in patient samples was 20.37%. Methylation of HRK was significantly related to transcriptional downregulation (P=0.023). The difference in frequency of hypermethylated HRK gene was significant between good (10%) and poor (71.42%) cytogenetic risk groups (P= 0.001), advanced stage MDS patients (66.66%) in comparison with early stage MDS patients (2.56%) (P= 0.00), higher- risk MDS group (61.53%) and lower- risk MDS group (7.31%) (P= 0.00). HRK hypermethylation was associated with advanced- stage MDS and downregulation of HRK gene may play a role in the progression of MDS. PMID:27478805

  12. Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene

    PubMed Central

    Morano, Annalisa; Angrisano, Tiziana; Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Bartollino, Silvia; Zuchegna, Candida; Babbio, Federica; Bonapace, Ian Marc; Allen, Brittany; Muller, Mark T.; Chiariotti, Lorenzo; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

    2014-01-01

    We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15–20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression. PMID:24137009

  13. Maternal tobacco use modestly alters correlated epigenome-wide placental DNA methylation and gene expression

    PubMed Central

    Suter, Melissa; Ma, Jun; Harris, Alan; Patterson, Lauren; Brown, Kathleen A; Shope, Cynthia; Showalter, Lori; Abramovici, Adi

    2011-01-01

    Several studies linking alterations in differential placental methylation with pregnancy disorders have implicated (de) regulation of the placental epigenome with fetal programming and later-in-life disease. We have previously demonstrated that maternal tobacco use is associated with alterations in promoter methylation of placental CYP1A1 and that these changes are correlated with CYP1A1 gene expression and fetal growth restriction. In this study we sought to expand our analysis of promoter methylation by correlating it to gene expression on a genome-wide scale. Employing side-by-side IlluminaHG-12 gene transcription with Infinium27K methylation arrays, we interrogated correlative changes in placental gene expression and DNA methylation associated with maternal tobacco smoke exposure at an epigenome-wide level and in consideration of signature gene pathways. We observed that the expression of 623 genes and the methylation of 1,024 CpG dinucleotides are significantly altered among smokers, with only 38 CpGs showing significant differential methylation (differing by a methylation level of ≥10%). We identified a significant Pearson correlation (≥0.7 or ≤-0.7) between placental transcriptional regulation and differential CpG methylation in only 25 genes among non-smokers but in 438 genes among smokers (18-fold increase, p < 0.0001), with a dominant effect among oxidative stress pathways. Differential methylation at as few as 6 sites was attributed to maternal smoking-mediated birth weight reduction in linear regression models with Bonferroni correction (p < 1.8 × 10−6). These studies suggest that a common perinatal exposure (such as maternal smoking) deregulates placental methylation in a CpG site-specific manner that correlates with meaningful alterations in gene expression along signature pathways. PMID:21937876

  14. Classification of Colon Cancer Patients Based on the Methylation Patterns of Promoters

    PubMed Central

    Choi, Wonyoung; Lee, Jungwoo; Lee, Jin-Young; Lee, Sun-Min; Kim, Da-Won

    2016-01-01

    Diverse somatic mutations have been reported to serve as cancer drivers. Recently, it has also been reported that epigenetic regulation is closely related to cancer development. However, the effect of epigenetic changes on cancer is still elusive. In this study, we analyzed DNA methylation data on colon cancer taken from The Caner Genome Atlas. We found that several promoters were significantly hypermethylated in colon cancer patients. Through clustering analysis of differentially methylated DNA regions, we were able to define subgroups of patients and observed clinical features associated with each subgroup. In addition, we analyzed the functional ontology of aberrantly methylated genes and identified the G-protein-coupled receptor signaling pathway as one of the major pathways affected epigenetically. In conclusion, our analysis shows the possibility of characterizing the clinical features of colon cancer subgroups based on DNA methylation patterns and provides lists of important genes and pathways possibly involved in colon cancer development. PMID:27445647

  15. Estrogen and promoter methylation in the regulation of PLA2G7 transcription.

    PubMed

    Jiang, Danjie; Wang, Yunliang; Shen, Yusheng; Xu, Yan; Zhu, Huangkai; Wang, Jinhua; Wang, Hongwei; Duan, Shiwei

    2016-10-10

    In the current study, cell lines including HEK293, SW480, HPASMC, HPCASMC and HAEC were cultured with 5-aza-2-deoxycytidine (DAC) and 17-β-estradiol to investigate whether PLA2G7 transcription was under the control of promoter methylation and 17-β-estradiol. Luciferase reporter gene assays were used to evaluate whether reporter gene activity was enhanced by PLA2G7 promoter fragment. Gene expression and methylation were detected using RT-PCR and pyrosequencing methods, respectively. Endogenous PLA2G7 transcription levels were found to be significantly lower in vascular related cell lines than in the other cell lines. Luciferase reporter gene assays indicated that gene activity was significantly enhanced by PLA2G7 promoter fragment. PLA2G7 transcription was found to be up-regulated with the treatment of DAC. The 17-β-estradiol was found to down-regulate PLA2G7 transcription in all the cell lines. However, 17-β-estradiol did not have significant effect on PLA2G7 methylation. Further chromatin immunoprecipitation assay showed that 17-β-estradiol might regulate gene transcription by affecting the acetylated histone H3 and H4 marks on PLA2G7 promoter. Our results showed that PLA2G7 gene expression was co-regulated by 17-β-estradiol and promoter methylation. Our findings might provide molecular clues for gender disparity in the contribution of PLA2G7 to vascular related diseases such as coronary heart disease. PMID:27450918

  16. Racial variation in breast tumor promoter methylation in the Carolina Breast Cancer Study

    PubMed Central

    Conway, Kathleen; Edmiston, Sharon N.; Tse, Chiu-Kit; Bryant, Christopher; Kuan, Pei Fen; Hair, Brionna Y.; Parrish, Eloise A.; May, Ryan; Swift-Scanlan, Theresa

    2015-01-01

    Background African American (AA) women are diagnosed with more advanced breast cancers and have worse survival than white women, but a comprehensive understanding of the basis for this disparity remains unclear. Analysis of DNA methylation, an epigenetic mechanism that can regulate gene expression, could help to explain racial differences in breast tumor clinical biology and outcomes. Methods DNA methylation was evaluated at 1287 CpGs in the promoters of cancer-related genes in 517 breast tumors of AA (n=216) or non-AA (n=301) cases in the Carolina Breast Cancer Study. Results Multivariable linear regression analysis of all tumors, controlling for age, menopausal status, stage, intrinsic subtype, and multiple comparisons (FDR), identified 7 CpG probes that showed significant (adjusted p<0.05) differential methylation between AAs and non-AAs. Stratified analyses detected an additional 4 CpG probes differing by race within hormone receptor-negative (HR−) tumors. Genes differentially methylated by race included DSC2, KCNK4, GSTM1, AXL, DNAJC15, HBII-52, TUSC3 and TES; the methylation state of several of these genes may be associated with worse survival in AAs. TCGA breast tumor data confirmed the differential methylation by race and negative correlations with expression for most of these genes. Several loci also showed racial differences in methylation in peripheral blood leukocytes (PBLs) from CBCS cases, indicating that these variations were not necessarily tumor-specific. Conclusions Racial differences in the methylation of cancer-related genes are detectable in both tumors and PBLs from breast cancer cases. Impact Epigenetic variation could contribute to differences in breast tumor development and outcomes between AAs and non-AAs. PMID:25809865

  17. Differential methylation of the TRPA1 promoter in pain sensitivity.

    PubMed

    Bell, J T; Loomis, A K; Butcher, L M; Gao, F; Zhang, B; Hyde, C L; Sun, J; Wu, H; Ward, K; Harris, J; Scollen, S; Davies, M N; Schalkwyk, L C; Mill, J; Williams, F M K; Li, N; Deloukas, P; Beck, S; McMahon, S B; Wang, J; John, S L; Spector, T D

    2014-01-01

    Chronic pain is a global public health problem, but the underlying molecular mechanisms are not fully understood. Here we examine genome-wide DNA methylation, first in 50 identical twins discordant for heat pain sensitivity and then in 50 further unrelated individuals. Whole-blood DNA methylation was characterized at 5.2 million loci by MeDIP sequencing and assessed longitudinally to identify differentially methylated regions associated with high or low pain sensitivity (pain DMRs). Nine meta-analysis pain DMRs show robust evidence for association (false discovery rate 5%) with the strongest signal in the pain gene TRPA1 (P=1.2 × 10(-13)). Several pain DMRs show longitudinal stability consistent with susceptibility effects, have similar methylation levels in the brain and altered expression in the skin. Our approach identifies epigenetic changes in both novel and established candidate genes that provide molecular insights into pain and may generalize to other complex traits. PMID:24496475

  18. A Feature Selection Algorithm to Compute Gene Centric Methylation from Probe Level Methylation Data

    PubMed Central

    Baur, Brittany; Bozdag, Serdar

    2016-01-01

    DNA methylation is an important epigenetic event that effects gene expression during development and various diseases such as cancer. Understanding the mechanism of action of DNA methylation is important for downstream analysis. In the Illumina Infinium HumanMethylation 450K array, there are tens of probes associated with each gene. Given methylation intensities of all these probes, it is necessary to compute which of these probes are most representative of the gene centric methylation level. In this study, we developed a feature selection algorithm based on sequential forward selection that utilized different classification methods to compute gene centric DNA methylation using probe level DNA methylation data. We compared our algorithm to other feature selection algorithms such as support vector machines with recursive feature elimination, genetic algorithms and ReliefF. We evaluated all methods based on the predictive power of selected probes on their mRNA expression levels and found that a K-Nearest Neighbors classification using the sequential forward selection algorithm performed better than other algorithms based on all metrics. We also observed that transcriptional activities of certain genes were more sensitive to DNA methylation changes than transcriptional activities of other genes. Our algorithm was able to predict the expression of those genes with high accuracy using only DNA methylation data. Our results also showed that those DNA methylation-sensitive genes were enriched in Gene Ontology terms related to the regulation of various biological processes. PMID:26872146

  19. DNA methylation does not stably lock gene expression but instead serves as a molecular mark for gene silencing memory

    PubMed Central

    Raynal, Noël J.-M.; Si, Jiali; Taby, Rodolphe F.; Gharibyan, Vazganush; Ahmed, Saira; Jelinek, Jaroslav; Estécio, Marcos R.H.; Issa, Jean-Pierre J.

    2012-01-01

    DNA methylation is commonly thought of as a "molecular lock" that leads to permanent gene silencing. To investigate this notion, we tested 24 different HDAC inhibitors (HDACi) on colon cancer cells that harbor a GFP locus stably integrated and silenced by a hypermethylated CMV promoter. We found that HDACi efficiently reactivated expression of GFP and many other endogenous genes silenced by DNA hypermethylation. After treatment, all promoters were marked with active chromatin, yet DNA hypermethylation did not change. Thus, DNA methylation could not prevent gene reactivation by drug-induced resetting of the chromatin state. In evaluating the relative contribution of DNA methylation and histone modifications to stable gene silencing, we followed expression levels of GFP and other genes silenced by DNA hypermethylation over time after treatment with HDACi or DNA demethylating drugs. Reactivation of methylated loci by HDACi was detectable for only 2 weeks, whereas DNA demethylating drugs induced permanent epigenetic reprogramming. Therefore, DNA methylation cannot be considered as a lock for gene expression, but rather as a memory signal for long-term maintenance of gene silencing. These findings define chromatin as an important druggable target for cancer epigenetic therapy and suggest that removal of DNA methylation signals is required to achieve long-term gene reactivation. PMID:22219169

  20. Prognostic Role of Methylation Status of the MGMT Promoter Determined Quantitatively by Pyrosequencing in Glioblastoma Patients

    PubMed Central

    Kim, Dae Cheol; Kim, Ki Uk

    2016-01-01

    Objective This study investigated whether pyrosequencing can be used to determine the methylation status of the MGMT promoter as a clinical biomarker using relatively old archival tissue samples of glioblastoma. We also examined other prognostic factors for survival of glioblastoma patients. Methods The available study set included formalin-fixed paraffin-embedded (FFPE) tissue from 104 patients at two institutes from 1997 to 2012, all of which were diagnosed histopathologically as glioblastoma. Clinicopathologic data were collected by review of medical records. For pyrosequencing analysis, the PyroMark Q96 CpG MGMT kit (Qiagen, Hilden, Germany) was used to detect the level of methylation at exon 1 positions 17–39 of the MGMT gene, which contains 5 CpGs. Results Methylation of the MGMT promoter was detected in 43 (41.3%) of 104 samples. The average percentage methylation was 14.0±16.8% overall and 39.0±14.7% for methylated cases. There was no significant pattern of linear increase or decrease according to the age of the FFPE block (p=0.687). In multivariate analysis, age, performance status, extent of surgery, method of adjuvant therapy, and methylation status estimated by pyrosequencing were independently associated with overall survival. Additionally, patients with a high level of methylation survived longer than those with low methylation (p=0.016). Conclusion In this study, the status and extent of methylation of the MGMT promoter analyzed by pyrosequencing were associated with overall survival in glioblastoma patients. Pyrosequencing is a quantitative method that overcomes the problems of MSP and a simple technique for accurate analysis of DNA sequences. PMID:26885283

  1. Comparison of Different Promoter Methylation Assays in Breast Cancer

    PubMed Central

    Suijkerbuijk, Karijn P. M.; Pan, Xiaojuan; van der Wall, Elsken; van Diest, Paul J.; Vooijs, Marc

    2010-01-01

    Background: Promoter hypermethylation has emerged as a promising cancer biomarker. Currently, a large variety of quantitative and non-quantitative techniques is used to measure methylation in clinical specimens. Here we directly compared three commonly used methylation assays and assessed the influence of tissue fixation, target sequence location and the amount of DNA on their performance. Methods: We used Methylation-Specific PCR (MSP), Quantitative Multiplex MSP (QM-MSP) and Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) to compare methylation of CCND2, SCGB3A1, RARB and RASSF1 on DNA from 40 breast carcinomas. Results: A comparison between MSP and QM-MSP on the same samples showed a high discrepancy: 20% of tumors that showed no methylation in MSP gave >10% methylation in QM-MSP. In contrast, QM-MSP correlated strongly with MS-MLPA when targeting the same sequence in DNA from paraffin embedded as well as fresh frozen tissue. This correlation declined when target sequences were non-overlapping. In titration experiments, MSP and MS-MLPA performed robust with 10 ng of DNA, while QM-MSP was at least ten-fold more sensitive. Conclusion: Despite the difference in molecular basis, QM-MSP and MS-MLPA showed moderate to strong correlations. In contrast, there was a poor concordance between either of these techniques and non-quantitative MSP. For biological samples with scarce DNA, QM-MSP is the method of choice. PMID:20978321

  2. Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.

    PubMed

    Singal, Rakesh; vanWert, Jane M; Ferdinand, Larry

    2002-12-01

    The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex. PMID:12393573

  3. Endothelial glucocorticoid receptor promoter methylation according to dexamethasone sensitivity.

    PubMed

    Mata-Greenwood, Eugenia; Jackson, P Naomi; Pearce, William J; Zhang, Lubo

    2015-10-01

    We have previously shown that in vitro sensitivity to dexamethasone (DEX) stimulation in human endothelial cells is positively regulated by the glucocorticoid receptor (NR3C1, GR). The present study determined the role of differential GR transcriptional regulation in glucocorticoid sensitivity. We studied 25 human umbilical vein endothelial cells (HUVECs) that had been previously characterized as DEX-sensitive (n=15), or resistant (n=10). Real-time PCR analysis of GR 5'UTR mRNA isoforms showed that all HUVECs expressed isoforms 1B, 1C, 1D, 1F, and 1H, and isoforms 1B and 1C were predominantly expressed. DEX-resistant cells expressed higher basal levels of the 5'UTR mRNA isoforms 1C and 1D, but lower levels of the 5'UTR mRNA isoform 1F than DEX-sensitive cells. DEX treatment significantly decreased GRα and GR-1C mRNA isoform expression in DEX-resistant cells only. Reporter luciferase assays indicated that differential GR mRNA isoform expression was not due to differential promoter usage between DEX-sensitive and DEX-resistant cells. Analysis of promoter methylation, however, showed that DEX-sensitive cells have higher methylation levels of promoter 1D and lower methylation levels of promoter 1F than DEX-resistant cells. Treatment with 5-aza-2-deoxycytidine abolished the differential 5'UTR mRNA isoform expression between DEX-sensitive and DEX-resistant cells. Finally, both GRα overexpression and 5-aza-2-deoxycytidine treatment eliminated the differences between sensitivity groups to DEX-mediated downregulation of endothelial nitric oxide synthase (NOS3), and upregulation of plasminogen activator inhibitor 1 (SERPINE1). In sum, human endothelial GR 5'UTR mRNA expression is regulated by promoter methylation with DEX-sensitive and DEX-resistant cells having different GR promoter methylation patterns. PMID:26242202

  4. Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma.

    PubMed

    Gonzalez-Gomez, Pilar; Bello, M Josefa; Inda, M Mar; Alonso, M Eva; Arjona, Dolores; Amiñoso, Cinthia; Lopez-Marin, Isabel; de Campos, Jose M; Sarasa, Jose L; Castresana, Javier S; Rey, Juan A

    2004-09-01

    Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm. PMID:15289853

  5. Methylation of B-hordein genes in barley endosperm is inversely correlated with gene activity and affected by the regulatory gene Lys3.

    PubMed Central

    Sørensen, M B

    1992-01-01

    The methylation status of B-hordein genes in the developing barley endosperm was analyzed by digestion with methylation-sensitive restriction enzymes. Southern blotting revealed specific demethylation of Hpa II sites in DNA from wild-type endosperm, whereas leaf DNA and lys3a mutant endosperm DNA were highly methylated at these sites. Similar methylation patterns were observed at an Ava I site situated at position -260 in the B-hordein promoter. This differential methylation was confirmed by genomic sequencing with ligation-mediated PCR. The analyzed sequence covers most of the B-hordein promoter and includes 10 CpGs from the promoter and 4 CpGs from the adjacent coding region. These sites were all hypomethylated in wild-type endosperm, whereas--except for three partially methylated sites--full methylation was seen in leaf DNA. The four sites in the coding region were partially methylated in lys3a endosperm DNA, but the promoter sites remained highly methylated. The possible role of methylation in the regulatory function of the Lys3 gene product is discussed. Images PMID:1570338

  6. Promoter Methylation of PTEN Is a Significant Prognostic Factor in Melanoma Survival.

    PubMed

    Roh, Mi Ryung; Gupta, Sameer; Park, Kyu-Hyun; Chung, Kee Yang; Lauss, Martin; Flaherty, Keith T; Jönsson, Göran; Rha, Sun Young; Tsao, Hensin

    2016-05-01

    Structural compromise of the tumor suppressor gene, phosphatase and tensin homolog (PTEN), occurs in 10% of melanoma specimens, and loss of PTEN expression through DNA methylation of the PTEN promoter region has also been reported in a number of other malignancies. However, the role of PTEN promoter methylation in melanoma is not well understood. We thus sought to elucidate the prevalence of PTEN promoter methylation in melanoma specimens, its relationship to clinical features, and its impact on the outcome of patients with melanoma. PTEN promoter methylation data were acquired from an archived primary Korean melanoma cohort (KMC) of 158 patients and, for validation, 234 patients from The Cancer Genome Atlas melanoma (TCGA-MEL) cohort. Hierarchical clustering was performed to identify PTEN "high methylated" and "low methylated" samples. Subsequently, differences in clinical features and outcomes based on PTEN promoter methylation status were then analyzed using SPSS and R. In the KMC, all tumors were acquired from primary tumors and 65.7% (n = 105) were acral or mucosal by site, whereas in the TCGA-MEL cohort, 90.5% of the tumors were from regional lymph node and distant metastatic lesions. Overall, 17.7% and 45.7% of the specimens harbored BRAF mutations in the KMC and TCGA-MEL cohort, respectively. Neuroblastoma RAS viral oncogene homolog was mutated in 12.2% and 26.9% of the tumors in the KMC and TCGA-MEL cohort, respectively. In the KMC, 31 cases (19.6%) were included in the high methylated group versus 142 cases (60.7%) in the TCGA-MEL cohort (P < 0.001). Multivariate Cox-regression analysis revealed promoter methylation of PTEN to be an independent negative prognostic factor for survival in both the KMC (hazard ratio 3.76, 95% confidence interval = 1.24-11.12, P = 0.017) and TCGA-MEL cohort (HR 1.88, 95% confidence interval = 1.13-3.12, P = 0.015). Our results indicate that PTEN promoter methylation is an independent predictor for impaired survival in

  7. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    PubMed

    Siegel, Erin M; Riggs, Bridget M; Delmas, Amber L; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated. PMID:25826459

  8. Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

    PubMed Central

    Siegel, Erin M.; Riggs, Bridget M.; Delmas, Amber L.; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D.

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated. PMID:25826459

  9. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  10. The Homeobox Gene MEIS1 Is Methylated in BRAFp.V600E Mutated Colon Tumors

    PubMed Central

    Dihal, Ashwin A.; Boot, Arnoud; van Roon, Eddy H.; Schrumpf, Melanie; Fariña-Sarasqueta, Arantza; Fiocco, Marta; Zeestraten, Eliane C. M.; Kuppen, Peter J. K.; Morreau, Hans; van Wezel, Tom; Boer, Judith M.

    2013-01-01

    Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAFp.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAFp.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAFp.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAFp.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAFp.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAFp.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAFp.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression. PMID:24244575

  11. Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

    PubMed Central

    2011-01-01

    Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease. PMID:21669002

  12. Inflammatory and steroid receptor gene methylation in the human amnion and decidua.

    PubMed

    Mitchell, Carolyn M; Sykes, Shane D; Pan, Xin; Pringle, Kirsty G; Lumbers, Eugenie R; Hirst, Jonathan J; Zakar, Tamas

    2013-04-01

    Correct timing of parturition requires inflammatory gene activation in the gestational tissues at term and repression during pregnancy. Promoter methylation at CpG dinucleotides represses gene activity; therefore, we examined the possibility that DNA methylation is involved in the regulation of labour-associated genes in human pregnancy. Amnion and decidua were collected at 11-17 weeks of gestation and at term following elective Caesarean delivery or spontaneous labour. Methylation of the inflammatory genes PTGS2, BMP2, NAMPT and CXCL2 was analysed using the Methyl-Profiler PCR System and bisulphite sequencing. Methylation of the glucocorticoid, progesterone and oestrogen receptor genes, involved in the hormonal regulation of gestational tissue function, and the expression of the DNA methyltransferases DNMT1, -3A and -3B were also determined. Variable proportions of inflammatory and steroid receptor gene copies, to a maximum of 50.9%, were densely methylated in both tissues consistent with repression. Densely methylated copy proportions were significantly different between genes showing no relationship with varying expression during pregnancy, between tissues and in individuals. Methylated copy proportions of all genes in amnion and most genes in decidua were highly correlated in individuals. DNMT1 and -3A were expressed in both tissues with significantly higher levels in the amnion at 11-17 weeks than at term. We conclude that the unmethylated portion of gene copies is responsible for the full range of regulated expression in the amnion and decidua during normal pregnancy. Dense methylation of individually variable gene copy proportions happens in the first trimester amnion influenced by sequence context and affected strongly by individual circumstances. PMID:23393306

  13. MGMT promoter methylation and glioblastoma: a comparison of analytical methods and of tumor specimens.

    PubMed

    Lattanzio, Laura; Borgognone, Marzia; Mocellini, Cristina; Giordano, Fabrizio; Favata, Ermanno; Fasano, Gaetano; Vivenza, Daniela; Monteverde, Martino; Tonissi, Federica; Ghiglia, Annalisa; Fillini, Claudia; Bernucci, Claudio; Merlano, Marco; Lo Nigro, Cristiana

    2015-01-01

    It is already well known that hypermethylation of the O6-methylguanine DNA methyltransferase (MGMT) gene promoter is a predictive biomarker of response to temozolomide treatment and of favorable outcomes in terms of overall survival (OS) and progression-free survival (PFS) in glioblastoma (GBM) patients. Nevertheless, MGMT methylation status has not currently been introduced into routine clinical practice, as the choice of the ideal technique and tissue sample specimen is still controversial. The aim of this study was to compare 2 analytical methods, methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), and their use on 2 different tissue type samples, snap-frozen and formalin-fixed paraffin-embedded (FFPE), obtained from a single-center and uniformly treated cohort of 46 GBM patients. We obtained methylation data from all frozen tissues, while no results were obtained for 5 FFPE samples. The highest concordance for methylation was found on frozen tissues (88.5%, 23/26 samples), using PSQ (76.7%, 23/30 samples). Moreover, we confirmed that OS and PFS for patients carrying methylation of the MGMT promoter were longer than for patients with an unmethylated promoter. In conclusion, we considered MSP a limited technique for FFPE tissues due to the high risk of false-positive results; in contrast, our data indicated PSQ as the most powerful method to stratify methylated/unmethylated patients as it allows reaching quantitative results with high sensitivity and specificity. Furthermore, frozen tumor tissues were shown to be the best specimens for MGMT methylation analysis, due to the low DNA degradation and homogeneity in methylation throughout the tumor. PMID:25588856

  14. BDNF promoter methylation and genetic variation in late-life depression.

    PubMed

    Januar, V; Ancelin, M-L; Ritchie, K; Saffery, R; Ryan, J

    2015-01-01

    The regulation of the brain-derived neurotrophic factor (BDNF) is important for depression pathophysiology and epigenetic regulation of the BDNF gene may be involved. This study investigated whether BDNF methylation is a marker of depression. One thousand and twenty-four participants were recruited as part of a longitudinal study of psychiatric disorders in general population elderly (age ⩾ 65). Clinical levels of depression were assessed using the Mini International Neuropsychiatric Interview for the diagnosis of major depressive disorder according to the Diagnostic and Statistical Manual of Mental Disorder IV criteria, and the Centre for Epidemiologic Studies Depression Scale (CES-D) for assessment of moderate to severe depressive symptoms. Buccal DNA methylation at the two most widely studied BDNF promoters, I and IV, was investigated using the Sequenom MassARRAY platform that allows high-throughput investigation of methylation at individual CpG sites within defined genomic regions. In multivariate linear regression analyses adjusted for a range of participant characteristics including antidepressant use, depression at baseline, as well as chronic late-life depression over the 12-year follow-up, were associated with overall higher BDNF methylation levels, with two sites showing significant associations (promoter I, Δ mean = 0.4%, P = 0.0002; promoter IV, Δ mean = 5.4%, P = 0.021). Three single-nucleotide polymorphisms (rs6265, rs7103411 and rs908867) were also found to modify the association between depression and promoter I methylation. As one of the largest epigenetic studies of depression, and the first investigating BDNF methylation in buccal tissue, our findings highlight the potential for buccal BDNF methylation to be a biomarker of depression. PMID:26285129

  15. Global and gene specific DNA methylation changes during zebrafish development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA methylation is dynamic through the life of an organism. In this study, we measured the global and gene specific DNA methylation changes in zebrafish at different developmental stages. We found that the methylation percentage of cytosines was 11.75 ± 0.96% in 3.3 hour post fertilization (hpf) zeb...

  16. Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates.

    PubMed

    Long, Hannah K; Sims, David; Heger, Andreas; Blackledge, Neil P; Kutter, Claudia; Wright, Megan L; Grützner, Frank; Odom, Duncan T; Patient, Roger; Ponting, Chris P; Klose, Robert J

    2013-01-01

    Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation on chromatin. In cold-blooded vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution. DOI:http://dx.doi.org/10.7554/eLife.00348.001. PMID:23467541

  17. Incomplete methylation of the FMR gene in amniotic cells

    SciTech Connect

    Skare, J.C.; Townes, P.L.

    1994-09-01

    Fragile X mental retardation is usually caused by expansion of triplet repeats near the 5{prime} end of the FMR gene. It has been reported that expansions over 600 bp (full mutations) result in mental retardation of males. Furthermore, FMR genes with full mutations have methylation of certain CpG dinucleotides upstream of the gene, one of which is in an Eag I recognition site. Methylation of the Eag I site correlates with transcriptional inactivation. We report a pregnancy with twin males which were shown to be dizygotic by RFLP analysis. The mother possessed an expansion of 150 bp in one of her FMR genes. Amniocentesis was performed. One fetus had an FMR gene with a 600 bp expansion and the other had a heterogeneous expansion with an average of 1100 bp. The gene with a 600 bp expansion had no methylation of its Eag I site, while about half of the FMR genes with the 1100 bp expansion had methylated Eag I sites. At birth, peripheral blood DNA was examined. The extent of methylation in the newborn with the 600 bp expansion had increased to about 50%. The newborn with the 1000 bp expansion was almost completely methylated. Therefore, methylation of FMR genes progressed prenatally in both, fetuses, and the larger expansion was methylated earliest. Furthermore, it would appear that methylation analysis is of limited value in prenatal diagnosis of fragile X mental retardation.

  18. Arabidopsis PAI gene arrangements, cytosine methylation and expression.

    PubMed Central

    Melquist, S; Luff, B; Bender, J

    1999-01-01

    Previous analysis of the PAI tryptophan biosynthetic gene family in Arabidopsis thaliana revealed that the Wassilewskija (WS) ecotype has four PAI genes at three unlinked sites: a tail-to-tail inverted repeat at one locus (PAI1-PAI4) plus singlet genes at two other loci (PAI2 and PAI3). The four WS PAI genes are densely cytosine methylated over their regions of DNA identity. In contrast, the Columbia (Col) ecotype has three singlet PAI genes at the analogous loci (PAI1, PAI2, and PAI3) and no cytosine methylation. To understand the mechanism of PAI gene duplication at the polymorphic PAI1 locus, and to investigate the relationship between PAI gene arrangement and PAI gene methylation, we analyzed 39 additional ecotypes of Arabidopsis. Six ecotypes had PAI arrangements similar to WS, with an inverted repeat and dense PAI methylation. All other ecotypes had PAI arrangements similar to Col, with no PAI methylation. The novel PAI-methylated ecotypes provide insights into the mechanisms underlying PAI gene duplication and methylation, as well as the relationship between methylation and gene expression. PMID:10471722

  19. Arabidopsis PAI gene arrangements, cytosine methylation and expression.

    PubMed

    Melquist, S; Luff, B; Bender, J

    1999-09-01

    Previous analysis of the PAI tryptophan biosynthetic gene family in Arabidopsis thaliana revealed that the Wassilewskija (WS) ecotype has four PAI genes at three unlinked sites: a tail-to-tail inverted repeat at one locus (PAI1-PAI4) plus singlet genes at two other loci (PAI2 and PAI3). The four WS PAI genes are densely cytosine methylated over their regions of DNA identity. In contrast, the Columbia (Col) ecotype has three singlet PAI genes at the analogous loci (PAI1, PAI2, and PAI3) and no cytosine methylation. To understand the mechanism of PAI gene duplication at the polymorphic PAI1 locus, and to investigate the relationship between PAI gene arrangement and PAI gene methylation, we analyzed 39 additional ecotypes of Arabidopsis. Six ecotypes had PAI arrangements similar to WS, with an inverted repeat and dense PAI methylation. All other ecotypes had PAI arrangements similar to Col, with no PAI methylation. The novel PAI-methylated ecotypes provide insights into the mechanisms underlying PAI gene duplication and methylation, as well as the relationship between methylation and gene expression. PMID:10471722

  20. Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

    PubMed Central

    LING, SHIZHANG; RETTIG, ELENI M.; TAN, MARIETTA; CHANG, XIAOFEI; WANG, ZHIMING; BRAIT, MARIANA; BISHOP, JUSTIN A.; FERTIG, ELANA J.; CONSIDINE, MICHAEL; WICK, MICHAEL J.; HA, PATRICK K.

    2016-01-01

    Salivary gland adenoid cystic carcinoma (ACC) is a rare head and neck malignancy without molecular biomarkers that can be used to predict the chemotherapeutic response or prognosis of ACC. The regulation of gene expression of oncogenes and tumor suppressor genes (TSGs) through DNA promoter methylation may play a role in the carcinogenesis of ACC. To identify differentially methylated genes in ACC, a global demethylating agent, 5-aza-2′-deoxycytidine (5-AZA) was utilized to unmask putative TSG silencing in ACC xenograft models in mice. Fresh xenografts were passaged, implanted in triplicate in mice that were treated with 5-AZA daily for 28 days. These xenografts were then evaluated for genome-wide DNA methylation patterns using the Illumina Infinium HumanMethylation27 BeadChip array. Validation of the 32 candidate genes was performed by bisulfite sequencing (BS-seq) in a separate cohort of 6 ACC primary tumors and 6 normal control salivary gland tissues. Hypermethylation was identified in the HCN2 gene promoter in all 6 control tissues, but hypomethylation was found in all 6 ACC tumor tissues. Quantitative validation of HCN2 promoter methylation level in the region detected by BS-seq was performed in a larger cohort of primary tumors (n=32) confirming significant HCN2 hypomethylation in ACCs compared with normal samples (n=10; P=0.04). HCN2 immunohistochemical staining was performed on an ACC tissue microarray. HCN2 staining intensity and H-score, but not percentage of the positively stained cells, were significantly stronger in normal tissues than those of ACC tissues. With our novel screening and sequencing methods, we identified several gene candidates that were methylated. The most significant of these genes, HCN2, was actually hypomethylated in tumors. However, promoter methylation status does not appear to be a major determinant of HCN2 expression in normal and ACC tissues. HCN2 hypomethylation is a biomarker of ACC and may play an important role in the

  1. The impact of endurance exercise on global and AMPK gene-specific DNA methylation.

    PubMed

    King-Himmelreich, Tanya S; Schramm, Stefanie; Wolters, Miriam C; Schmetzer, Julia; Möser, Christine V; Knothe, Claudia; Resch, Eduard; Peil, Johannes; Geisslinger, Gerd; Niederberger, Ellen

    2016-05-27

    Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression. PMID:27103439

  2. DNA methylation as a regulatory mechanism in rat gamma-crystallin gene expression.

    PubMed Central

    Peek, R; Niessen, R W; Schoenmakers, J G; Lubsen, N H

    1991-01-01

    We have investigated the methylation state of the rat gamma-crystallin genes in DNA from lens cells at different developmental stages as well as from kidney and heart cells. A clear correlation between the extent of demethylation of the promoter and 5' gene regions and the expression of these genes was observed. No change in the methylation state of the far upstream or 3' regions of the genes was seen. The demethylation of the promoter region was shown to occur during the differentiation from the lens epithelial to the lens fiber cell. The effect of cytosine methylation on gamma-crystallin promoter activity was tested by measuring gamma-crystallin promoter/chloramphenicol acetyltransferase fusion gene expression after in vitro primed repair synthesis of the promoter region in the presence of either dCTP or 5mdCTP. The hemimethylated promoter was no longer capable of promoting high CAT activity after introduction into lens-like cells. Taken together, our data suggest that DNA demethylation may be the determining step in the developmental stage-specific expression of the rat gamma-crystallin genes. Images PMID:2011513

  3. Implication of Reprimo and hMLH1 gene methylation in early diagnosis of gastric carcinoma

    PubMed Central

    Liu, Lianhua; Yang, Xiaofeng

    2015-01-01

    DNA methylation has been recently recognized as a novel tumor marker. This study investigated the methylation status of Reprimo and hMLH1 gene in both plasma and tissue samples from gastric cancer patients, in an attempt to investigate their diagnostic implications in gastric cancer. A total of 180 tissue and plasma samples (including 50 cases of gastric cancer, 50 dysplasia, 50 chronic atrophic gastritis with intestinal metaplasia and 30 normal controls) were collected for detecting DNA methylation status of Reprimo and hMLH1 genes using MSP method. Tissue protein expression levels were further tested by immunohistochemical (IHC) staining. The positive rate of DNA methylation rate was, in ascending sequence, gastritis tissue, dysplasia tissue and gastric carcinoma tissue. All those tissues had significantly elevated DNA methylation level compared to normal group (P < 0.05). Expression level of Reprimo and hMLH1 proteins were, however, decreased in pathological tissues compared to normal ones (P < 0.05). A significantly negative relationship existed between protein level and promoter region methylation level. The DNA methylation occurred in promoter regions of both Reprimo and hMLH1 genes depressed the protein expression, and may participate in the occurrence and progression and gastric cancer. The combined assay of serum Reprimo and hMLH1 DNA methylation levels thus had critical importance in the early diagnosis and gastric cancer. PMID:26823831

  4. A mechanistic role for DNA methylation in endothelial cell (EC)-enriched gene expression: relationship with DNA replication timing

    PubMed Central

    Shirodkar, Apurva V.; St. Bernard, Rosanne; Gavryushova, Anna; Kop, Anna; Knight, Britta J.; Yan, Matthew Shu-Ching; Man, Hon-Sum Jeffrey; Sud, Maneesh; Hebbel, Robert P.; Oettgen, Peter; Aird, William C.; Marsden, Philip A.

    2013-01-01

    Proximal promoter DNA methylation has been shown to be important for regulating gene expression. However, its relative contribution to the cell-specific expression of endothelial cell (EC)-enriched genes has not been defined. We used methyl-DNA immunoprecipitation and bisulfite conversion to analyze the DNA methylation profile of EC-enriched genes in ECs vs nonexpressing cell types, both in vitro and in vivo. We show that prototypic EC-enriched genes exhibit functional differential patterns of DNA methylation in proximal promoter regions of most (eg, CD31, von Willebrand factor [vWF], VE-cadherin, and intercellular adhesion molecule-2), but not all (eg, VEGFR-1 and VEGFR-2), EC-enriched genes. Comparable findings were evident in cultured ECs, human blood origin ECs, and murine aortic ECs. Promoter-reporter episomal transfection assays for endothelial nitric oxide synthase, VE-cadherin, and vWF indicated functional promoter activity in cell types where the native gene was not active. Inhibition of DNA methyltransferase activity indicated important functional relevance. Importantly, profiling DNA replication timing patterns indicated that EC-enriched gene promoters with differentially methylated regions replicate early in S-phase in both expressing and nonexpressing cell types. Collectively, these studies highlight the functional importance of promoter DNA methylation in controlling vascular EC gene expression. PMID:23449636

  5. DNA Methylation is Developmentally Regulated for Genes Essential for Cardiogenesis

    PubMed Central

    Chamberlain, Alyssa A.; Lin, Mingyan; Lister, Rolanda L.; Maslov, Alex A.; Wang, Yidong; Suzuki, Masako; Wu, Bingruo; Greally, John M.; Zheng, Deyou; Zhou, Bin

    2014-01-01

    Background DNA methylation is a major epigenetic mechanism altering gene expression in development and disease. However, its role in the regulation of gene expression during heart development is incompletely understood. The aim of this study is to reveal DNA methylation in mouse embryonic hearts and its role in regulating gene expression during heart development. Methods and Results We performed the genome‐wide DNA methylation profiling of mouse embryonic hearts using methyl‐sensitive, tiny fragment enrichment/massively parallel sequencing to determine methylation levels at ACGT sites. The results showed that while global methylation of 1.64 million ACGT sites in developing hearts remains stable between embryonic day (E) 11.5 and E14.5, a small fraction (2901) of them exhibit differential methylation. Gene Ontology analysis revealed that these sites are enriched at genes involved in heart development. Quantitative real‐time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2). Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer. Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co‐expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function. Conclusions DNA methylation is developmentally regulated for genes essential to heart development, and abnormal DNA methylation may contribute to congenital heart disease. PMID:24947998

  6. Divergence of Gene Body DNA Methylation and Evolution of Plant Duplicate Genes

    PubMed Central

    Wang, Jun; Marowsky, Nicholas C.; Fan, Chuanzhu

    2014-01-01

    It has been shown that gene body DNA methylation is associated with gene expression. However, whether and how deviation of gene body DNA methylation between duplicate genes can influence their divergence remains largely unexplored. Here, we aim to elucidate the potential role of gene body DNA methylation in the fate of duplicate genes. We identified paralogous gene pairs from Arabidopsis and rice (Oryza sativa ssp. japonica) genomes and reprocessed their single-base resolution methylome data. We show that methylation in paralogous genes nonlinearly correlates with several gene properties including exon number/gene length, expression level and mutation rate. Further, we demonstrated that divergence of methylation level and pattern in paralogs indeed positively correlate with their sequence and expression divergences. This result held even after controlling for other confounding factors known to influence the divergence of paralogs. We observed that methylation level divergence might be more relevant to the expression divergence of paralogs than methylation pattern divergence. Finally, we explored the mechanisms that might give rise to the divergence of gene body methylation in paralogs. We found that exonic methylation divergence more closely correlates with expression divergence than intronic methylation divergence. We show that genomic environments (e.g., flanked by transposable elements and repetitive sequences) of paralogs generated by various duplication mechanisms are associated with the methylation divergence of paralogs. Overall, our results suggest that the changes in gene body DNA methylation could provide another avenue for duplicate genes to develop differential expression patterns and undergo different evolutionary fates in plant genomes. PMID:25310342

  7. Distributional changes in gene-specific methylation associated with temperature.

    PubMed

    Bind, Marie-Abele C; Coull, Brent A; Baccarelli, Andrea; Tarantini, Letizia; Cantone, Laura; Vokonas, Pantel; Schwartz, Joel

    2016-10-01

    Temperature has been related to mean differences in DNA methylation. However, heterogeneity in these associations may exist across the distribution of methylation outcomes. This study examined whether the association between three-week averaged of temperature and methylation differs across quantiles of the methylation distributions in nine candidate genes. We measured gene-specific blood methylation repeatedly in 777 elderly men participating in the Normative Aging Study (1999-2010). We fit quantile regressions for longitudinal data to investigate whether the associations of temperature on methylation (expressed as %5mC) varied across the distribution of the methylation outcomes. We observed heterogeneity in the associations of temperature across percentiles of methylation in F3, TLR-2, CRAT, iNOS, and ICAM-1 genes. For instance, an increase in three-week temperature exposure was associated with a longer left-tail of the F3 methylation distribution. A 5°C increase in temperature was associated with a 0.15%5mC (95% confidence interval (CI): -0.27,-0.04) decrease on the 20th quantile of F3 methylation, but was not significantly related to the 80th quantile of this distribution (Estimate:0.06%5mC, 95%CI: -0.22, 0.35). Individuals with low values of F3, TLR-2, CRAT, and iNOS methylation, as well as a high value of ICAM-1 methylation, may be more susceptible to temperature effects on systemic inflammation. PMID:27236570

  8. NANOG promoter methylation and expression correlation during normal and malignant human germ cell development

    PubMed Central

    Nettersheim, Daniel; Bierman, Katharina; Gillis, Ad JM; Steger, Klaus; Looijenga, Leendert HJ

    2011-01-01

    Testicular germ cell tumors are the most frequent malignant tumors in young Caucasian males, with increasing incidence. The actual model of tumorigenesis is based on the theory that a block in maturation of fetal germ cells lead to formation of the intratubular germ cell neoplasia unclassified. Early fetal germ cells and undifferentiated germ cell tumors express pluripotency markers such as the transcription factor NANOG. It has been demonstrated that epigenetic modifications, such as promoter DNA methylation, are able to silence gene expression in normal and cancer cells. Here we show that OCT3/4-SOX2 mediated expression of NANOG can be silenced by methylation of promoter CpG-sites. We found that global methylation of DNA decreased from fetal spermatogonia to mature sperm. In contrast, CpGs in the NANOG promoter were found hypomethylated in spermatogonia and hypermethylated in sperm. This selective repression might reflect the cells need to suppress pluripotency in order to prevent malignant transformation. Finally, methylation of CpGs in the NANOG promoter in germ cell tumors and derived cell lines correlated to differentiation state. PMID:20930529

  9. Genome-wide methylation profiling identifies novel methylated genes in neuroblastoma tumors

    PubMed Central

    Olsson, Maja; Beck, Stephan; Kogner, Per; Martinsson, Tommy; Carén, Helena

    2016-01-01

    ABSTRACT Neuroblastoma is a very heterogeneous tumor of childhood. The clinical spectra range from very aggressive metastatic disease to spontaneous regression, even without therapy. Aberrant DNA methylation pattern is a common feature of most cancers. For neuroblastoma, it has been demonstrated both for single genes as well as genome-wide, where a so-called methylator phenotype has been described. Here, we present a study using Illumina 450K methylation arrays on 60 neuroblastoma tumors. We show that aggressive tumors, characterized by International Neuroblastoma Risk Group (INRG) as stage M, are hypermethylated compared to low-grade tumors. On the contrary, INRG stage L tumors display more non-CpG methylation. The genes with the highest number of hypermethylated CpG sites in INRG M tumors are TERT, PCDHGA4, DLX5, and DLX6-AS1. Gene ontology analysis showed a representation of neuronal tumor relevant gene functions among the differentially methylated genes. For validation, we used a set of independent tumors previously analyzed with the Illumina 27K methylation arrays, which confirmed the differentially methylated sites. Top candidate genes with aberrant methylation were analyzed for altered gene expression through the R2 platform (http://r2.amc.nl), and for correlations between methylation and gene expression in a public dataset. Altered expression in nonsurvivors was found for the genes B3GALT4 and KIAA1949, CLIC5, DLX6-AS, TERT, and PIRT, and strongest correlations were found for TRIM36, KIAA0513, and PIRT. Our data indicate that methylation profiling can be used for patient stratification and informs on epigenetically deregulated genes with the potential of increasing our knowledge about the underlying mechanisms of tumor development. PMID:26786290

  10. A Genome-Wide Methylation Approach Identifies a New Hypermethylated Gene Panel in Ulcerative Colitis.

    PubMed

    Kang, Keunsoo; Bae, Jin-Han; Han, Kyudong; Kim, Eun Soo; Kim, Tae-Oh; Yi, Joo Mi

    2016-01-01

    The cause of inflammatory bowel disease (IBD) is still unknown, but there is growing evidence that environmental factors such as epigenetic changes can contribute to the disease etiology. The aim of this study was to identify newly hypermethylated genes in ulcerative colitis (UC) using a genome-wide DNA methylation approach. Using an Infinium HumanMethylation450 BeadChip array, we screened the DNA methylation changes in three normal colon controls and eight UC patients. Using these methylation profiles, 48 probes associated with CpG promoter methylation showed differential hypermethylation between UC patients and normal controls. Technical validations for methylation analyses in a larger series of UC patients (n = 79) were performed by methylation-specific PCR (MSP) and bisulfite sequencing analysis. We finally found that three genes (FAM217B, KIAA1614 and RIBC2) that were significantly elevating the promoter methylation levels in UC compared to normal controls. Interestingly, we confirmed that three genes were transcriptionally silenced in UC patient samples by qRT-PCR, suggesting that their silencing is correlated with the promoter hypermethylation. Pathway analyses were performed using GO and KEGG databases with differentially hypermethylated genes in UC. Our results highlight that aberrant hypermethylation was identified in UC patients which can be a potential biomarker for detecting UC. Moreover, pathway-enriched hypermethylated genes are possibly implicating important cellular function in the pathogenesis of UC. Overall, this study describes a newly hypermethylated gene panel in UC patients and provides new clinical information that can be used for the diagnosis and therapeutic treatment of IBD. PMID:27517910

  11. A Genome-Wide Methylation Approach Identifies a New Hypermethylated Gene Panel in Ulcerative Colitis

    PubMed Central

    Kang, Keunsoo; Bae, Jin-Han; Han, Kyudong; Kim, Eun Soo; Kim, Tae-Oh; Yi, Joo Mi

    2016-01-01

    The cause of inflammatory bowel disease (IBD) is still unknown, but there is growing evidence that environmental factors such as epigenetic changes can contribute to the disease etiology. The aim of this study was to identify newly hypermethylated genes in ulcerative colitis (UC) using a genome-wide DNA methylation approach. Using an Infinium HumanMethylation450 BeadChip array, we screened the DNA methylation changes in three normal colon controls and eight UC patients. Using these methylation profiles, 48 probes associated with CpG promoter methylation showed differential hypermethylation between UC patients and normal controls. Technical validations for methylation analyses in a larger series of UC patients (n = 79) were performed by methylation-specific PCR (MSP) and bisulfite sequencing analysis. We finally found that three genes (FAM217B, KIAA1614 and RIBC2) that were significantly elevating the promoter methylation levels in UC compared to normal controls. Interestingly, we confirmed that three genes were transcriptionally silenced in UC patient samples by qRT-PCR, suggesting that their silencing is correlated with the promoter hypermethylation. Pathway analyses were performed using GO and KEGG databases with differentially hypermethylated genes in UC. Our results highlight that aberrant hypermethylation was identified in UC patients which can be a potential biomarker for detecting UC. Moreover, pathway-enriched hypermethylated genes are possibly implicating important cellular function in the pathogenesis of UC. Overall, this study describes a newly hypermethylated gene panel in UC patients and provides new clinical information that can be used for the diagnosis and therapeutic treatment of IBD. PMID:27517910

  12. P04.18PROGNOSIS IMPACT OF THE REGIONAL DISTRIBUTION OF MGMT GENE METHYLATION ACCORDING TO THE CPGISLAND METHYLATOR PHENOTYPE AND AGE IN HIGH-GRADE GLIOMAS

    PubMed Central

    Mur, P.; de Lope, A. Rodriguez; Hernandez-Iglesias, T.; Diaz, F.; Ribalta, T.; Fiaño, C.; Garcia, J.F.; Rey, J.A.; Mollejo, M.; Meléndez, B.

    2014-01-01

    Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP) and promoter methylation of the O6-methylguanine DNA-methyltransferase (MGMT) gene, among others. Clinical trials supported the predictive value of MGMT promoter methylation for benefit from alkylating chemotherapy in elderly GBM patients. In this study, methylation data were obtained from 46 oligodendroglial samples with the Illumina 450K platform, and were analyzed with external data to reach a total 247 glioma samples. MGMT gene methylation analysis with this platform revealed two significant survival-associated CpG regions, one within the promoter (cg12981137) and the other within the gene body (cg07933035), both significantly associated with better overall survival (OS) and strongly correlated with the G-CIMP+ status. However, although around 50% of G-CIMP- tumors were MGMT methylated on these CpG sites, their prognostic relevance were not observed in these patients. Only the gene body methylation was prognostic, but in the context of age, showing significant differences of OS in elderly patients. The absence of the MGMT promoter prognostic value in G-CIMP- tumors was validated in an independent series of 59 chemoradiated GBM patients by MSP and qMSP assays. Our study suggests that the prognostic value of MGMT methylation should be reviewed in the context of specific G-CIMP profiles and age groups. Further analysis on the impact of MGMT methylation on gene and protein expression is necessary for better clinical treatment settings. The routine use of MGMT methylation for the individual treatment of patients should be still viewed with caution.

  13. Chromatin inactivation precedes de novo dna methylation during the progressive epigenetic silencing of the rassf1a promoter

    SciTech Connect

    Strunnikova Maria; Schagdarsurengin, Undraga; Kehlen, Astrid; Garbe, James C.; Stampfer, Martha R.; Dammann, Reinhard

    2005-02-23

    Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1 A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.

  14. CMTM5 exhibits tumor suppressor activity through promoter methylation in oral squamous cell carcinoma

    SciTech Connect

    Zhang, Heyu; Nan, Xu; Li, Xuefen; Chen, Yan; Zhang, Jianyun; Sun, Lisha; Han, Wenlin; Li, Tiejun

    2014-05-02

    Highlights: • Down-regulation of CMTM5 expression in OSCC tissues was found. • The promoter methylation status of CMTM5 was measured. • CMTM5-v1 inhibited cell proliferation and migration and induced apoptosis. • CMTM5 might act as a putative tumor suppressor gene in OSCC. - Abstract: Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancies in the head and neck region. CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) has been recently implicated as a tumor suppressor gene in several cancer types. Herein, we examined the expression and function of CMTM5 in oral squamous cell carcinoma. CMTM5 was down-regulated in oral squamous cell lines and tumor samples from patients with promoter methylation. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored CMTM5 expression. In the OSCC cell lines CAL27 and GNM, the ectopic expression of CMTM5-v1 strongly inhibited cell proliferation and migration and induced apoptosis. In addition, CMTM5-v1 inhibited tumor formation in vivo. Therefore, CMTM5 might act as a putative tumor suppressor gene through promoter methylation in oral squamous cell carcinoma.

  15. Identification of a DNA methylation-dependent activator sequence in the pseudoxanthoma elasticum gene, ABCC6.

    PubMed

    Arányi, Tamás; Ratajewski, Marcin; Bardóczy, Viola; Pulaski, Lukasz; Bors, András; Tordai, Attila; Váradi, András

    2005-05-13

    ABCC6 encodes MRP6, a member of the ABC protein family with an unknown physiological role. The human ABCC6 and its two pseudogenes share 99% identical DNA sequence. Loss-of-function mutations of ABCC6 are associated with the development of pseudoxanthoma elasticum (PXE), a recessive hereditary disorder affecting the elastic tissues. Various disease-causing mutations were found in the coding region; however, the mutation detection rate in the ABCC6 coding region of bona fide PXE patients is only approximately 80%. This suggests that polymorphisms or mutations in the regulatory regions may contribute to the development of the disease. Here, we report the first characterization of the ABCC6 gene promoter. Phylogenetic in silico analysis of the 5' regulatory regions revealed the presence of two evolutionarily conserved sequence elements embedded in CpG islands. The study of DNA methylation of ABCC6 and the pseudogenes identified a correlation between the methylation of the CpG island in the proximal promoter and the ABCC6 expression level in cell lines. Both activator and repressor sequences were uncovered in the proximal promoter by reporter gene assays. The most potent activator sequence was one of the conserved elements protected by DNA methylation on the endogenous gene in non-expressing cells. Finally, in vitro methylation of this sequence inhibits the transcriptional activity of the luciferase promoter constructs. Altogether these results identify a DNA methylation-dependent activator sequence in the ABCC6 promoter. PMID:15760889

  16. RING finger protein 4 (RNF4) derepresses gene expression from DNA methylation.

    PubMed

    Wang, Yu

    2014-12-01

    RNF4 is an E3 ubiquitin ligase originally identified as a transcription co-activator. The mechanism by which RNF4 promotes transcription remains unclear. In this study, I found that RNF4 antagonizes transcriptional repression mediated by DNA methylation. RNF4 does not promote DNA demethylation, but mediates the ubiquitination of MeCP2, a methyl-CpG-binding domain (MBD) protein. Removal of MeCP2 from gene promoters activates transcription. This study thus not only uncovers how RNF4 functions as a transcription activator, but also reveals the mechanism by which MeCP2 protein stability is regulated. PMID:25355316

  17. Association of Cigarette Smoking with Aberrant Methylation of the Tumor Suppressor Gene RARβ2 in Papillary Thyroid Cancer.

    PubMed

    Kiseljak-Vassiliades, Katja; Xing, Mingzhao

    2011-01-01

    Aberrant gene methylation is often seen in thyroid cancer, a common endocrine malignancy. Tobacco smoking has been shown to be associated with aberrant gene methylation in several cancers, but its relationship with gene methylation in thyroid cancer has not been examined. In the present study, we investigated the relationship between smoking of patients and aberrant methylation of tumor suppressor genes for TIMP3, SLC5A8, death-associated protein kinase, and retinoic acid receptor β2 (RARβ2) in papillary thyroid cancer (PTC), the most common type of thyroid cancer. The promoter methylation status of these genes was analyzed using quantitative real-time methylation-specific PCR on bisulfite-treated genomic DNA isolated from tumor tissues and correlated with smoking history of the patients. Among the four genes, methylation of the RARβ2 gene was significantly associated with smoking and other three genes showed a trend of association. Specifically, among the 138 patients investigated, 13/42 (31.0%) ever smokers vs. 10/96 (10.4%) never smokers harbored methylation of the RARβ2 gene (P = 0.003). This association was highly significant also in the subset of conventional variant PTC (P = 0.005) and marginally significant in follicular variant PTC (P = 0.06). The results demonstrate that smoking-associated aberrant methylation of the RARβ2 gene is a specific molecular event that may represent an important mechanism in thyroid tumorigenesis in smokers. PMID:22649395

  18. Towards understanding the breast cancer epigenome: a comparison of genome-wide DNA methylation and gene expression data

    PubMed Central

    Michiels, Stefan; Metzger-Filho, Otto; Saini, Kamal S.

    2016-01-01

    Until recently, an elevated disease risk has been ascribed to a genetic predisposition, however, exciting progress over the past years has discovered alternate elements of inheritance that involve epigenetic regulation. Epigenetic changes are heritably stable alterations that include DNA methylation, histone modifications and RNA-mediated silencing. Aberrant DNA methylation is a common molecular basis for a number of important human diseases, including breast cancer. Changes in DNA methylation profoundly affect global gene expression patterns. What is emerging is a more dynamic and complex association between DNA methylation and gene expression than previously believed. Although many tools have already been developed for analyzing genome-wide gene expression data, tools for analyzing genome-wide DNA methylation have not yet reached the same level of refinement. Here we provide an in-depth analysis of DNA methylation in parallel with gene expression data characteristics and describe the particularities of low-level and high-level analyses of DNA methylation data. Low-level analysis refers to pre-processing of methylation data (i.e. normalization, transformation and filtering), whereas high-level analysis is focused on illustrating the application of the widely used class comparison, class prediction and class discovery methods to DNA methylation data. Furthermore, we investigate the influence of DNA methylation on gene expression by measuring the correlation between the degree of CpG methylation and the level of expression and to explore the pattern of methylation as a function of the promoter region. PMID:26657508

  19. Methylation of the RARB Gene Increases Prostate Cancer Risk in Black Americans

    PubMed Central

    Tang, Deliang; Kryvenko, Oleksandr N.; Mitrache, Nicoleta; Do, Kieu C.; Jankowski, Michelle; Chitale, Dhananjay A.; Trudeau, Sheri; Rundle, Andrew; Belinsky, Steven A.; Rybicki, Benjamin A.

    2013-01-01

    Purpose Gene promoter hypermethylation may be useful as a biomarker for cancer risk in histopathologically benign prostate specimens. Materials and Methods We performed a nested case-control study of gene promoter methylation status for 5 genes (APC, RARB, CCND2, RASSF1 and MGMT) measured in benign biopsy specimens from 511 prostate cancer case-control pairs. We estimated the overall and race stratified risk of subsequent prostate cancer associated with methylation status. Results On race stratified analysis RARB methylation was associated with a higher cancer risk in black American men (OR 2.18, 95% CI 1.39–3.44). APC methylation was associated with an increased risk of high grade tumors (OR 2.43, 95% CI 1.20–4.90), which was higher in black than in white men (OR 3.21 vs 2.04). In cases RARB and APC gene methylation in benign prostate samples persisted in matched malignant specimens. In black cases the combined risk associated with RARB and APC methylation (OR 3.04, 95% CI 1.44–6.42) was greater than the individual risk of each gene and significantly different from that in white cases (OR 1.14, 95% CI 0.56–2.30). Conclusions RARB gene methylation in histopathologically benign prostate samples was associated with a statistically significant increased risk of subsequent prostate cancer in black men. Methylation data on additional genes may improve risk stratification and clinical decision making algorithms for cancer screening and diagnosis. PMID:23376149

  20. Methylation impact analysis of erythropoietin (EPO) Gene to hypoxia inducible factor-1α (HIF-1α) activity.

    PubMed

    Dewi, Firli Rahmah Primula; Fatchiyah, Fatchiyah

    2013-01-01

    Erythropoietin (EPO) is a glycoprotein hormone that play a role as key regulator in the production of red blood cells. The promoter region of EPO is methylated in normoxic (non-hypoxia) condition, but not in hypoxic condition. Methylation of the EPO enhancer region decline the transcription activity of EPO gene. The aim of this study is to investigate how different methylation percentage affected on the regulation and transcriptional activity of EPO gene. The DNA sequence of erythropoietin gene and protein sequence was retrieved from the sequence database of NCBI. DNA structure was constructed using 3D-DART web server and modeling structure of HIF1 predicted using SWISS-MODEL web server. Methylated DNA sequence of EPO gene using performed with YASARA View software and docking of EPO gene and transcription factor HIF1 analyzed by using HADDOCK webserver. Our result showed that binding energy in 46% methylated DNA was higher (-161,45 kcal/mol) than in unmethylated DNA (-194,16 kcal/mol) and 8% methylated DNA (-175,94 kcal/mol). So, we presume that a silencing mechanism of the Epo gene by methylation is correlated with the binding energy, which is required for interaction. A higher methylation percentage correlates with a higher binding energy which can cause an unstable interaction between DNA and transcription factor. In conclution, methylation of promoter and enhancer region of Epo gene leads to silencing. PMID:24023421

  1. γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells.

    PubMed

    Kim, Sung-Eun; Hinoue, Toshinori; Kim, Michael S; Sohn, Kyoung-Jin; Cho, Robert C; Cole, Peter D; Weisenberger, Daniel J; Laird, Peter W; Kim, Young-In

    2015-01-01

    γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways. PMID:25502219

  2. The Promoter Methylation Status and mRNA Expression Levels of CTCF and SIRT6 in Sporadic Breast Cancer

    PubMed Central

    Wang, Da; Zhang, Xuemei

    2014-01-01

    Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. This study was to detect promoter methylation status and mRNA expression levels of CCCTC-binding factor (CTCF) and sirtuin 6 (SIRT6), and to explore the relationship between methylation and mRNA expression in breast cancer patient samples. Promoter methylation analysis and expression profile analysis of two genes were performed by methylation-specific PCR, bisulfite sequencing PCR, and quantitative real-time PCR in cancer lesions and matched normal tissues. The promoter region of CTCF has not been hypermethylated in all patient samples. In contrast, methylation of SIRT6 gene was present in invasive cancers (93.5%) and matched normal tissues (96.8%) from 62 patients. Promoter hypermethylation of SIRT6 was also observed in ductal carcinoma in situ (three of three) and matched normal tissues (two of three). mRNA expression of CTCF and SIRT6 in invasive tumors showed a lower level than that in paired normal tissues (p=0.008 and p=0.030, respectively). The fold change values of CTCF expression were significantly lower in invasive ductal cancer lesions with Ki-67-positive status (p=0.042). In conclusion, our data showed that the methylation status of CTCF and SIRT6 promoter regions was not statistically different in cancer lesions compared with matched normal tissues. No significant association between promoter methylation status and expression profiles of CTCF and SIRT6 was found in invasive breast cancers. PMID:24842653

  3. Tissue-specific Leptin promoter DNA methylation is associated with maternal and infant perinatal factors.

    PubMed

    Lesseur, Corina; Armstrong, David A; Paquette, Alison G; Koestler, Devin C; Padbury, James F; Marsit, Carmen J

    2013-12-01

    Leptin a regulator of body weight is involved in reproductive and developmental functions. Leptin promoter DNA methylation (LEP) regulates gene expression in a tissue-specific manner and has been linked to adverse pregnancy outcomes. In non-pathologic human pregnancies, we assessed LEP methylation, genotyped the single nucleotide polymorphism (SNP) rs2167270 in placental (n=81), maternal and cord blood samples (n=60), and examined the association between methylation, genotype, and perinatal factors. Maternal blood LEP methylation was lower in pre-pregnancy obese women (P=0.01). Cord blood LEP methylation was higher in small for gestational age (SGA) (P=4.6×10(-3)) and A/A genotype (P=1.6×10(-4)), lower (-1.47, P=0.03) in infants born to pre-pregnancy obese mothers and correlated (P=0.01) with maternal blood LEP. Gender was associated with placental LEP methylation (P=0.05). These results suggest that LEP epigenetic control may be influenced by perinatal factors including: maternal obesity, infant growth, genotype and gender in a tissue-specific manner and may have multigenerational implications. PMID:23911897

  4. Correlation of CCNA1 promoter methylation with malignant tumors: a meta-analysis introduction.

    PubMed

    Yang, Bin; Miao, Shuai; Zhang, Le-Ning; Sun, Hong-Bin; Xu, Zhe-Nan; Han, Chun-Shan

    2015-01-01

    Epigenetic silencing of tumor suppressor genes by promoter methylation plays vital roles in the process of carcinogenesis. The purpose of this meta-analysis was to determine whether the aberrant methylation of cyclin A1 (CCNA1) may be of great significance to human malignant tumors. By searching both English and Chinese language-based electronic databases carefully, we tabulated and analyzed parameters from each study. All human-associated case-control studies were included providing available data for CCNA1 methylation and reporting the adjusted odds ratios (ORs) and 95% confidence intervals (CI) conducted with the use of Version 12.0 STATA software. A total of 10 case-control studies (619 patients with cancers and 292 healthy controls) were included for the following statistical analysis. Pooled OR values from all articles revealed that the frequency of CCNA1 methylation in cancer tissues was significantly higher than those of normal tissues (P < 0.001). Further ethnicity indicated that the frequency of CCNA1 methylation was correlated with the development of malignant tumors among all those included experimental subgroups (all P < 0.05). These data from results indicated a significant connection of CCNA1 methylation with poor progression in human malignant tumors among both Caucasian and Asian populations. PMID:25654082

  5. Transcriptional activity of acetylcholinesterase gene is regulated by DNA methylation during C2C12 myogenesis.

    PubMed

    Lau, Kei M; Gong, Amy G W; Xu, Miranda L; Lam, Candy T W; Zhang, Laura M L; Bi, Cathy W C; Cui, D; Cheng, Anthony W M; Dong, Tina T X; Tsim, Karl W K; Lin, Huangquan

    2016-07-01

    The expression of acetylcholinesterase (AChE), an enzyme hydrolyzes neurotransmitter acetylcholine at vertebrate neuromuscular junction, is regulated during myogenesis, indicating the significance of muscle intrinsic factors in controlling the enzyme expression. DNA methylation is essential for temporal control of myogenic gene expression during myogenesis; however, its role in AChE regulation is not known. The promoter of vertebrate ACHE gene carries highly conserved CG-rich regions, implying its likeliness to be methylated for epigenetic regulation. A DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), was applied onto C2C12 cells throughout the myotube formation. When DNA methylation was inhibited, the promoter activity, transcript expression and enzymatic activity of AChE were markedly increased after day 3 of differentiation, which indicated the putative role of DNA methylation. By bisulfite pyrosequencing, the overall methylation rate was found to peak at day 3 during C2C12 cell differentiation; a SP1 site located at -1826bp upstream of mouse ACHE gene was revealed to be heavily methylated. The involvement of transcriptional factor SP1 in epigenetic regulation of AChE was illustrated here: (i) the SP1-driven transcriptional activity was increased in 5-Aza-treated C2C12 culture; (ii) the binding of SP1 onto the SP1 site of ACHE gene was fully blocked by the DNA methylation; and (iii) the sequence flanking SP1 sites of ACHE gene was precipitated by chromatin immuno-precipitation assay. The findings suggested the role of DNA methylation on AChE transcriptional regulation and provided insight in elucidating the DNA methylation-mediated regulatory mechanism on AChE expression during muscle differentiation. PMID:27021952

  6. FOXA1 positively regulates gene expression by changing gene methylation status in human breast cancer MCF-7 cells

    PubMed Central

    Zheng, Lu; Qian, Bo; Tian, Duo; Tang, Tong; Wan, Shengyun; Wang, Lei; Zhu, Lixin; Geng, Xiaoping

    2015-01-01

    Objective: DNA methylation is an important epigenetic modification with tumor suppressor gene silencing in cancer. The mechanisms underlying DNA methylation patterns are still poorly understood. This study aims to evaluate the potential value of FOXA1 for controlling gene CpG island methylation in breast cancer. Methods: FOXA1 was down-regulated by transfection with siRNA and up-regulated by transfection with plasmid in MCF-7 cell lines. The DNA methylation and mRNA levels were examined by qMSP and qRT-PCR. The cell proliferation and apoptosis was detected by MTT and Flow cytometry. Results: Suppression of FOXA1 enhanced the methylation status of DAPK, MGMT, RASSF1A, p53, and depressed mRNA levels of these tumor suppressor genes, whereas over-expression of FOXA1 showed the opposite effects. DNMT1, DNMT3A and DNMT3B mRNA were up-regulated by siRNA knock-down of FOXA1. At the same time, FOXA1 suppression promoted cell growth and inhibited apoptosis. Conclusions: FOXA1 may be associated with methylation of the tumor suppressor genes promoter through changing DNMTs expression. FOXA1 could be a potential demethylation target for prevention and treatment of breast cancer. PMID:25755696

  7. Methylation of microRNA genes regulates gene expression in bisexual flower development in andromonoecious poplar

    PubMed Central

    Song, Yuepeng; Tian, Min; Ci, Dong; Zhang, Deqiang

    2015-01-01

    Previous studies showed sex-specific DNA methylation and expression of candidate genes in bisexual flowers of andromonoecious poplar, but the regulatory relationship between methylation and microRNAs (miRNAs) remains unclear. To investigate whether the methylation of miRNA genes regulates gene expression in bisexual flower development, the methylome, microRNA, and transcriptome were examined in female and male flowers of andromonoecious poplar. 27 636 methylated coding genes and 113 methylated miRNA genes were identified. In the coding genes, 64.5% of the methylated reads mapped to the gene body region; by contrast, 60.7% of methylated reads in miRNA genes mainly mapped in the 5′ and 3′ flanking regions. CHH methylation showed the highest methylation levels and CHG showed the lowest methylation levels. Correlation analysis showed a significant, negative, strand-specific correlation of methylation and miRNA gene expression (r=0.79, P <0.05). The methylated miRNA genes included eight long miRNAs (lmiRNAs) of 24 nucleotides and 11 miRNAs related to flower development. miRNA172b might play an important role in the regulation of bisexual flower development-related gene expression in andromonoecious poplar, via modification of methylation. Gynomonoecious, female, and male poplars were used to validate the methylation patterns of the miRNA172b gene, implying that hyper-methylation in andromonoecious and gynomonoecious poplar might function as an important regulator in bisexual flower development. Our data provide a useful resource for the study of flower development in poplar and improve our understanding of the effect of epigenetic regulation on genes other than protein-coding genes. PMID:25617468

  8. Methionine-dependent histone methylation at developmentally important gene loci in mouse preimplantation embryos.

    PubMed

    Kudo, Mari; Ikeda, Shuntaro; Sugimoto, Miki; Kume, Shinichi

    2015-12-01

    The involvement of specific nutrients in epigenetic gene regulation is a possible mechanism underlying nutrition-directed phenotypic alteration. However, the involvement of nutrients in gene-specific epigenetic regulation remains poorly understood. Methionine has been received attention as a possible nutrient involved in epigenetic modifications, as it is a precursor of the universal methyl donor for epigenetic methylation of DNA and histones. In the present study, the disruption of methionine metabolism by ethionine, an antimetabolite of methionine, induced abnormally higher expression of genes related to cell lineage differentiation and resulted in impaired blastocyst development of mouse preimplantation embryos in vitro. These effects were mitigated by the presence of methionine. Importantly, ethionine treatment induced lower trimethylation of histone H3 lysine 9 but did not affect methylation of DNA in the promoter regions of the examined genes. These results demonstrated that intact methionine metabolism is required for proper epigenetic histone modifications and normal expression of developmentally important genes during preimplantation development. PMID:26372092

  9. Increased DNA methylation of neuropsychiatric genes occurs in borderline personality disorder.

    PubMed

    Dammann, Gerhard; Teschler, Stefanie; Haag, Tanja; Altmüller, Franziska; Tuczek, Frederik; Dammann, Reinhard H

    2011-12-01

    Borderline personality disorder (BPD) is a complex psychiatric disease of increasing importance. Epigenetic alterations are hallmarks for altered gene expression and could be involved in the etiology of BPD. In our study we analyzed DNA methylation patterns of 14 neuropsychiatric genes (COMT, DAT1, GABRA1, GNB3, GRIN2B, HTR1B, HTR2A, 5-HTT, MAOA, MAOB, NOS1, NR3C1, TPH1 and TH). DNA methylation was analyzed by bisulfite restriction analysis and pyrosequencing in whole blood samples of patients diagnosed with DSM-IV BPD and in controls. Aberrant methylation was not detectable using bisulfite restriction analysis, but a significantly increased methylation of HTR2A, NR3C1, MAOA, MAOB and soluble COMT (S-COMT) was revealed for BPD patients using pyrosequencing. For HTR2A the average methylation of four CpG sites was 0.8% higher in BPD patients compared to controls (p = 0.002). The average methylation of NR3C1 was 1.8% increased in BPD patients compared to controls (p = 0.0003) and was higher at 2 out of 8 CpGs (p ≤ 0.04). In females, an increased average methylation (1.5%) of MAOA was observed in BPD patients compared to controls (p = 0.046). A similar trend (1.4% higher methylation) was observed for MAOB in female BPD patients and increased methylation was significant for 1 out of 6 CpG sites. For S-COMT, a higher methylation of 2 out of 4 CpG sites was revealed in BPD patients (p ≤ 0.02). In summary, methylation signatures of several promoter regions were established and a significant increased average methylation (1.7%) occurred in blood samples of BPD patients (p < 0.0001). Our data suggest that aberrant epigenetic regulation of neuropsychiatric genes may contribute to the pathogenesis of BPD. PMID:22139575

  10. Histone Methyltransferase Enhancer of Zeste Homolog 2-Mediated ABCA1 Promoter DNA Methylation Contributes to the Progression of Atherosclerosis

    PubMed Central

    Wan, Wei; Yao, Feng; He, Ping-Ping; Xie, Wei; Mo, Zhong-Cheng; Shi, Jin-Feng; Wu, Jian-Feng; Peng, Juan; Liu, Dan; Cayabyab, Francisco S.; Zheng, Xi-Long; Tang, Xiang-Yang; Ouyang, Xin-Ping; Tang, Chao-Ke

    2016-01-01

    ATP-binding cassette transporter A1 (ABCA1) plays a critical role in maintaining cellular cholesterol homeostasis. The purpose of this study is to identify the molecular mechanism(s) underlying ABCA1 epigenetic modification and determine its potential impact on ABCA1 expression in macrophage-derived foam cell formation and atherosclerosis development. DNA methylation induced foam cell formation from macrophages and promoted atherosclerosis in apolipoprotein E-deficient (apoE−/−) mice. Bioinformatics analyses revealed a large CpG island (CGI) located in the promoter region of ABCA1. Histone methyltransferase enhancer of zeste homolog 2 (EZH2) downregulated ABCA1 mRNA and protein expression in THP-1 and RAW264.7 macrophage-derived foam cells. Pharmacological inhibition of DNA methyltransferase 1 (DNMT1) with 5-Aza-dC or knockdown of DNMT1 prevented the downregulation of macrophage ABCA1 expression, suggesting a role of DNA methylation in ABCA1 expression. Polycomb protein EZH2 induced DNMT1 expression and methyl-CpG-binding protein-2 (MeCP2) recruitment, and stimulated the binding of DNMT1 and MeCP2 to ABCA1 promoter, thereby promoting ABCA1 gene DNA methylation and atherosclerosis. Knockdown of DNMT1 inhibited EZH2-induced downregulation of ABCA1 in macrophages. Conversely, EZH2 overexpression stimulated DNMT1-induced ABCA1 gene promoter methylation and atherosclerosis. EZH2-induced downregulation of ABCA1 gene expression promotes foam cell formation and the development of atherosclerosis by DNA methylation of ABCA1 gene promoter. PMID:27295295

  11. Histone Methyltransferase Enhancer of Zeste Homolog 2-Mediated ABCA1 Promoter DNA Methylation Contributes to the Progression of Atherosclerosis.

    PubMed

    Lv, Yun-Cheng; Tang, Yan-Yan; Zhang, Ping; Wan, Wei; Yao, Feng; He, Ping-Ping; Xie, Wei; Mo, Zhong-Cheng; Shi, Jin-Feng; Wu, Jian-Feng; Peng, Juan; Liu, Dan; Cayabyab, Francisco S; Zheng, Xi-Long; Tang, Xiang-Yang; Ouyang, Xin-Ping; Tang, Chao-Ke

    2016-01-01

    ATP-binding cassette transporter A1 (ABCA1) plays a critical role in maintaining cellular cholesterol homeostasis. The purpose of this study is to identify the molecular mechanism(s) underlying ABCA1 epigenetic modification and determine its potential impact on ABCA1 expression in macrophage-derived foam cell formation and atherosclerosis development. DNA methylation induced foam cell formation from macrophages and promoted atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice. Bioinformatics analyses revealed a large CpG island (CGI) located in the promoter region of ABCA1. Histone methyltransferase enhancer of zeste homolog 2 (EZH2) downregulated ABCA1 mRNA and protein expression in THP-1 and RAW264.7 macrophage-derived foam cells. Pharmacological inhibition of DNA methyltransferase 1 (DNMT1) with 5-Aza-dC or knockdown of DNMT1 prevented the downregulation of macrophage ABCA1 expression, suggesting a role of DNA methylation in ABCA1 expression. Polycomb protein EZH2 induced DNMT1 expression and methyl-CpG-binding protein-2 (MeCP2) recruitment, and stimulated the binding of DNMT1 and MeCP2 to ABCA1 promoter, thereby promoting ABCA1 gene DNA methylation and atherosclerosis. Knockdown of DNMT1 inhibited EZH2-induced downregulation of ABCA1 in macrophages. Conversely, EZH2 overexpression stimulated DNMT1-induced ABCA1 gene promoter methylation and atherosclerosis. EZH2-induced downregulation of ABCA1 gene expression promotes foam cell formation and the development of atherosclerosis by DNA methylation of ABCA1 gene promoter. PMID:27295295

  12. Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy

    PubMed Central

    Miller-Delaney, Suzanne F.C.; Bryan, Kenneth; Das, Sudipto; McKiernan, Ross C.; Bray, Isabella M.; Reynolds, James P.; Gwinn, Ryder; Stallings, Raymond L.

    2015-01-01

    Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain. PMID

  13. Increased methylation of interleukin 6 gene is associated with obesity in Korean women.

    PubMed

    Na, Yeon Kyung; Hong, Hae Sook; Lee, Won Kee; Kim, Young Hun; Kim, Dong Sun

    2015-05-01

    Obesity is the fifth leading risk for death globally, and a significant challenge to global health. It is a common, complex, non-malignant disease and develops due to interactions between the genes and the environment. DNA methylation can act as a downstream effector of environmental signals; analysis of this process therefore holds substantial promise for identifying mechanisms through which genetic and environmental factors jointly contribute to disease risk. To assess the effects of excessive weight and obesity on gene-specific methylation levels of promoter regions, we determined the methylation status of four genes involved in inflammation and oxidative stress [interleukin 6 (IL6), tumor necrosis factor α (TNFα), mitochondrial transcription factor A (TFAM), and glucose transport 4 (GLUT4)] in blood cell-derived DNA from healthy women volunteers with a range of body mass indices (BMIs) by methylation-specific PCR. Interestingly, the samples from obese individuals (BMI ≥ 30 kg/m(2)) showed significantly increased hypermethylation for IL6 gene compared to normal weight (BMI < 23 kg/m(2)) and overweight samples (23 kg/m(2) ≤ BMI < 30 kg/m(2)) (P = 0.034 and P = 0.026). However, there was no statistically significant difference in promoter methylation of the other 3 genes between each group. These findings suggest that aberrant DNA methylation of IL6 gene promoter may play an important role in the etiology and pathogenesis of obesity and IL6 methylation could be used as molecular biomarker for obesity risk assessment. Further studies are required to elucidate the potential mechanisms underlying this relationship. PMID:25921605

  14. Global Methylation Patterns and Their Relationship with Gene Expression and Small RNA in Rice Lines with Different Ploidy

    PubMed Central

    Zhang, Hong-Yu; Zhao, Hui-Xia; Wu, Shao-Hua; Huang, Fang; Wu, Kai-Ting; Zeng, Xiu-Feng; Chen, Xiao-Qiong; Xu, Pei-Zhou; Wu, Xian-Jun

    2016-01-01

    Whole genome duplication (WGD) is a major force in angiosperm evolution. Whether WGD is accompanied by the evolution of epigenetic regulators remains to be explored. Here we investigate whole genome methylation, gene expression, and miRNA regulation among monoploid, diploid, and triploid rice plants isolated from a twin-seedling population. The DNA methylation patterns in the three different ploidy plants were highly similar, with DNA methylation primarily enriched in the promoters. We examined the methylation of single genes and detected around 25,500 methylated genes, of which 22,751 were methylated in all three lines. Significantly divergent DNA methylation patterns between each pair of three lines were only detected in 64 genes, though more genes were found to exhibit differential expression. Analysis of DNA methylation and expression patterns showed that higher DNA methylation levels upstream of the transcription start sites are correlated with higher levels of expression of related genes; whereas higher DNA methylation levels in gene body regions are correlated with lower levels of expression. We also carried out high-throughput sequencing of small RNA libraries and identified 36 new miRNAs. These miRNAs have different expression levels depending on the ploidy. PMID:27493648

  15. Association between MGMT Promoter Methylation and Non-Small Cell Lung Cancer: A Meta-Analysis

    PubMed Central

    Gu, Changmei; Lu, Jiachun; Cui, Tianpen; Lu, Cheng; Shi, Hao; Xu, Wenmao; Yuan, Xueli; Yang, Xiaobo; Huang, Yangxin; Lu, Meixia

    2013-01-01

    Background O6-methylguanine-DNA methyltransferase (MGMT) is one of most important DNA repair enzyme against common carcinogens such as alkylate and tobacco. Aberrant promoter methylation of the gene is frequently observed in non-small cell lung cancer (NSCLC). However, the importance of epigenetic inactivation of the gene in NSCLC published in the literature showed inconsistence. We quantified the association between MGMT promoter methylation and NSCLC using a meta-analysis method. Methods We systematically reviewed studies of MGMT promoter methylation and NSCLC in PubMed, EMBASE, Ovid, ISI Web of Science, Elsevier and CNKI databases and quantified the association between MGMT promoter methylation and NSCLC using meta-analysis method. Odds ratio (OR) and corresponding 95% confidence interval (CI) were calculated to evaluate the strength of association. Potential sources of heterogeneity were assessed by subgroup analysis and meta-regression. Results A total of 18 studies from 2001 to 2011, with 1, 160 tumor tissues and 970 controls, were involved in the meta-analysis. The frequencies of MGMT promote methylation ranged from 1.5% to 70.0% (median, 26.1%) in NSCLC tissue and 0.0% to 55.0% (median, 2.4%) in non-cancerous control, respectively. The summary of OR was 4.43 (95% CI: 2.85, 6.89) in the random-effects model. With stratification by potential source of heterogeneity, the OR was 20.45 (95% CI: 5.83, 71.73) in heterogeneous control subgroup, while it was 4.16 (95% CI: 3.02, 5.72) in the autologous control subgroup. The OR was 5.31 (95% CI: 3.00, 9.41) in MSP subgroup and 3.06 (95% CI: 1.75, 5.33) in Q-MSP subgroup. Conclusion This meta-analysis identified a strong association between methylation of MGMT gene and NSCLC. Prospective studies should be required to confirm the results in the future. PMID:24086261

  16. Expression of DNA methylation genes in secondary progressive multiple sclerosis.

    PubMed

    Fagone, Paolo; Mangano, Katia; Di Marco, Roberto; Touil-Boukoffa, Chafia; Chikovan, Tinatin; Signorelli, Santo; Lombardo, Giuseppe A G; Patti, Francesco; Mammana, Santa; Nicoletti, Ferdinando

    2016-01-15

    Multiple sclerosis (MS) is an immunoinflammatory disease of the central nervous system that seems to be influenced by DNA methylation. We sought to explore the expression pattern of genes involved in the control of DNA methylation in Secondary Progressive (SP) MS patients' PBMCs. We have found that SP MS is characterized by a significant upregulation of two genes belonging to the MBD family genes, MBD2 and MBD4, and by a downregulation of TDG and TET3. PMID:26711572

  17. Effects on specific promoter DNA methylation in zebrafish embryos and larvae following benzo[a]pyrene exposure☛

    PubMed Central

    Corrales, J.; Fang, X.; Thornton, C.; Mei, W.; Barbazuk, W.B.; Duke, M.; Scheffler, B.E.; Willett, K.L.

    2014-01-01

    Benzo[a]pyrene (BaP) is an established carcinogen and reproductive and developmental toxicant. BaP exposure in humans and animals has been linked to infertility and multigenerational health consequences. DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and mapping of methylation patterns has become an important tool for understanding pathologic gene expression events. The goal of this study was to investigate aberrant changes in promoter DNA methylation in zebrafish embryos and larvae following a parental and continued embryonic waterborne BaP exposure. A total of 21 genes known for their role in human diseases were selected to measure percent methylation by multiplex deep sequencing. At 96 hours post fertilization (hpf) compared to 3.3 hpf, dazl, nqo1, sox3, cyp1b1, and gstp1 had higher methylation percentages while c-fos and cdkn1a had decreased CG methylation. BaP exposure significantly reduced egg production and offspring survival. Moreover, BaP decreased global methylation and altered CG, CHH, and CHG methylation both at 3.3 and 96 hpf. CG methylation changed by 10% or more due to BaP in six genes (c-fos, cdkn1a, dazl, nqo1, nrf2, and sox3) at 3.3 hpf and in ten genes (c-fos, cyp1b1, dazl, gstp1, mlh1, nqo1, pten, p53, sox2, and sox3) at 96 hpf. BaP also induced gene expression of cyp1b1 and gstp1 at 96 hpf which were found to be hypermethylated. Further studies are needed to link aberrant CG, CHH, and CHG methylation to heritable epigenetic consequences associated with disease in later life. PMID:24576477

  18. KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    PubMed Central

    2014-01-01

    Background Pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function. Results Here, we show that KDM5B, which demethylates lysine 4 of histone H3, co-localizes with H3K4me3 near promoters and enhancers of active genes in ES cells; its depletion leads to spreading of H3K4 methylation into gene bodies and enhancer shores, indicating that KDM5B functions to focus H3K4 methylation at promoters and enhancers. Spreading of H3K4 methylation to gene bodies and enhancer shores is linked to defects in gene expression programs and enhancer activity, respectively, during self-renewal and differentiation of KDM5B-depleted ES cells. KDM5B critically regulates H3K4 methylation at bivalent genes during differentiation in the absence of LIF or Oct4. We also show that KDM5B and LSD1, another H3K4 demethylase, co-regulate H3K4 methylation at active promoters but they retain distinct roles in demethylating gene body regions and bivalent genes. Conclusions Our results provide global and functional insight into the role of KDM5B in regulating H3K4 methylation marks near promoters, gene bodies, and enhancers in ES cells and during differentiation. PMID:24495580

  19. Flow-Dependent Epigenetic DNA Methylation in Endothelial Gene Expression and Atherosclerosis.

    PubMed

    Dunn, Jessilyn; Thabet, Salim; Jo, Hanjoong

    2015-07-01

    Epigenetic mechanisms that regulate endothelial cell gene expression are now emerging. DNA methylation is the most stable epigenetic mark that confers persisting changes in gene expression. Not only is DNA methylation important in rendering cell identity by regulating cell type-specific gene expression throughout differentiation, but it is becoming clear that DNA methylation also plays a key role in maintaining endothelial cell homeostasis and in vascular disease development. Disturbed blood flow causes atherosclerosis, whereas stable flow protects against it by differentially regulating gene expression in endothelial cells. Recently, we and others have shown that flow-dependent gene expression and atherosclerosis development are regulated by mechanisms dependent on DNA methyltransferases (1 and 3A). Disturbed blood flow upregulates DNA methyltransferase expression both in vitro and in vivo, which leads to genome-wide DNA methylation alterations and global gene expression changes in a DNA methyltransferase-dependent manner. These studies revealed several mechanosensitive genes, such as HoxA5, Klf3, and Klf4, whose promoters were hypermethylated by disturbed blood flow, but rescued by DNA methyltransferases inhibitors such as 5Aza-2-deoxycytidine. These findings provide new insight into the mechanism by which flow controls epigenomic DNA methylation patterns, which in turn alters endothelial gene expression, regulates vascular biology, and modulates atherosclerosis development. PMID:25953647

  20. Quantitation of CDH1 promoter methylation in formalin-fixed paraffin-embedded tissues of breast cancer patients using differential high resolution melting analysis

    PubMed Central

    Naghitorabi, Mojgan; Mohammadi-Asl, Javad; Sadeghi, Hamid Mir Mohammad; Rabbani, Mohammad; Jafarian-Dehkordi, Abbas; Javanmard, Shaghayegh Haghjooy

    2016-01-01

    Background: E-cadherin (CDH1) plays an important role in cell–cell adhesion of epithelial tissues. Loss of E-cadherin expression can lead to loss of tissue integrity, metastasis, and cancer progression. Also loss of E-cadherin expression might be related to aberrant promoter methylation of the CDH1 gene. Many studies have been performed on CDH1 promoter methylation, especially in breast cancer. Although most of the studies have used qualitative methods for methylation analysis, this study is designed to quantitatively investigate CDH1 promoter methylation in breast cancer and its correlation with patients’ clinicopathological features. Materials and Methods: Using differential high resolution melting analysis (D-HRMA), the methylation level of the CDH1 gene promoter was quantified in 98 breast cancer formalin-fixed paraffin-embedded (FFPE) tissues and also 10 fresh frozen normal breast tissues. Results: All samples were detected to be methylated at the CDH1 promoter region. About 74.5% of the breast cancer samples were hypermethylated with an average methylation level of around 60%, while 25.5% of the patients were methylated with the mean methylation level of about 33%, and 90% of the normal samples had a mean methylation level of about 18%. Statistical analyses represented a significant correlation between CDH1 promoter methylation and cancer progression hallmarks, such as, clinical stage, nodal involvement, tumor size, and histological grade. Conclusion: In summary, quantitation of CDH1 promoter methylation can serve as a diagnostic and prognostic tool in breast cancer. Also D-HRMA can be used as a fast and reliable method for quantitation of promoter methylation. PMID:27308263

  1. Methyl-accepting chemotaxis protein III and transducer gene trg.

    PubMed Central

    Hazelbauer, G L; Engström, P; Harayama, S

    1981-01-01

    A comparison of the two-dimensional gel patterns of methyl-3H- and 35S-labeled membrane proteins from trg+ and trg null mutant strains of Escherichia coli indicated that the product of trg is probably methyl-accepting chemotaxis protein III. Like the other known methyl-accepting chemotaxis proteins, the trg product is a membrane protein that migrates as more than one species in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that it too is multiple methylated. It appears likely that all chemoreceptors are linked to the tumble regulator through a single class of membrane protein transducers which are methyl-accepting proteins. Three transducers are coded for by genes tsr, tar, and, probably, trg. Another methyl-accepting protein, which is not related to any of these genes, was observed. Images PMID:7007323

  2. Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes

    PubMed Central

    Lin, I-Hsuan; Chen, Dow-Tien; Chang, Yi-Feng; Lee, Yu-Ling; Su, Chia-Hsin; Cheng, Ching; Tsai, Yi-Chien; Ng, Swee-Chuan; Chen, Hsiao-Tan; Lee, Mei-Chen; Chen, Hong-Wei; Suen, Shih-Hui; Chen, Yu-Cheng; Liu, Tze-Tze; Chang, Chuan-Hsiung; Hsu, Ming-Ta

    2015-01-01

    Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation. PMID:25706888

  3. Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.

    PubMed

    Galamb, Orsolya; Kalmár, Alexandra; Péterfia, Bálint; Csabai, István; Bodor, András; Ribli, Dezső; Krenács, Tibor; Patai, Árpád V; Wichmann, Barnabás; Barták, Barbara Kinga; Tóth, Kinga; Valcz, Gábor; Spisák, Sándor; Tulassay, Zsolt; Molnár, Béla

    2016-08-01

    The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis. PMID:27245242

  4. DNA methylation of oxidative stress genes and cancer risk in the Normative Aging Study

    PubMed Central

    Gao, Tao; Joyce, Brian Thomas; Liu, Lei; Zheng, Yinan; Dai, Qi; Zhang, Zhou; Zhang, Wei; Shrubsole, Martha J; Tao, Meng-Hua; Schwartz, Joel; Baccarelli, Andrea; Hou, Lifang

    2016-01-01

    Oxidative stress (OS) is a primary mechanism of carcinogenesis, and methylation of genes related to it may play a role in cancer development. In this study, we examined the prospective association between blood DNA methylation of four oxidative stress genes and cancer incidence. Our study population included a total of 582 participants in the Normative Aging Study (NAS) who had blood drawn during 1-4 visits from 1999-2012 (mean follow up 9.0 years). Promoter DNA methylation of CRAT, iNOS, OGG1 and GCR in blood leukocytes was measured using pyrosequencing. We used Cox regression models to examine prospective associations between cancer incidence and both methylation at the baseline visit and methylation rate of changes over time. Baseline OGG1 methylation was associated with higher risk of all-cancer (HR: 1.43, 95% CI: 1.15-1.78) and prostate cancer (HR: 1.52, 95% CI: 1.03-2.25) incidence. Compared with participants remaining cancer-free, those who eventually developed cancer had significantly accelerated CRAT methylation (p = 0.04) and decelerated iNOS methylation (p<0.01) over time prior to cancer diagnosis. Accelerated CRAT methylation was associated with higher all-cancer incidence (HR: 3.88, 95% CI: 1.06-14.30), whereas accelerated iNOS methylation was associated with lower all-cancer incidence (HR: 0.08, 95% CI 0.02-0.38). Our results suggest that methylation and its dynamic change over time in OS-related genes, including OGG1, CRAT and iNOS, may play an important role in carcinogenesis. These results can potentially facilitate the development of early detection biomarkers and new treatments for a variety of cancers. PMID:27186424

  5. The role of CpG methylation in cell type-specific expression of the aquaporin-5 gene.

    PubMed

    Nomura, Johji; Hisatsune, Akinori; Miyata, Takeshi; Isohama, Yoichiro

    2007-02-23

    Aquaporin-5 (AQP5) is expressed in a cell type-specific manner. Here, we show that the AQP5 gene is regulated by CpG methylation. The AQP5 promoter containing a putative CpG island was highly methylated in NIH-3T3 or freshly isolated alveolar epithelial cells, correlating with the repression of this gene in these cells. In contrast, the AQP5 promoter was hypo-methylated in MLE-12 or cultured alveolar epithelial cells, which express high levels of AQP5. Repression of AQP5 transcription in NIH-3T3 cells could be relieved with 5-azacytidine, and in vitro methylation of the AQP5 promoter resulted in inhibition of transcription of the reporter gene in MLE-12 cells. Chromatin immunoprecipitation assays showed that endogenous Sp1 bound to the hypo-methylated, but not highly methylated, AQP5 promoter region. These results demonstrate that the hypo-methylated state of the AQP5 promoter leading to increased Sp1 binding may play a role in regulation of cell type-specific expression of the AQP5 gene. PMID:17198683

  6. Targeted Methylation of the Epithelial Cell Adhesion Molecule (EpCAM) Promoter to Silence Its Expression in Ovarian Cancer Cells

    PubMed Central

    Nunna, Suneetha; Reinhardt, Richard; Ragozin, Sergey; Jeltsch, Albert

    2014-01-01

    The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers. PMID:24489952

  7. Oral decitabine reactivates expression of the methylated gamma-globin gene in Papio anubis.

    PubMed

    Lavelle, Donald; Chin, Janet; Vaitkus, Kestis; Redkar, Sanjeev; Phiasivongsa, Pasit; Tang, Chunlin; Will, Roselle; Hankewych, Maria; Roxas, Bryan; Singh, Mahipal; Saunthararajah, Yogen; Desimone, Joseph

    2007-11-01

    The silencing of tumor suppressor genes associated with increased DNA methylation of the promoter regions is a frequent observation in many forms of cancer. Reactivation of these genes using pharmacological inhibitors of DNA methyltransferase such as 5-aza-2'-deoxycytidine (decitabine) is a worthwhile therapeutic goal. The effectiveness and tolerability of low-dose intravenous and subcutaneous decitabine regimens to demethylate and reactivate expression of the methylated gamma-globin gene in baboons and in patients with sickle cell disease led to successful trials of low-dose regimens of this drug in patients with myelodysplastic syndrome. Since these low-dose regimens are well-tolerated with minimal toxicity, they are suitable for chronic dosing to maintain promoter hypomethylation and expression of target genes. The development of an orally administered therapy using DNA methyltransferase inhibitors would facilitate such chronic approaches to therapy. We tested the ability of decitabine and a new salt derivative, decitabine mesylate, to reactivate the methylated gamma-globin gene in baboons when administered orally. Our results demonstrate that oral administration of these drugs at doses 17-34 times optimal subcutaneous doses of decitabine reactivates fetal hemoglobin, demethylates the epsilon- and gamma-globin gene promoters, and increases histone acetylation of these promoters in baboons (Papio anubis). PMID:17696208

  8. Loss of CDKN2A Promoter Methylation Coincides With the Epigenetic Transdifferentiation of Uterine Myosarcomatous Cells.

    PubMed

    Roncati, Luca; Barbolini, Giuseppe; Sartori, Giuliana; Siopis, Elena; Pusiol, Teresa; Maiorana, Antonio

    2016-07-01

    Leiomyosarcoma is the most common type of uterine sarcoma and usually displays typical morphology. Heterologous leiomyosarcoma is the rarest variant, in which the tumor contains liposarcomatous, osteosarcomatous, or rhabdomyosarcomatous components. We have investigated the largest series of uterine leiomyosarcoma with a rhabdomyosarcomatous component and we have disclosed a molecular finding, which coincides to the process of transdifferentiation from smooth muscle into striated muscle phenotype. The surgical specimens of 5 rare cases of uterine leiomyosarcoma with a rhabdomyosarcomatous component were formalin fixed and paraffin embedded. In addition to hematoxylin/eosin stains, phosphotungstic acid hematoxylin staining, immunohistochemistry, and methylation-specific polymerase chain reaction the CDKN2A promoter region were performed. Leiomyosarcomatous cells were found to be strongly immunoreactive for both desmin and α-smooth muscle actin. Rhabdomyosarcomatous cells were immunoreactive for sarcomeric actin, desmin, vimentin, CD10, and p16. The methylation-specific polymerase chain reaction revealed the presence of a methylated allele and an unmethylated allele in the microdissected samples, coming from leiomyosarcomatous cells. On the contrary, 2 unmethylated alleles, molecular expression of a loss of heterozygosity, were detected in all the microdissected samples in the rhabdomyosarcomatous cells. The loss of heterozygosity methylation in the promoter region of the CDKN2A gene, occurred only in the rhabdomyosarcomatous cells with increases in both p16 and p14 expression. This event may result in an inhibition of cdk4/cdk6 activity, stabilizes the tumor suppressor protein p53, and coincides with the transdifferentiation from smooth muscle into striated muscle. PMID:27276112

  9. Lead exposure in pheochromocytoma cells induces persistent changes in amyloid precursor protein gene methylation patterns.

    PubMed

    Li, Yuan-Yuan; Chen, Tian; Wan, Yanjian; Xu, Shun-qing

    2012-08-01

    It has been suggested that lead (Pb) exposure in early life may increase amyloid precursor protein (APP) expression and promote the pathogenesis of Alzheimer's disease in old age. The current study examined whether the DNA methylation patterns of APP gene in rat pheochromocytoma (PC12) cells changed after Pb acetate exposure. Undifferentiated PC12 cells were exposed to three doses of Pb acetate (50, 250, and 500 nM) and one control for 2 days or 1 week. The methylation patterns of APP promoter and global DNA methylation were analyzed. The DNA methyltransferase 1 (DNMT1) expression and the level of amyloid β peptide (Aβ) were also investigated. The results showed that the exposure of the three concentrations of Pb acetate could make the APP promoter hypomethylated. The global DNA methylation level and the expression of DNMT1 were changed in the 500 nM group after 2 days exposure and in the 250 and 500 nM group after 7 days exposure. Thus, Pb may exert neurotoxic effects through mechanisms that alter the global and promoter methylation patterns of APP gene. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012. PMID:22764079

  10. Air pollution and gene-specific methylation in the Normative Aging Study

    PubMed Central

    Bind, Marie-Abele; Lepeule, Johanna; Zanobetti, Antonella; Gasparrini, Antonio; Baccarelli, Andrea A; Coull, Brent A; Tarantini, Letizia; Vokonas, Pantel S; Koutrakis, Petros; Schwartz, Joel

    2014-01-01

    The mechanisms by which air pollution has multiple systemic effects in humans are not fully elucidated, but appear to include inflammation and thrombosis. This study examines whether concentrations of ozone and components of fine particle mass are associated with changes in methylation on tissue factor (F3), interferon gamma (IFN-γ), interleukin 6 (IL-6), toll-like receptor 2 (TLR-2), and intercellular adhesion molecule 1 (ICAM-1). We investigated associations between air pollution exposure and gene-specific methylation in 777 elderly men participating in the Normative Aging Study (1999–2009). We repeatedly measured methylation at multiple CpG sites within each gene’s promoter region and calculated the mean of the position-specific measurements. We examined intermediate-term associations between primary and secondary air pollutants and mean methylation and methylation at each position with distributed-lag models. Increase in air pollutants concentrations was significantly associated with F3, ICAM-1, and TLR-2 hypomethylation, and IFN-γ and IL-6 hypermethylation. An interquartile range increase in black carbon concentration averaged over the four weeks prior to assessment was associated with a 12% reduction in F3 methylation (95% CI: -17% to -6%). For some genes, the change in methylation was observed only at specific locations within the promoter region. DNA methylation may reflect biological impact of air pollution. We found some significant mediated effects of black carbon on fibrinogen through a decrease in F3 methylation, and of sulfate and ozone on ICAM-1 protein through a decrease in ICAM-1 methylation. PMID:24385016

  11. Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

    PubMed Central

    Williams, Ben P.; Pignatta, Daniela; Henikoff, Steven; Gehring, Mary

    2015-01-01

    Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability. PMID:25826366

  12. Functional epigenetic approach identifies frequently methylated genes in Ewing sarcoma.

    PubMed

    Alholle, Abdullah; Brini, Anna T; Gharanei, Seley; Vaiyapuri, Sumathi; Arrigoni, Elena; Dallol, Ashraf; Gentle, Dean; Kishida, Takeshi; Hiruma, Toru; Avigad, Smadar; Grimer, Robert; Maher, Eamonn R; Latif, Farida

    2013-11-01

    Using a candidate gene approach we recently identified frequent methylation of the RASSF2 gene associated with poor overall survival in Ewing sarcoma (ES). To identify effective biomarkers in ES on a genome-wide scale, we used a functionally proven epigenetic approach, in which gene expression was induced in ES cell lines by treatment with a demethylating agent followed by hybridization onto high density gene expression microarrays. After following a strict selection criterion, 34 genes were selected for expression and methylation analysis in ES cell lines and primary ES. Eight genes (CTHRC1, DNAJA4, ECHDC2, NEFH, NPTX2, PHF11, RARRES2, TSGA14) showed methylation frequencies of>20% in ES tumors (range 24-71%), these genes were expressed in human bone marrow derived mesenchymal stem cells (hBMSC) and hypermethylation was associated with transcriptional silencing. Methylation of NPTX2 or PHF11 was associated with poorer prognosis in ES. In addition, six of the above genes also showed methylation frequency of>20% (range 36-50%) in osteosarcomas. Identification of these genes may provide insights into bone cancer tumorigenesis and development of epigenetic biomarkers for prognosis and detection of these rare tumor types. PMID:24005033

  13. Differential promoter methylation of kinesin family member 1a in plasma is associated with breast cancer and DNA repair capacity.

    PubMed

    Guerrero-Preston, Rafael; Hadar, Tal; Ostrow, Kimberly Laskie; Soudry, Ethan; Echenique, Miguel; Ili-Gangas, Carmen; Pérez, Gabriela; Perez, Jimena; Brebi-Mieville, Priscilla; Deschamps, José; Morales, Luisa; Bayona, Manuel; Sidransky, David; Matta, Jaime

    2014-08-01

    Methylation alterations of CpG islands, CpG island shores and first exons are key events in the formation and progression of human cancer, and an increasing number of differentially methylated regions and genes have been identified in breast cancer. Recent studies of the breast cancer methylome using deep sequencing and microarray platforms are providing a novel insight on the different roles aberrant methylation plays in molecular subtypes of breast cancer. Accumulating evidence from a subset of studies suggests that promoter methylation of tumor-suppressor genes associated with breast cancer can be quantified in circulating DNA. However, there is a paucity of studies that examine the combined presence of genetic and epigenetic alterations associated with breast cancer using blood-based assays. Dysregulation of DNA repair capacity (DRC) is a genetic risk factor for breast cancer that has been measured in lymphocytes. We isolated plasma DNA from 340 participants in a breast cancer case control project to study promoter methylation levels of five genes previously shown to be associated with breast cancer in frozen tissue and in cell line DNA: MAL, KIF1A, FKBP4, VGF and OGDHL. Methylation of at least one gene was found in 49% of the cases compared to 20% of the controls. Three of the four genes had receiver characteristic operator curve values of ≥ 0.50: MAL (0.64), KIF1A (0.51) and OGDHL (0.53). KIF1A promoter methylation was associated with breast cancer and inversely associated with DRC. This is the first evidence of a significant association between genetic and epigenetic alterations in breast cancer using blood-based tests. The potential diagnostic utility of these biomarkers and their relevance for breast cancer risk prediction should be examined in larger cohorts. PMID:24927296

  14. Host-cell-determined methylation of specific Epstein-Barr virus promoters regulates the choice between distinct viral latency programs.

    PubMed Central

    Schaefer, B C; Strominger, J L; Speck, S H

    1997-01-01

    Epstein-Barr virus (EBV) is capable of adopting three distinct forms of latency: the type III latency program, in which six EBV-encoded nuclear antigens (EBNAs) are expressed, and the type I and type II latency programs, in which only a single viral nuclear protein, EBNA1, is produced. Several groups have reported heavy CpG methylation of the EBV genome in Burkitt's lymphoma cell lines which maintain type I latency, and loss of viral genome methylation in tumor cell lines has been correlated with a switch to type III latency. Here, evidence that the type III latency program must be inactivated by methylation to allow EBV to enter the type I or type II restricted latency program is provided. The data demonstrates that the EBNA1 gene promoter, Qp, active in types I and II latency, is encompassed by a CpG island which is protected from methylation. CpG methylation inactivates the type III latency program and consequently allows the type I or II latency program to operate by alleviating EBNA1-mediated repression of Qp. Methylation of the type III latency EBNA gene promoter, Cp, appears to be essential to prevent type III latency, since EBNA1 is expressed in all latently infected cells and, as shown here, is the only viral antigen required for activation of Cp. EBV is thus a pathogen which subverts host-cell-determined methylation to regulate distinct genetic programs. PMID:8972217

  15. Association between early promoter-specific DNA methylation changes and outcome in older acute myeloid leukemia patients.

    PubMed

    Achille, Nicholas J; Othus, Megan; Phelan, Kathleen; Zhang, Shubin; Cooper, Kathrine; Godwin, John E; Appelbaum, Frederick R; Radich, Jerald P; Erba, Harry P; Nand, Sucha; Zeleznik-Le, Nancy J

    2016-03-01

    Treatment options for older patients with acute myeloid leukemia (AML) range from supportive care alone to full-dose chemotherapy. Identifying factors that predict response to therapy may help increase efficacy and avoid toxicity. The phase II SWOG S0703 study investigated the use of hydroxyurea and azacitidine with gemtuzumab ozogamicin in the elderly AML population and found survival rates similar to those expected with standard AML regimens, with less toxicity. As part of this study, global DNA methylation along with promoter DNA methylation and expression analysis of six candidate genes (CDKN2A, CDKN2B, HIC1, RARB, CDH1 and APAF1) were determined before and during therapy to investigate whether very early changes are prognostic for clinical response. Global DNA methylation was not associated with a clinical response. Samples after 3 or 4 days of treatment with azacitidine showed significantly decreased CDKN2A promoter DNA methylation in patients achieving complete remission (CR) compared to those who did not. Samples from day 7 of treatment showed significantly decreased RARB, CDKN2B and CDH1 promoter DNA methylation in responders compared to nonresponders. Gene-specific DNA methylation analysis of peripheral blood samples may help early identification of those older AML patients most likely to benefit from demethylating agent therapy. PMID:26818573

  16. Correlation between methylation of the E-Cadherin gene and malignancy of prostate cancer.

    PubMed

    Zhang, S Q; Zhang, G Q; Zhang, L

    2016-01-01

    Prostate cancer is a common malignant tumor in males with an unclear pathogenic mechanism. As one epigenetic regulation mechanism, DNA methylation of the whole genome and specific gene(s) plays critical roles in pathogenesis, progression, diagnosis, and treatment of prostate cancer. The E-Cadherin gene is involved in cell metabolism and has been suggested to be related with malignancy of multiple tumors. This study investigated the correlation between E-Cadherin methylation and malignancy of prostate cancer. Gradient concentrations of 5-Aza-CdR (5, 10, and 20 mM) were used to treat the prostate cancer cell line (LNCaP), and mRNA level of E-Cadherin was detected by reverse transcription-polymerase chain reaction (RT-PCR). A total of 82 prostate cancer patients were recruited to detect the methylation status of the promoter region of the E-Cadherin gene by pyrophosphate sequencing. Real-time fluorescent quantitative PCR (qRT-PCR) was employed to determine mRNA levels of E-Cadherin. Methylation and mRNA levels of E-Cadherin were analyzed by the SPSS software. With elevated concentrations of 5-Aza-CdR, mRNA levels of E-Cadherin gradually increased. DNA methylation levels of tumor tissues were significantly elevated with increased Gleason score (P < 0.05) and tumor-node-metastasis stage (P < 0.05) but were not related to age, smoking habits, or alcohol consumption (P > 0.05). DNA methylation level was negatively correlated with mRNA expression of the E-Cadherin gene. Methylation in tumor tissues was significantly higher than that in tumor adjacent tissues (P < 0.05). DNA methylation level of the E-Cadherin gene could be an important predictive index for malignancy of prostate cancer. PMID:27420993

  17. An NF-Y-Dependent Switch of Positive and Negative Histone Methyl Marks on CCAAT Promoters

    PubMed Central

    Dolfini, Diletta; Fossati, Andrea; Ceribelli, Michele; Mantovani, Roberto

    2008-01-01

    Background Histone tails have a plethora of different post-translational modifications, which are located differently in “open” and “closed” parts of genomes. H3K4me3/H3K79me2 and H4K20me3 are among the histone marks associated with the early establishment of active and inactive chromatin, respectively. One of the most widespread promoter elements is the CCAAT box, bound by the NF-Y trimer. Two of NF-Y subunits have an H2A-H2B-like structure. Principal findings We established the causal relationship between NF-Y binding and positioning of methyl marks, by ChIP analysis of mouse and human cells infected with a dominant negative NF-YA: a parallel decrease in NF-Y binding, H3K4me3, H3K79me2 and transcription was observed in promoters that are dependent upon NF-Y. On the contrary, changes in the levels of H3K9-14ac were more subtle. Components of the H3K4 methylating MLL complex are not recruited in the absence of NF-Y. As for repressed promoters, NF-Y removal leads to a decrease in the H4K20me3 mark and deposition of H3K4me3. Conclusions Two relevant findings are reported: (i) NF-Y gains access to its genomic locations independently from the presence of methyl histone marks, either positive or negative; (ii) NF-Y binding has profound positive or negative consequences on the deposition of histone methyl marks. Therefore NF-Y is a fundamental switch at the heart of decision between gene activation and repression in CCAAT regulated genes. PMID:18446193

  18. MAOA promoter methylation and susceptibility to carotid atherosclerosis: role of familial factors in a monozygotic twin sample

    PubMed Central

    2012-01-01

    Background Atherosclerosis is a complex process involving both genetic and epigenetic factors. The monoamine oxidase A (MAOA) gene regulates the metabolism of key neurotransmitters and has been associated with cardiovascular risk factors. This study investigates whether MAOA promoter methylation is associated with atherosclerosis, and whether this association is confounded by familial factors in a monozygotic (MZ) twin sample. Methods We studied 84 monozygotic (MZ) twin pairs drawn from the Vietnam Era Twin Registry. Carotid intima-media thickness (IMT) was measured by ultrasound. DNA methylation in the MAOA promoter region was quantified by bisulfite pyrosequencing using genomic DNA isolated from peripheral blood leukocytes. The association between DNA methylation and IMT was first examined by generalized estimating equation, followed by matched pair analyses to determine whether the association was confounded by familial factors. Results When twins were analyzed as individuals, increased methylation level was associated with decreased IMT at four of the seven studied CpG sites. However, this association substantially reduced in the matched pair analyses. Further adjustment for MAOA genotype also considerably attenuated this association. Conclusions The association between MAOA promoter methylation and carotid IMT is largely explained by familial factors shared by the twins. Because twins reared together share early life experience, which may leave a long-lasting epigenetic mark, aberrant MAOA methylation may represent an early biomarker for unhealthy familial environment. Clarification of familial factors associated with DNA methylation and early atherosclerosis will provide important information to uncover clinical correlates of disease. PMID:23116433

  19. DNA Methylation Profile and Expression of Surfactant Protein A2 gene in Lung Cancer

    PubMed Central

    Grageda, Melissa; Silveyra, Patricia; Thomas, Neal J.; DiAngelo, Susan L.; Floros, Joanna

    2014-01-01

    Knowledge of the methylation profile of genes allow for the identification of biomarkers that may guide diagnosis and effective treatment of disease. Human surfactant protein A (SP-A) plays an important role in lung homeostasis and immunity, and is encoded by two genes (SFTPA1 and SFTPA2). The goal of this study was to identify differentially methylated CpG sites in the promoter region of the SFTPA2 gene in lung cancer tissue, and to determine the correlation between the promoter’s methylation profile and gene expression. For this, we collected 28 pairs of cancerous human lung tissue and adjacent non-cancerous (NC) lung tissue: 17 adenocarcinoma (AC), 9 squamous cell carcinoma (SCC), and 2 AC with SCC features, and we evaluated DNA methylation of the SFTPA2 promoter region by bisulfite conversion. Our results identified a higher methylation ratio in one CpG site of the SFTPA2 gene in cancerous tissue vs. NC tissue (0.36 vs. 0.11, p=0.001). When assessing AC samples, we also found cancerous tissues associated with a higher methylation ratio (0.43 vs. 0.10, p=0.02). In the SCC group, although cancerous tissue showed a higher methylation ratio (0.22 vs. 0.11), this difference was not statistically significant (p=0.35). Expression of SFTPA2 mRNA and total SP-A protein was significantly lower in cancer tissue when compared to adjacent NC tissue (p<0.001), and correlated with the hypermethylated status of a SFTPA2 CpG site in AC samples. The findings of this pilot study may hold promise for future use of SFTPA2 as a biomarker for the diagnosis of lung cancer. PMID:25514367

  20. CG gene body DNA methylation changes and evolution of duplicated genes in cassava

    PubMed Central

    Wang, Haifeng; Beyene, Getu; Zhai, Jixian; Feng, Suhua; Fahlgren, Noah; Taylor, Nigel J.; Bart, Rebecca; Carrington, James C.; Jacobsen, Steven E.; Ausin, Israel

    2015-01-01

    DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits. PMID:26483493

  1. CG gene body DNA methylation changes and evolution of duplicated genes in cassava.

    PubMed

    Wang, Haifeng; Beyene, Getu; Zhai, Jixian; Feng, Suhua; Fahlgren, Noah; Taylor, Nigel J; Bart, Rebecca; Carrington, James C; Jacobsen, Steven E; Ausin, Israel

    2015-11-01

    DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits. PMID:26483493

  2. Methylation changes of H{sub 19} gene in sperms of X-irradiated mouse and maintenance in offspring

    SciTech Connect

    Zhu Bin; Huang Xinghua; Chen Jindong; Lu Yachao; Chen Ying; Zhao Jingyong . E-mail: sudazhaojy@hotmail.com

    2006-02-03

    The nature of imprinting is just differential methylation of imprinted genes. Unlike the non-imprinted genes, the methylation pattern of imprinted genes established during the period of gametogenesis remains unchangeable after fertilization and during embryo development. It implies that gametogenesis is the key stage for methylation pattern of imprinted genes. The imprinting interfered by exogenous factors during this stage could be inherited to offspring and cause genetic effect. Now many studies have proved that ionizing irradiation could disturb DNA methylation. Here we choose BALB/c mice as a research model and X-ray as interfering source to further clarify it. We discovered that the whole-body irradiation of X-ray to male BALB/c mice could influence the methylation pattern of H{sub 19} gene in sperms, which resulted in some cytosines of partial CpG islands in the imprinting control region could not transform to methylated cytosines. Furthermore, by copulating the interfered male mice with normal female, we analyzed the promoter methylation pattern of H{sub 19} in offspring fetal liver and compared the same to the pattern of male parent in sperms. We found that the majority of methylation changes in offspring liver were related to the ones in their parent sperms. Our data proved that the changes of the H{sub 19} gene methylation pattern interfered by X-ray irradiation could be transmitted and maintained in First-generation offspring.

  3. Promoter DNA Methylation of Farnesoid X Receptor and Pregnane X Receptor Modulates the Intrahepatic Cholestasis of Pregnancy Phenotype

    PubMed Central

    Cabrerizo, Romina; Castaño, Gustavo O.; Burgueño, Adriana L.; Fernández Gianotti, Tomas; Gonzalez Lopez Ledesma, María Mora; Flichman, Diego; Pirola, Carlos J.; Sookoian, Silvia

    2014-01-01

    The intrahepatic cholestasis of pregnancy (ICP) is a multifactorial liver disorder which pathogenesis involves the interplay among abnormal bile acid (BA) levels, sex hormones, environmental factors, and genetic susceptibility. The dynamic nature of ICP that usually resolves soon after delivery suggests the possibility that its pathobiology is under epigenetic modulation. We explored the status of white blood peripheral cells-DNA methylation of CpG-enriched sites at the promoter of targeted genes (FXR/NR1H4, PXR/NR1I2, NR1I3, ESR1, and ABCC2) in a sample of 88 ICP patients and 173 healthy pregnant women in the third trimester of their pregnancies. CpG dinucleotides at the gene promoter of nuclear receptors subfamily 1 members and ABCC2 transporter were highly methylated during healthy pregnancy. We observed significant differences at the distal (−1890) and proximal promoter (−358) CpG sites of the FXR/NR1H4 and at the distal PXR/NR1I2 (−1224) promoter, which were consistently less methylated in ICP cases when compared with controls. In addition, we observed that methylation at FXR/NR1H4-1890 and PXR/NR1I2-1224 promoter sites was highly and positively correlated with BA profiling, particularly, conjugated BAs. Conversely, methylation level at the proximal FXR/NR1H4-358 CpG site was significantly and negatively correlated with the primary cholic and secondary deoxycholic acid. In vitro exploration showed that epiallopregnanolone sulfate, a reported FXR inhibitor, regulates the transcriptional activity of FXR/NR1H4 but seems to be not involved in the methylation changes. In conclusion, the identification of epigenetic marks in target genes provides a basis for the understanding of adverse liver-related pregnancy outcomes, including ICP. PMID:24498169

  4. Interindividual concordance of methylation profiles in human genes for tumor necrosis factors alpha and beta.

    PubMed Central

    Kochanek, S; Toth, M; Dehmel, A; Renz, D; Doerfler, W

    1990-01-01

    The DNA in mammalian genomes is characterized by complex patterns of DNA methylation that reflect the states of all genetic activities of that genome. The modified nucleotide 5-methyldeoxycytidine (5mdC) can affect the interactions of specific proteins with DNA sequence motifs. The most extensively studied effect of sequence-specific methylations is that of the long-term silencing of eukaryotic (mammalian) promoters. We have initiated studies on the methylation status of parts of the human genome to view patterns of DNA methylation as indicators for genetic activities. In this report, analyses using both restriction enzyme--Southern blotting and the very precise genomic sequencing technique have been done. The genes for tumor necrosis factors (TNF) alpha and beta--in particular, their 5'-upstream and promoter regions--have been investigated in DNA isolated from human lymphocytes, granulocytes, and sperm. The results are characterized by a remarkable interindividual concordance of DNA methylation in specific human cell types. The patterns are identical in the DNA from one cell type for different individuals even of different genetic origins but different in the DNA from different cell types. As an example, in the DNA from human granulocytes of 15 different individuals (ages 20-48 yr, both sexes), 5mdC residues have been localized by the genomic sequencing technique in three identical sequence positions in the 5'-upstream region and in one downstream position of the gene encoding TNF-alpha. The promoter of this gene is free of 5mdC, and TNF-alpha is expressed in human granulocytes. The TNF-beta promoter is methylated in granulocytes from 9 different individuals, and TNF-beta is not expressed. In human lymphocytes, the main source of TNF-beta, the TNF-beta promoter is free of 5mdC residues. All 5'-CG-3' sites studied in the TNF-alpha and -beta genes are methylated in DNA from human sperm. In human cell lines HL-60, Jurkat, and RPMI 1788, the extent of DNA methylation

  5. H19ICR mediated transcriptional silencing does not require target promoter methylation.

    PubMed

    Gebert, Claudia; Rong, Qi; Jeong, Sangkyun; Iben, James; Pfeifer, Karl

    2016-07-29

    Transcription of the reciprocally imprinted genes Insulin-like growth factor 2 (Igf2) and H19 is orchestrated by the 2.4-kb H19 Imprinting Control Region (H19ICR) located upstream of H19. Three known functions are associated with the H19ICR: (1) it is a germline differentially methylated region, (2) it is a transcriptional insulator, and (3) it is a transcriptional silencer. The molecular mechanisms of the DMR and insulator functions have been well characterized but the basis for the ICR's silencer function is less well understood. In order to study the role the H19ICR intrinsically plays in gene silencing, we transferred the 2.4-kb H19ICR to a heterologous non-imprinted location on chromosome 5, upstream of the alpha fetoprotein (Afp) promoter. Independent of its orientation, the 2.4-kb H19ICR silences transcription from the paternal Afp promoter. Thus silencing is a function intrinsic to this DNA element. Further, ICR mediated silencing is a developmental process that, unexpectedly, does not occur through DNA methylation at the target promoter. PMID:27178213

  6. Epigenetics of human myometrium: DNA methylation of genes encoding contraction-associated proteins in term and preterm labor.

    PubMed

    Mitsuya, Kohzoh; Singh, Natasha; Sooranna, Suren R; Johnson, Mark R; Myatt, Leslie

    2014-05-01

    Preterm birth involves the interaction of societal and environmental factors potentially modulating the length of gestation via the epigenome. An established form of epigenetic regulation is DNA methylation where promoter hypermethylation is associated with gene repression. We hypothesized we would find differences in DNA methylation in the myometrium of women with preterm labor of different phenotypes versus normal term labor. Myometrial tissue was obtained at cesarean section at term with or without labor, preterm without labor, idiopathic preterm labor, and twin gestations with labor. Genomic DNA was isolated, and samples in each group were combined and analyzed on a NimbleGen 2.1M human DNA methylation array. Differences in methylation from -8 to +3 kb of transcription start sites of 22 contraction-associated genes were determined. Cytosine methylation was not present in CpG islands of any gene but was present outside of CpG islands in shores and shelves in 19 genes. No differential methylation was found across the tissue groups for six genes (PTGES3L, PTGER2, PTGER4, PTGFRN, ESR2, and GJA1). For 13 genes, differential methylation occurred in several patterns between tissue groups. We find a correlation between hypomethylation and increased mRNA expression of PTGES/mPGES-1, indicating potential functional relevance of methylation, but no such correlation for PTGS2/COX-2, suggesting other regulatory mechanisms for PTGS2 at labor. The majority of differential DNA methylation of myometrial contraction-associated genes with different labor phenotypes occurs outside of CpG islands in gene promoters, suggesting that the entirety of DNA methylation across the genome should be considered. PMID:24571989

  7. Methyl jasmonate affects phenolic metabolism and gene expression in blueberry (Vaccinium corymbosum).

    PubMed

    Cocetta, Giacomo; Rossoni, Mara; Gardana, Claudio; Mignani, Ilaria; Ferrante, Antonio; Spinardi, Anna

    2015-02-01

    Blueberry (Vaccinium corymbosum) is a fruit very much appreciated by consumers for its antioxidant potential and health-promoting traits. Its beneficial potential properties are mainly due to a high content of anthocyanins and their amount can change after elicitation with methyl jasmonate. The aim of this work is to evaluate the changes in expression of several genes, accumulation of phenolic compounds and alterations in antioxidant potential in two different blueberry cultivars ('Duke' and 'Blueray') in response to methyl jasmonate (0.1 mM). Results showed that 9 h after treatment, the expression of phenylalanine ammonium lyase, chalcone synthase and anthocyanidin synthase genes was stimulated more in the 'Blueray' variety. Among the phenols measured an increase was recorded also for epicatechin and anthocyanin concentrations. 'Duke' is a richer sourche of anthocyanins compared to 'Blueray', treatment with methyl jasmonate promoted in 'Blueray' an increase in pigments as well as in the antioxidant potential, especially in fully ripe berries, but treated 'Duke' berries had greater levels, which were not induced by methyl jasmonate treatment. In conclusion, methyl jasmonate was, in some cases, an effective elicitor of phenolic metabolism and gene expression in blueberry, though with different intensity between cultivars. PMID:24943920

  8. Recurrent epimutations activate gene body promoters in primary glioblastoma.

    PubMed

    Nagarajan, Raman P; Zhang, Bo; Bell, Robert J A; Johnson, Brett E; Olshen, Adam B; Sundaram, Vasavi; Li, Daofeng; Graham, Ashley E; Diaz, Aaron; Fouse, Shaun D; Smirnov, Ivan; Song, Jun; Paris, Pamela L; Wang, Ting; Costello, Joseph F

    2014-05-01

    Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein. PMID:24709822

  9. Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas

    PubMed Central

    Patai, Árpád V.; Valcz, Gábor; Hollósi, Péter; Kalmár, Alexandra; Péterfia, Bálint; Patai, Árpád; Wichmann, Barnabás; Spisák, Sándor; Barták, Barbara Kinga; Leiszter, Katalin; Tóth, Kinga; Sipos, Ferenc; Kovalszky, Ilona; Péter, Zoltán; Miheller, Pál; Tulassay, Zsolt; Molnár, Béla

    2015-01-01

    Microarray analysis of promoter hypermethylation provides insight into the role and extent of DNA methylation in the development of colorectal cancer (CRC) and may be co-monitored with the appearance of driver mutations. Colonic biopsy samples were obtained endoscopically from 10 normal, 23 adenoma (17 low-grade (LGD) and 6 high-grade dysplasia (HGD)), and 8 ulcerative colitis (UC) patients (4 active and 4 inactive). CRC samples were obtained from 24 patients (17 primary, 7 metastatic (MCRC)), 7 of them with synchronous LGD. Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC. Tissue materials were studied for DNA methylation status using a 96 gene panel and for KRAS and BRAF mutations. Expression levels were assayed using whole genomic mRNA arrays. SFRP1 was further examined by immunohistochemistry. HT29 cells were treated with 5-aza-2’ deoxycytidine to analyze the reversal possibility of DNA methylation. More than 85% of tumor samples showed hypermethylation in 10 genes (SFRP1, SST, BNC1, MAL, SLIT2, SFRP2, SLIT3, ALDH1A3, TMEFF2, WIF1), whereas the frequency of examined mutations were below 25%. These genes distinguished precancerous and cancerous lesions from inflamed and healthy tissue. The mRNA alterations that might be caused by systematic methylation could be partly reversed by demethylation treatment. Systematic changes in methylation patterns were observed early in CRC carcinogenesis, occuring in precursor lesions and CRC. Thus we conclude that DNA hypermethylation is an early and systematic event in colorectal carcinogenesis, and it could be potentially reversed by systematic demethylation therapy, but it would need more in vitro and in vivo experiments to support this theory. PMID:26291085

  10. Methylation of DAPK and THBS1 genes in esophageal gastric-type columnar metaplasia

    PubMed Central

    Herrera-Goepfert, Roberto; Oñate-Ocaña, Luis F; Mosqueda-Vargas, José Luis; Herrera, Luis A; Castro, Clementina; Mendoza, Julia; González-Barrios, Rodrigo

    2016-01-01

    AIM: To explore methylation of DAPK, THBS1, CDH-1, and p14 genes, and Helicobacter pylori (H. pylori) status in individuals harboring esophageal columnar metaplasia. METHODS: Distal esophageal mucosal samples obtained by endoscopy and histologically diagnosed as gastric-type (non-specialized) columnar metaplasia, were studied thoroughly. DNA was extracted from paraffin blocks, and methylation status of death-associated protein kinase (DAPK), thrombospondin-1 (THBS1), cadherin-1 (CDH1), and p14 genes, was examined using a methyl-sensitive polymerase chain reaction (MS-PCR) and sodium bisulfite modification protocol. H. pylori cagA status was determined by PCR. RESULTS: In total, 68 subjects (33 females and 35 males), with a mean age of 52 years, were included. H. pylori cagA positive was present in the esophageal gastric-type metaplastic mucosa of 18 individuals. DAPK, THSB1, CDH1, and p14 gene promoters were methylated by MS-PCR in 40 (58.8%), 33 (48.5%), 46 (67.6%), and 23 (33.8%) cases of the 68 esophageal samples. H. pylori status was associated with methylation of DAPK (P = 0.003) and THBS1 (P = 0.019). CONCLUSION: DNA methylation occurs in cases of gastric-type (non-specialized) columnar metaplasia of the esophagus, and this modification is associated with H. pylori cagA positive infection. PMID:27182166

  11. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer

    PubMed Central

    MATTHAIOS, DIMITRIOS; BALGKOURANIDOU, IOANNA; KARAYIANNAKIS, ANASTASIOS; BOLANAKI, HELEN; XENIDIS, NIKOLAOS; AMARANTIDIS, KYRIAKOS; CHELIS, LEONIDAS; ROMANIDIS, KONSTANTINOS; CHATZAKI, AIKATERINI; LIANIDOU, EVI; TRYPSIANIS, GRIGORIOS; KAKOLYRIS, STYLIANOS

    2016-01-01

    DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with

  12. Prenatal stress-induced programming of genome-wide promoter DNA methylation in 5-HTT-deficient mice

    PubMed Central

    Schraut, K G; Jakob, S B; Weidner, M T; Schmitt, A G; Scholz, C J; Strekalova, T; El Hajj, N; Eijssen, L M T; Domschke, K; Reif, A; Haaf, T; Ortega, G; Steinbusch, H W M; Lesch, K P; Van den Hove, D L

    2014-01-01

    The serotonin transporter gene (5-HTT/SLC6A4)-linked polymorphic region has been suggested to have a modulatory role in mediating effects of early-life stress exposure on psychopathology rendering carriers of the low-expression short (s)-variant more vulnerable to environmental adversity in later life. The underlying molecular mechanisms of this gene-by-environment interaction are not well understood, but epigenetic regulation including differential DNA methylation has been postulated to have a critical role. Recently, we used a maternal restraint stress paradigm of prenatal stress (PS) in 5-HTT-deficient mice and showed that the effects on behavior and gene expression were particularly marked in the hippocampus of female 5-Htt+/− offspring. Here, we examined to which extent these effects are mediated by differential methylation of DNA. For this purpose, we performed a genome-wide hippocampal DNA methylation screening using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip Mouse Promoter 1.0 R arrays. Using hippocampal DNA from the same mice as assessed before enabled us to correlate gene-specific DNA methylation, mRNA expression and behavior. We found that 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a subset of which showed overlap with the expression profiles of the corresponding transcripts. For example, a differentially methylated region in the gene encoding myelin basic protein (Mbp) was associated with its expression in a 5-Htt-, PS- and 5-Htt × PS-dependent manner. Subsequent fine-mapping of this Mbp locus linked the methylation status of two specific CpG sites to Mbp expression and anxiety-related behavior. In conclusion, hippocampal DNA methylation patterns and expression profiles of female prenatally stressed 5-Htt+/− mice suggest that distinct molecular mechanisms, some of which are promoter methylation-dependent, contribute to the behavioral effects of the 5-Htt

  13. Methylation of miR-145a-5p promoter mediates adipocytes differentiation.

    PubMed

    Du, Jingjing; Cheng, Xiao; Shen, Linyuan; Tan, Zhendong; Luo, Jia; Wu, Xiaoqian; Liu, Chendong; Yang, Qiong; Jiang, Yanzhi; Tang, Guoqing; Li, Xuewei; Zhang, Shunhua; Zhu, Li

    2016-06-17

    MicroRNAs (miRNAs, miR) play important roles in adipocyte development. Recent studies showed that the expression of several miRNAs is closely related with promoter methylation. However, it is not known whether miRNA mediates adipocytes differentiation by means of DNA methylation. Here, we showed that miR-145a-5p was poorly expressed in adipose tissue from mice fed a high fat diet (HFD). Overexpression or inhibition of miR-145a-5p was unfavorable or beneficial, respectively, for adipogenesis, and these effects were achieved by regulating adipocyte-specific genes involved in lipogenic transcription, fatty acid synthesis, and fatty acid transportation. Particularly, we first suggested that miR-145a-5p mimics or inhibitors promoted or repressed adipocytes proliferation by regulating p53 and p21, which act as cell cycle regulating factors. Surprisingly, the miR-145a-5p-repressed adipocyte differentiation was enhanced or rescued when cells treated with 5-Aza-dC were transfected with miR-145a-5p mimics or inhibitors, respectively. These data indicated that, as a new mean to positively regulate adipocyte proliferation, the process of miR-145a-5p-inhibited adipogenesis may be regulated by DNA methylation. PMID:27179777

  14. Psychological factors and DNA methylation of genes related to immune/inflammatory system markers: the VA Normative Aging Study

    PubMed Central

    Kim, Daniel; Kubzansky, Laura D; Baccarelli, Andrea; Sparrow, David; Spiro, Avron; Tarantini, Letizia; Cantone, Laura; Vokonas, Pantel; Schwartz, Joel

    2016-01-01

    Objectives Although psychological factors have been associated with chronic diseases such as coronary heart disease (CHD), the underlying pathways for these associations have yet to be elucidated. DNA methylation has been posited as a mechanism linking psychological factors to CHD risk. In a cohort of community-dwelling elderly men, we explored the associations between positive and negative psychological factors with DNA methylation in promoter regions of multiple genes involved in immune/inflammatory processes related to atherosclerosis. Design Prospective cohort study. Setting Greater Boston, Massachusetts area. Participants Samples of 538 to 669 men participating in the Normative Aging Study cohort with psychological measures and DNA methylation measures, collected on 1–4 visits between 1999 and 2006 (mean age=72.7 years at first visit). Outcome measures We examined anxiety, depression, hostility and life satisfaction as predictors of leucocyte gene-specific DNA methylation. We estimated repeated measures linear mixed models, controlling for age, smoking, education, history of heart disease, stroke or diabetes, % lymphocytes, % monocytes and plasma folate. Results Psychological distress measured by anxiety, depression and hostility was positively associated, and happiness and life satisfaction were inversely associated with average Intercellular Adhesion Molecule-1 (ICAM-1) and coagulation factor III (F3) promoter methylation levels. There was some evidence that hostility was positively associated with toll-like receptor 2 (TLR-2) promoter methylation, and that life satisfaction was inversely associated with TLR-2 and inducible nitric oxide synthase (iNOS) promoter methylation. We observed less consistent and significant associations between psychological factors and average methylation for promoters of the genes for glucocorticoid receptor (NR3C1), interferon-γ (IFN-γ) and interleukin 6 (IL-6). Conclusions These findings suggest that positive and negative

  15. DNA Methylation in the Neuropeptide S Receptor 1 (NPSR1) Promoter in Relation to Asthma and Environmental Factors

    PubMed Central

    Reinius, Lovisa E.; Gref, Anna; Sääf, Annika; Acevedo, Nathalie; Joerink, Maaike; Kupczyk, Maciej; D'Amato, Mauro; Bergström, Anna; Melén, Erik; Scheynius, Annika; Dahlén, Sven-Erik; Pershagen, Göran; Söderhäll, Cilla; Kere, Juha

    2013-01-01

    Asthma and allergy are complex disorders influenced by both inheritance and environment, a relationship that might be further clarified by epigenetics. Neuropeptide S Receptor 1 (NPSR1) has been associated with asthma and allergy and a study suggested modulation of the genetic risk by environmental factors. We aimed to study DNA methylation in the promoter region of NPSR1 in relation to asthma and environmental exposures. Electrophoretic Mobility Shift Assay (EMSA) was used to investigate potential functional roles of both genotypes and methylation status in the NPSR1 promoter. DNA methylation was analysed using EpiTYPER in blood samples from two well-characterized cohorts; the BIOAIR study of severe asthma in adults and the Swedish birth cohort BAMSE. We observed that DNA methylation and genetic variants in the promoter influenced the binding of nuclear proteins to DNA, suggesting functional relevance. Significant, although small, differences in methylation were related to both adult severe asthma (p = 0.0001) and childhood allergic asthma (p = 0.01). Furthermore, DNA methylation was associated with exposures such as current smoking in adults for two CpG sites (p = 0.005 and 0.04), parental smoking during infancy in the children (p = 0.02) and in which month the sample was taken (p = 0.01). In summary, DNA methylation levels in the promoter of NPSR1 showed small but significant associations with asthma, both in adults and in children, and to related traits such as allergy and certain environmental exposures. Both genetic variation and the methylated state of CpG sites seem to have an effect on the binding of nuclear proteins in the regulatory region of NPSR1 suggesting complex regulation of this gene in asthma and allergy. PMID:23372674

  16. Promoter CpG island methylation of RET predicts poor prognosis in stage II colorectal cancer patients.

    PubMed

    Draht, Muriel X G; Smits, Kim M; Tournier, Benjamin; Jooste, Valerie; Chapusot, Caroline; Carvalho, Beatriz; Cleven, Arjen H G; Derks, Sarah; Wouters, Kim A D; Belt, Eric J T; Stockmann, Hein B A C; Bril, Herman; Weijenberg, Matty P; van den Brandt, Piet A; de Bruïne, Adriaan P; Herman, James G; Meijer, Gerrit A; Piard, Françoise; Melotte, Veerle; van Engeland, Manon

    2014-05-01

    Improved prognostic stratification of patients with TNM stage II colorectal cancer (CRC) is desired, since 20-30% of high-risk stage II patients may die within five years of diagnosis. This study was conducted to investigate REarranged during Transfection (RET) gene promoter CpG island methylation as a possible prognostic marker for TNM stage II CRC patients. The utility of RET promoter CpG island methylation in tumors of stage II CRC patients as a prognostic biomarker for CRC related death was studied in three independent series (including 233, 231, and 294 TNM stage II patients, respectively) by using MSP and pyrosequencing. The prognostic value of RET promoter CpG island methylation was analyzed by using Cox regression analysis. In the first series, analyzed by MSP, CRC stage II patients (n = 233) with RET methylated tumors had a significantly worse overall survival as compared to those with unmethylated tumors (HRmultivariable = 2.51, 95%-CI: 1.42-4.43). Despite a significant prognostic effect of RET methylation in stage III patients of a second series, analyzed by MSP, the prognostic effect in stage II patients (n = 231) was not statistically significant (HRmultivariable = 1.16, 95%-CI 0.71-1.92). The third series (n = 294), analyzed by pyrosequencing, confirmed a statistically significant association between RET methylation and poor overall survival in stage II patients (HRmultivariable = 1.91, 95%-CI: 1.04-3.53). Our results show that RET promoter CpG island methylation, analyzed by two different techniques, is associated with a poor prognosis in stage II CRC in two independent series and a poor prognosis in stage III CRC in one series. RET methylation may serve as a useful and robust tool for clinical practice to identify high-risk stage II CRC patients with a poor prognosis. This merits further investigation. PMID:24560444

  17. Epigenetic Heterogeneity of B-Cell Lymphoma: DNA Methylation, Gene Expression and Chromatin States.

    PubMed

    Hopp, Lydia; Löffler-Wirth, Henry; Binder, Hans

    2015-01-01

    Mature B-cell lymphoma is a clinically and biologically highly diverse disease. Its diagnosis and prognosis is a challenge due to its molecular heterogeneity and diverse regimes of biological dysfunctions, which are partly driven by epigenetic mechanisms. We here present an integrative analysis of DNA methylation and gene expression data of several lymphoma subtypes. Our study confirms previous results about the role of stemness genes during development and maturation of B-cells and their dysfunction in lymphoma locking in more proliferative or immune-reactive states referring to B-cell functionalities in the dark and light zone of the germinal center and also in plasma cells. These dysfunctions are governed by widespread epigenetic effects altering the promoter methylation of the involved genes, their activity status as moderated by histone modifications and also by chromatin remodeling. We identified four groups of genes showing characteristic expression and methylation signatures among Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma and multiple myeloma. These signatures are associated with epigenetic effects such as remodeling from transcriptionally inactive into active chromatin states, differential promoter methylation and the enrichment of targets of transcription factors such as EZH2 and SUZ12. PMID:26371046

  18. Epigenetic Heterogeneity of B-Cell Lymphoma: DNA Methylation, Gene Expression and Chromatin States

    PubMed Central

    Hopp, Lydia; Löffler-Wirth, Henry; Binder, Hans

    2015-01-01

    Mature B-cell lymphoma is a clinically and biologically highly diverse disease. Its diagnosis and prognosis is a challenge due to its molecular heterogeneity and diverse regimes of biological dysfunctions, which are partly driven by epigenetic mechanisms. We here present an integrative analysis of DNA methylation and gene expression data of several lymphoma subtypes. Our study confirms previous results about the role of stemness genes during development and maturation of B-cells and their dysfunction in lymphoma locking in more proliferative or immune-reactive states referring to B-cell functionalities in the dark and light zone of the germinal center and also in plasma cells. These dysfunctions are governed by widespread epigenetic effects altering the promoter methylation of the involved genes, their activity status as moderated by histone modifications and also by chromatin remodeling. We identified four groups of genes showing characteristic expression and methylation signatures among Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma and multiple myeloma. These signatures are associated with epigenetic effects such as remodeling from transcriptionally inactive into active chromatin states, differential promoter methylation and the enrichment of targets of transcription factors such as EZH2 and SUZ12. PMID:26371046

  19. H19 gene methylation status is associated with male infertility

    PubMed Central

    LI, XIAO-PING; HAO, CHAO-LIANG; WANG, QIAN; YI, XIAO-MEI; JIANG, ZHI-SHENG

    2016-01-01

    The present study investigated the H19 gene methylation status in male infertility. Between March 2013 and June 2014, semen samples were collected from 15 normal fertile males and 15 males experiencing infertility, and routine analysis and sperm morphological assessment were performed. The semen samples were subjected to density gradient centrifugation to separate the sperm fraction, and genomic DNA from the sperms was extracted and treated for bisulfite modification. Following in vitro amplification by polymerase chain reaction (PCR), the purified PCR products were cloned into pMD®18-T vectors and successful cloning was confirmed by restriction enzyme digestion. Positive clones were sequenced and the DNA methylation status was analyzed. The overall methylation rate in the normal fertile group was 100% (270/270), whereas in the infertile group the methylation rate was lower at 94.1% (525/558), revealing a statistically significant decrease in overall methylation rate in the infertile patients compared with the control group (χ2=15.12; P<0.001). The average methylation rates of CpG 1, 3 and 6 in the infertile group were statistically different from those in the normal control group (all P<0.05). The abnormal methylation of imprinted gene H19 is associated with male infertility, suggesting that H19 may serve as a biomarker for the detection of defects in human spermiogenesis. PMID:27347077

  20. Validation study of genes with hypermethylated promoter regions associated with prostate cancer recurrence

    PubMed Central

    Stott-Miller, Marni; Zhao, Shanshan; Wright, Jonathan L.; Kolb, Suzanne; Bibikova, Marina; Klotzle, Brandy; Ostrander, Elaine A.; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L.

    2014-01-01

    Background One challenge in prostate cancer (PCa) is distinguishing indolent from aggressive disease at diagnosis. DNA promoter hypermethylation is a frequent epigenetic event in PCa, but few studies of DNA methylation in relation to features of more aggressive tumors or PCa recurrence have been completed. Methods We used the Infinium® HumanMethylation450 BeadChip to assess DNA methylation in tumor tissue from 407 patients with clinically localized PCa who underwent radical prostatectomy. Recurrence status was determined by follow-up patient surveys, medical record review, and linkage with the SEER registry. The methylation status of 14 genes for which promoter hypermethylation was previously correlated with advanced disease or biochemical recurrence was evaluated. Average methylation level for promoter region CpGs in patients who recurred compared to those with no evidence of recurrence was analyzed. For two genes with differential methylation, time to recurrence was examined. Results During an average follow-up of 11.7 years, 104 (26%) patients recurred. Significant promoter hypermethylation in at least 50% of CpG sites in two genes, ABHD9 and HOXD3, was found in tumors from patients who recurred compared to those without recurrence. Evidence was strongest for HOXD3 (lowest P = 9.46x10−6), with higher average methylation across promoter region CpGs associated with reduced recurrence-free survival (P = 2×10−4). DNA methylation profiles did not differ by recurrence status for the other genes. Conclusions These results validate the association between promoter hypermethylation of ADHB9 and HOXD3 and PCa recurrence. Impact Tumor DNA methylation profiling may help distinguish PCa patients at higher risk for disease recurrence. PMID:24718283

  1. Association of DNA methylation and monoamine oxidase A gene expression in the brains of different dog breeds.

    PubMed

    Eo, JungWoo; Lee, Hee-Eun; Nam, Gyu-Hwi; Kwon, Yun-Jeong; Choi, Yuri; Choi, Bong-Hwan; Huh, Jae-Won; Kim, Minkyu; Lee, Sang-Eun; Seo, Bohyun; Kim, Heui-Soo

    2016-04-15

    The monoamine oxidase A (MAOA) gene is an important candidate gene for human behavior that encodes an enzyme regulating the metabolism of key neurotransmitters. The regulatory mechanisms of the MAOA gene in dogs are yet to be elucidated. We measured MAOA gene transcription and analyzed the VNTR genotype and methylation status of the gene promoter region in different dog breeds to determine whether MAOA expression is correlated with the MAOA genotype or epigenetic modification in dogs. We found brain-specific expression of the MAOA gene and different transcription levels in different dog breeds including Beagle, Sapsaree, and German shepherd, and also a robust association of the DNA methylation of the gene promoter with mRNA levels. However, the 90 bp tandem repeats that we observed near the transcription start site were not variable, indicating no correlation with canine MAOA activity. These results show that differential DNA methylation in the MAOA promoter region may affect gene expression by modulating promoter activity. Moreover, the distinctive patterns of MAOA expression and DNA methylation may be involved in breed-specific or individual behavioral characteristics, such as aggression, because behavioral phenotypes are related to different physiological and neuroendocrine responses. PMID:26784655

  2. Light-regulated and cell-specific methylation of the maize PEPC promoter

    PubMed Central

    Tolley, Ben J.; Woodfield, Helen; Wanchana, Samart; Bruskiewich, Richard; Hibberd, Julian M.

    2012-01-01

    The molecular mechanisms governing PEPC expression in maize remain to be fully defined. Differential methylation of a region in the PEPC promoter has been shown to correlate with transcript accumulation, however, to date, investigations into the role of DNA methylation in maize PEPC expression have relied on the use of methylation-sensitive restriction enzymes. Bisulphite sequencing was used here to provide a single-base resolution methylation map of the maize PEPC promoter. It is shown that four cytosine residues in the PEPC promoter are heavily methylated in maize root tissue. In leaves, de-methylation of these cytosines is dependent on illumination and is coincident with elevated PEPC expression. Furthermore, light-regulated de-methylation of these cytosines occurs only in mesophyll cells. No methylation was discovered in the 0.6 kb promoter required for mesophyll-specific expression indicating that cytosine methylation is not required to direct the cell-specificity of PEPC expression. This raises interesting questions regarding the function of the cell-specific cytosine de-methylation observed in the upstream region of the PEPC promoter. PMID:22143916

  3. Placental leptin gene methylation and macrosomia during normal pregnancy.

    PubMed

    Xu, Xinyun; Yang, Xinjun; Liu, Ziwei; Wu, Kele; Liu, Zheng; Lin, Chong; Wang, Yuhuan; Yan, Hongtao

    2014-03-01

    The present study examined the placental leptin (LEP) DNA methylation and mRNA levels in macrosomic infants from normal pregnancies. In total, 49 neonates with macrosomia, i.e., high birth weights of ≥ 4,000 g, and 52 neonates with normal birth weights between 2,500 g and 4,000 g were recruited from The Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, Zhejiang) in China. Placental LEP promoter methylation and LEP transcript levels were determined by Sequenom MassARRAY and quantitative PCR, respectively. LEP promoter methylation and mRNA levels were not significantly different between the individuals with macrosomia and the controls. However, stratification revealed that individual CpG dinucleotides were hypermethylated in macrosomia (P<0.05) in primiparous females and at 39 weeks of gestation (P<0.05). Variations in methylation did not affect placental LEP expression. It was concluded that the methylation of the placental LEP promoter was altered during a specific gestational period in macrosomia following a normal pregnancy and under certain conditions. However, placental LEP expression was not affected. PMID:24452268

  4. DNA Methylation Analysis of the Angiotensin Converting Enzyme (ACE) Gene in Major Depression

    PubMed Central

    Zill, Peter; Baghai, Thomas C.; Schüle, Cornelius; Born, Christoph; Früstück, Clemens; Büttner, Andreas; Eisenmenger, Wolfgang; Varallo-Bedarida, Gabriella; Rupprecht, Rainer; Möller, Hans-Jürgen; Bondy, Brigitta

    2012-01-01

    Background The angiotensin converting enzyme (ACE) has been repeatedly discussed as susceptibility factor for major depression (MD) and the bi-directional relation between MD and cardiovascular disorders (CVD). In this context, functional polymorphisms of the ACE gene have been linked to depression, to antidepressant treatment response, to ACE serum concentrations, as well as to hypertension, myocardial infarction and CVD risk markers. The mostly investigated ACE Ins/Del polymorphism accounts for ∼40%–50% of the ACE serum concentration variance, the remaining half is probably determined by other genetic, environmental or epigenetic factors, but these are poorly understood. Materials and Methods The main aim of the present study was the analysis of the DNA methylation pattern in the regulatory region of the ACE gene in peripheral leukocytes of 81 MD patients and 81 healthy controls. Results We detected intensive DNA methylation within a recently described, functional important region of the ACE gene promoter including hypermethylation in depressed patients (p = 0.008) and a significant inverse correlation between the ACE serum concentration and ACE promoter methylation frequency in the total sample (p = 0.02). Furthermore, a significant inverse correlation between the concentrations of the inflammatory CVD risk markers ICAM-1, E-selectin and P-selectin and the degree of ACE promoter methylation in MD patients could be demonstrated (p = 0.01 - 0.04). Conclusion The results of the present study suggest that aberrations in ACE promoter DNA methylation may be an underlying cause of MD and probably a common pathogenic factor for the bi-directional relationship between MD and cardiovascular disorders. PMID:22808171

  5. Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors.

    PubMed

    Chen, Xiaofei; Liu, Liang; Mims, Jade; Punska, Elizabeth C; Williams, Kristin E; Zhao, Weiling; Arcaro, Kathleen F; Tsang, Allen W; Zhou, Xiaobo; Furdui, Cristina M

    2015-01-01

    Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC. PMID:25961636

  6. Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors

    PubMed Central

    Chen, Xiaofei; Liu, Liang; Mims, Jade; Punska, Elizabeth C; Williams, Kristin E; Zhao, Weiling; Arcaro, Kathleen F; Tsang, Allen W; Zhou, Xiaobo; Furdui, Cristina M

    2015-01-01

    Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC. PMID:25961636

  7. Cell-specific DNA methylation patterns of retina-specific genes.

    PubMed

    Merbs, Shannath L; Khan, Miriam A; Hackler, Laszlo; Oliver, Verity F; Wan, Jun; Qian, Jiang; Zack, Donald J

    2012-01-01

    Many studies have demonstrated that epigenetic mechanisms are important in the regulation of gene expression during embryogenesis, gametogenesis, and other forms of tissue-specific gene regulation. We sought to explore the possible role of epigenetics, specifically DNA methylation, in the establishment and maintenance of cell type-restricted gene expression in the retina. To assess the relationship between DNA methylation status and expression level of retinal genes, bisulfite sequence analysis of the 1000 bp region around the transcription start sites (TSS) of representative rod and cone photoreceptor-specific genes and gene expression analysis were performed in the WERI and Y79 human retinoblastoma cell lines. Next, the homologous genes in mouse were bisulfite sequenced in the retina and in non-expressing tissues. Finally, bisulfite sequencing was performed on isolated photoreceptor and non-photoreceptor retinal cells isolated by laser capture microdissection. Differential methylation of rhodopsin (RHO), retinal binding protein 3 (RBP3, IRBP) cone opsin, short-wave-sensitive (OPN1SW), cone opsin, middle-wave-sensitive (OPN1MW), and cone opsin, long-wave-sensitive (OPN1LW) was found in the retinoblastoma cell lines that inversely correlated with gene expression levels. Similarly, we found tissue-specific hypomethylation of the promoter region of Rho and Rbp3 in mouse retina as compared to non-expressing tissues, and also observed hypomethylation of retinal-expressed microRNAs. The Rho and Rbp3 promoter regions were unmethylated in expressing photoreceptor cells and methylated in non-expressing, non-photoreceptor cells from the inner nuclear layer. A third regional hypomethylation pattern of photoreceptor-specific genes was seen in a subpopulation of non-expressing photoreceptors (Rho in cones from the Nrl -/- mouse and Opn1sw in rods). These results demonstrate that a number of photoreceptor-specific genes have cell-specific differential DNA methylation that

  8. Association between the methylation status of the MGMT promoter in bone marrow specimens and chemotherapy outcomes of patients with acute myeloid leukemia

    PubMed Central

    HONG, QINGXIAO; CHEN, XIAOYING; YE, HUADAN; ZHOU, ANNAN; GAO, YUTING; JIANG, DANJIE; WU, XIAODONG; TIAN, BINGRU; CHEN, YOUFEN; WANG, MING; XIE, JIPING; XIA, YONGMING; DUAN, SHIWEI

    2016-01-01

    The O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a tumor suppressor gene that is associated with the risk of developing acute myeloid leukemia (AML). However, the association between the methylation status of the MGMT promoter and the chemotherapeutic outcomes of patients with AML remains unknown. In the present study, 30 bone marrow samples derived from patients with AML were collected prior and subsequent to chemotherapy. The methylation status of the MGMT promoter in the bone marrow specimens was determined by methylation-specific polymerase chain reaction. The results indicated that the methylation status of the MGMT promoter was influenced by different chemotherapeutic regimens. The MGMT methylation status of M4 patients (3 out of 6) were more chemosensitive, compared with that of patients with other AML subtypes (M1, 1 out of 3; M2, 0 out of 8; M3, 3 out of 7; M5, 0 out of 3; and M6, 1 out of 3). Age-based analysis revealed that the group aged ≤60 years (7 out of 24 patients) exhibited more methylation changes than patients aged >60 years (1 out of 6). Male patients (4 out of 13) were more susceptible to chemotherapy-induced methylation changes than female patients (4 out of 17). Thus, the methylation status of the MGMT promoter may serve as a potential biomarker to predict the therapeutic outcomes in male AML patients. However, further studies in larger sample sets are required to confirm the present findings. PMID:27073563

  9. Repetitive elements and enforced transcriptional repression co-operate to enhance DNA methylation spreading into a promoter CpG-island

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Repression of many tumor suppressor genes in cancer is concurrent with aberrantly increased DNA methylation levels at promoter CpG islands (CGIs). About one-fourth of empirically defined human promoters are surrounded by or contain clustered repetitive elements. It was previously observed that a sha...

  10. Identification of differentially methylated regions in new genes associated with knee osteoarthritis.

    PubMed

    Bonin, Carolina A; Lewallen, Eric A; Baheti, Saurabh; Bradley, Elizabeth W; Stuart, Michael J; Berry, Daniel J; van Wijnen, Andre J; Westendorf, Jennifer J

    2016-01-15

    Epigenetic changes in articular chondrocytes are associated with osteoarthritis (OA) disease progression. Numerous studies have identified differentially methylated cytosines in OA tissues; however, the consequences of altered CpG methylation at single nucleotides on gene expression and phenotypes are difficult to predict. With the objective of detecting novel genes relevant to OA, we conducted a genome-wide assessment of differentially methylated sites (DMSs) and differentially methylated regions (DMRs). DNA was extracted from visually damaged and normal appearing, non-damaged human knee articular cartilage from the same joint and then subjected to reduced representation bisulfite sequencing. DMRs were identified using a genome-wide systematic bioinformatics approach. A sliding-window of 500 bp was used for screening the genome for regions with clusters of DMSs. Gene expression levels were assessed and cell culture demethylation experiments were performed to further examine top candidate genes associated with damaged articular cartilage. More than 1000 DMRs were detected in damaged osteoarthritic cartilage. Nineteen of these contained five or more DMSs and were located in gene promoters or first introns and exons. Gene expression assessment revealed that hypermethylated DMRs in damaged samples were more consistently associated with gene repression than hypomethylated DMRs were with gene activation. Accordingly, a demethylation agent induced expression of most hypermethylated genes in chondrocytes. Our study revealed the utility of a systematic DMR search as an alternative to focusing on single nucleotide data. In particular, this approach uncovered promising candidates for functional studies such as the hypermethylated protein-coding genes FOXP4 and SHROOM1, which appear to be linked to OA pathology in humans and warrant further investigation. PMID:26484395

  11. Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

    PubMed Central

    Husseiny, Mohamed I.; Kaye, Alexander; Zebadua, Emily; Kandeel, Fouad; Ferreri, Kevin

    2014-01-01

    The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy. PMID:24722187

  12. Association of APC, GSTP1 and SOCS1 promoter methylation with the risk of hepatocellular carcinoma: a meta-analysis.

    PubMed

    Liu, Meng; Cui, Lian-Hua; Li, Cheng-Cheng; Zhang, Li

    2015-11-01

    Studies of the relationships of adenomatous polyposis coli (APC), glutathione-S-transferase P1 (GSTP1) and suppressor of the cytokine signalling 1 (SOCS1) promoter region methylation with the risk of hepatocellular carcinoma (HCC) have yielded inconsistent results. We carried out the current meta-analysis to comprehensively assess the associations between APC, GSTP1 and SOCS1 promoter methylation frequency and the risk of HCC. All relevant reports were identified by searching the PubMed, Embase, Web of Science, CNKI and the Chinese BioMedical Literature databases before 1 March 2014, with restriction to articles published in the Chinese and English languages. Pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to investigate the rates of APC, GSTP1 and SOCS1 promoter methylation and the risk of HCC. Our meta-analysis identified relationships of APC (12 studies with 592 HCC tumour tissues), GSTP1 (14 studies including 646 HCC tumour tissues) and SOCS1 (11 studies with 512 HCC tumour tissues) promoter methylation with the risk of HCC. Compared with paracancerous tissues, the pooled ORs of APC, GSTP1 and SOCS1 promoter region methylation in HCC cancer tissues were 5.32 (95% CI=2.96-9.56), 5.65, (95% CI=3.41-9.35) and 2.73 (95% CI=1.37-5.44), respectively. Compared with normal liver tissues as controls, the pooled ORs of APC, GSTP1 and SOCS1 promoter region methylation in HCC cancer tissues were 20.43 (95% CI=5.56-75.08), 18.78 (95% CI=5.76-61.19) and 13.00 (95% CI=5.20-32.47), respectively. Subgroup analysis by ethnicity showed that APC, GSTP1 and SOCS1 promoter methylation was associated significantly with the risk of HCC in both Asian and White populations (all P<0.05). Our meta-analysis suggested strong associations between APC, GSTP1 and SOCS1 gene promoter methylation and the risk of HCC, suggesting these to be promising biomarkers for HCC. PMID:25853848

  13. Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus.

    PubMed

    Saunderson, Emily A; Spiers, Helen; Mifsud, Karen R; Gutierrez-Mecinas, Maria; Trollope, Alexandra F; Shaikh, Abeera; Mill, Jonathan; Reul, Johannes M H M

    2016-04-26

    Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5'-cytosine-phosphate-guanine-3') sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental stimuli in

  14. The APOE Gene is Differentially Methylated in Alzheimer's Disease.

    PubMed

    Foraker, Jessica; Millard, Steven P; Leong, Lesley; Thomson, Zachary; Chen, Sunny; Keene, C Dirk; Bekris, Lynn M; Yu, Chang-En

    2015-01-01

    The ɛ4 allele of the human apolipoprotein E gene (APOE) is a well-proven genetic risk factor for the late onset form of Alzheimer's disease (AD). However, the biological mechanisms through which the ɛ4 allele contributes to disease pathophysiology are incompletely understood. The three common alleles of APOE, ɛ2, ɛ3 and ɛ4, are defined by two single nucleotide polymorphisms (SNPs) that reside in the coding region of exon 4, which overlaps with a well-defined CpG island (CGI). Both SNPs change not only the protein codon but also the quantity of CpG dinucleotides, primary sites for DNA methylation. Thus, we hypothesize that the presence of an ɛ4 allele changes the DNA methylation landscape of the APOE CGI and that such epigenetic alteration contributes to AD susceptibility. To explore the relationship between APOE genotype, AD risk, and DNA methylation of the APOE CGI, we applied bisulfite pyrosequencing and evaluated methylation profiles of postmortem brain from 15 AD and 10 control subjects. We observed a tissue-specific decrease in DNA methylation with AD and identified two AD-specific differentially methylated regions (DMRs), which were also associated with APOE genotype. We further demonstrated that one DMR was completely un-methylated in a sub-population of genomes, possibly due to a subset of brain cells carrying deviated APOE methylation profiles. These data suggest that the APOE CGI is differentially methylated in AD brain in a tissue- and APOE-genotype-specific manner. Such epigenetic alteration might contribute to neural cell dysfunction in AD brain. PMID:26402071

  15. P04.10PREDICTIVE VALUE OF MGMT PROMOTER METHYLATION QUANTITATIVE ASSESSMENT IN GLIOBLASTOMA, WHICH OPTIMAL CUT-POINT?

    PubMed Central

    de Saint-Denis, T.; Lerhun, E.; Ramirez, C.; Devos, P.; Maurage, C.A.; Dubois, F.; Reyns, N.; Escande, F.

    2014-01-01

    BACKGROUND: The methylation in the promoter region of the MGMT gene encoding the DNA repair protein O6-methyl guanine-DNA methyl transferase is a predictive marker for benefit from alkylating agent therapy and a prognostic factor in glioblastoma. Pyrosequencing-based assessement of MGMT promoter status is of particularly interest, but analytical methods and cut-points may varied from one center to another. We intend to establish a correlation between quantitative methylation of MGMT promoter values and overall survival (OS) or progression-free survival (PFS). METHODS: The clinical and biological data of 159 newly diagnosed glioblastoma recruited in Lille University Hospital between 2008 and 2011 were retrospectively collected. For the patients, the methylation status has been assessed by pyrosequencing on 5 CpG islands. Statistical analyses were performed using SAS software V9.3. RESULTS: Median age of the patients was 61 years. Sixty three patients were male. Of 159 patients, 156 underwent a surgery (40,4% of complete resection), 3 patients were diagnosed only by biopsies. 123 received concomitant radiotherapy and temozolomide and adjuvant temozolomide. The median PFS and the median OS were 9,9 and 15,9. The most significant cut-off methylation determined on 5 sites mean methylaion value was 12% for both predictive and pronostic evaluations. Site 1 methylation status was the best predictive value for both PFS and OS compared to the other CpG islands. On this site 1, a >3% methylation status was a significant predictive factor for reponse to standard treatment for PFS and a >25% site 1 methylation status was a strong pronostic factor for 2 years survival (OS) (47%). CONCLUSIONS: Even a low MGMT methylation (>3%) status on selected CpG site can already be a predictive factor for response to treatment in glioblastoma and allow us to reduce the cut-off values. Highest methylations status are correlated with the longest overall survival and therefore confirm the

  16. Effect of DR4 promoter methylation on the TRAIL-induced apoptosis in lung squamous carcinoma cell.

    PubMed

    Wang, Wenwu; Qi, Xiaoyan; Wu, Minghua

    2015-10-01

    The aim of the present study was to examine the relationship between DR4 methylation status and gene expression and to determine whether DR4 methylation status affects TRAIL-induced apoptosis in lung squamous carcinoma cells. MSP, RT-PCR and western blot analysis were applied to detect the methylation status and gene expression. An MTT assay was used to detect the cell proliferation inhibition rate and flow cytometry was utilized to detect the apoptotic rate. The results showed that there was no association of the apoptotic rate with the clinicopathological characteristics for 80.6% of 36 lung squamous carcinoma patients in the methylation status (P>0.05). In the lung squamous carcinoma patients, the probability of DR4 low expression was approximately 58.3%, which may be associated with DR4 promoter methylation. The results also showed that a low expression of DR4 was correlated with the prognosis of patients. The in vitro experiments suggested DR4 genes of H226 and SK-MES-1 cells were in the methylation status and their mRNA and proteins had a low expression. Following intervention with 5-Aza-CdR, the DR4 genes of H226 and SK-MES-1 cells were in the unmethylation status and their mRNA and protein expression was significantly upregulated compared with the pre-interference ones, with differences being statistically significant (P<0.05). In addition, following interference with 5-Aza-CdR, H226 and SK-MES-1 cells became significantly sensitive to TRAIL (P<0.05). The results revealed 5-Aza‑CdR was able to reverse DR4 methy-lation status to upregulate its expression, thereby increasing the TRAIL-induced apoptosis in lung squamous carcinoma cells. Therefore, combining 5-Aza-CdR and TRAIL is a new strategy for treating lung squamous carcinoma. PMID:26238205

  17. Folding-promoted methylation of a helical DMAP analogue.

    PubMed

    Heemstra, Jennifer M; Moore, Jeffrey S

    2004-02-18

    The methylation rate for a series of pyridine-containing phenylene ethynylene oligomers shows a nonlinear dependence on chain length, with a significant rate enhancement observed for oligomers that adopt a folded, helical conformation. The folded structure provides a microenvironment that lowers the energy barrier for the methylation reaction. Of these noncovalent interactions, the largest stabilization may arise from binding of methyl iodide in the hydrophobic cavity of the folded oligomer, in close proximity to the pyridine nucleophile. PMID:14871092

  18. Prognosis value of MGMT promoter methylation for patients with lung cancer: a meta-analysis

    PubMed Central

    Chen, Chao; Hua, Haiqing; Han, Chenglong; Cheng, Yuan; Cheng, Yin; Wang, Zhen; Bao, Jutao

    2015-01-01

    The role of MGMT promoter methylation in lung cancer (LC) remains controversial. To clarify the association of MGMT promoter methylation with survival in LC, we performed a meta-analysis of the literature with meta-analysis. Trials were selected for further analysis if they provided an independent assessment of MGMT promoter methylation in LC and reported the survival data in the context of MGMT promoter methylation status. Subgroup analyses were conducted according to the study characteristic. A total of 9 trials, which comprised 859 patients, were included in the meta-analysis. The combined hazard ratio (HR) of 1.27 [95% CI 0.88-1.82; test for heterogeneity P = 0.027] suggests that MGMT promoter methylation has none impact on patient survival. In Stage I-III or younger populations, a significant association was found for MGMT promoter methylation in the prognosis of LC. In addition, the heterogeneity disappeared when the analysis was restricted to Stage I-III LC. Our analysis indicates that MGMT promoter methylation in stage I-III or younger patients was significantly correlated with wore survival. Further study is needed to determine these specific subgroups of LC patients. PMID:26617891

  19. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure

    PubMed Central

    Chater-Diehl, Eric J.; Castellani, Christina A.; Alberry, Bonnie L.; Singh, Shiva M.

    2016-01-01

    The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse’s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as “Free radical scavenging”. We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was “Peroxisome biogenesis”; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD. PMID:27136348

  20. Nucleosomal promoter variation generates gene expression noise

    PubMed Central

    Brown, Christopher R.; Boeger, Hinrich

    2014-01-01

    Gene product molecule numbers fluctuate over time and between cells, confounding deterministic expectations. The molecular origins of this noise of gene expression remain unknown. Recent EM analysis of single PHO5 gene molecules of yeast indicated that promoter molecules stochastically assume alternative nucleosome configurations at steady state, including the fully nucleosomal and nucleosome-free configuration. Given that distinct configurations are unequally conducive to transcription, the nucleosomal variation of promoter molecules may constitute a source of gene expression noise. This notion, however, implies an untested conjecture, namely that the nucleosomal variation arises de novo or intrinsically (i.e., that it cannot be explained as the result of the promoter’s deterministic response to variation in its molecular surroundings). Here, we show—by microscopically analyzing the nucleosome configurations of two juxtaposed physically linked PHO5 promoter copies—that the configurational variation, indeed, is intrinsically stochastic and thus, a cause of gene expression noise rather than its effect. PMID:25468975

  1. Sox17 promoter methylation in plasma DNA is associated with poor survival and can be used as a prognostic factor in breast cancer.

    PubMed

    Fu, Deyuan; Ren, Chuanli; Tan, Haosheng; Wei, Jinli; Zhu, Yuxiang; He, Chunlan; Shao, Wenxi; Zhang, Jiaxin

    2015-03-01

    Aberrant DNA methylation that leads to the inactivation of tumor suppressor genes is known to play an important role in the development and progression of breast cancer. Methylation status of cancer-related genes is considered to be a promising biomarker for the early diagnosis and prognosis of tumors. This study investigated the methylation status of the Sox17 gene in breast cancer tissue and its corresponding plasma DNA to evaluate the association of methylation levels with clinicopathological parameters and prognosis.The methylation status of the Sox17 gene promoter was evaluated with methylation-specific polymerase chain reaction (MSP) in 155 paired breast cancer tissue and plasma samples and in 60 paired normal breast tissue and plasma samples. Association of Sox17 methylation status with clinicopathological parameters was analyzed by χ tests. Overall and disease-free survival (DFS) curves were calculated using Kaplan-Meier analysis, and the differences between curves were analyzed by log-rank tests.The frequency of Sox17 gene methylation was 72.9% (113/155) in breast cancer tissues and 58.1% (90/155) in plasma DNA. Sox17 gene methylation was not found in normal breast tissues or in their paired plasma DNA. There was a significant correlation of Sox17 methylation between corresponding tumor tissues and paired plasma DNA (r = 0.688, P < 0.001). Aberrant Sox17 methylation in cancer tissues and in plasma DNA was significantly associated with the tumor node metastasis stage (P = 0.035 and P = 0.001, respectively) and with lymph node metastasis (P < 0.001 and P = 0.001, respectively). Kaplan-Meier survival curves showed that aberrant Sox17 promoter methylation in cancer tissues and plasma DNA was associated with poor DFS (P < 0.005) and overall survival (OS) (P < 0.005). Multivariate analysis showed that Sox17 methylation in plasma DNA was an independent prognostic factor in breast cancer for both DFS (P = 0.020; hazard ratio [HR] = 2.142; 95% confidence

  2. Promoter methylation regulates the abundance of osa-miR393a in contrasting rice genotypes under salinity stress.

    PubMed

    Ganie, Showkat Ahmad; Dey, Narottam; Mondal, Tapan Kumar

    2016-01-01

    MicroRNAs (miRNAs) are important molecules that regulate gene expression under salinity stress. Despite their evolutionary conservation, these regulatory elements have been shown to behave differently in different plant species under a particular environmental stress. In this study, we investigated the behavior of salt responsive osa-miR393a and its target gene (TIR1, LOC_Os05g05800) in salt-tolerant (FL478) and salt-sensitive (IR29) rice genotypes. It was found that the mature and precursor sequences of osa-miR393a as well as its cleavage site in TIR1 were conserved among salt tolerant and sensitive genotypes. Promoters of different salt-responsive miRNAs were also found to be less variable between salt-tolerant and salt-susceptible genotypes. Analysis of gene expression, promoter methylation, and cis-element abundance showed that osa-miR393a behaves differently in FL478 and IR29. Salt stress altered the expression pattern of osa-miR393a-TIR1 module in a time-dependent manner in the roots and shoots of two genotypes. Promoter methylation of this regulatory module was also altered at different time points under salt stress. Expression analysis in two genotypes indicated the overall down-regulation of osa-miR393a and up-regulation of TIR1 in FL478 and their reciprocal regulation in IR29. The expression results were complemented by the differential promoter methylation and cis-element abundance of this regulatory module. Together, the results of transcript abundance and promoter methylation of osa-miR393a-TIR1 module signified the association between these two processes which is reported for the first time in plants to the best of our knowledge. PMID:26319531

  3. Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

    PubMed Central

    2012-01-01

    Background Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression. Methods CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter. Results Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2′-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2. Conclusion These data suggest that global and gene-specific changes in

  4. Evaluation of methylation status of the eNOS promoter at birth in relation to childhood bone mineral content

    PubMed Central

    Harvey, Nicholas C.; Lillycrop, Karen A.; Garratt, Emma; Sheppard, Allan; McLean, Cameron; Burdge, Graham; Slater-Jefferies, Jo; Rodford, Joanne; Crozier, Sarah; Inskip, Hazel; Emerald, Bright Starling; Gale, Catharine R; Hanson, Mark; Gluckman, Peter; Godfrey, Keith; Cooper, Cyrus

    2013-01-01

    Aim Our previous work has shown associations between childhood adiposity and perinatal methylation status of several genes in umbilical cord tissue, including endothelial nitric oxide synthase (eNOS). There is increasing evidence that eNOS is important in bone metabolism; we therefore related the methylation status of the eNOS gene promoter in stored umbilical cord to childhood bone size and density in a group of 9-year old children. Methods We used Sequenom MassARRAY to assess the methylation status of 2 CpGs in the eNOS promoter, identified from our previous study, in stored umbilical cords of 66 children who formed part of a Southampton birth cohort and who had measurements of bone size and density at age 9 years (Lunar DPXL DXA instrument). Results Percentage methylation varied greatly between subjects. For one of the two CpGs, eNOS chr7:150315553+, after taking account of age and sex there was a strong positive association between methylation status and the child’s whole body bone area (r=0.28,p=0.02), bone mineral content (r=0.34,p=0.005) and areal bone mineral density (r=0.34,p=0.005) at age 9 years. These associations were independent of previously documented maternal determinants of offspring bone mass. Conclusions Our findings suggest an association between methylation status at birth of a specific CpG within the eNOS promoter and bone mineral content in childhood. This supports a role for eNOS in bone growth and metabolism and implies that its contribution may at least in part occur during early skeletal development. PMID:22159788

  5. Child Abuse, Depression, and Methylation in Genes Involved with Stress, Neural Plasticity, and Brain Circuitry

    PubMed Central

    Weder, Natalie; Zhang, Huiping; Jensen, Kevin; Yang, Bao Zhu; Simen, Arthur; Jackowski, Andrea; Lipschitz, Deborah; Douglas-Palumberi, Heather; Ge, Margrat; Perepletchikova, Francheska; O’Laughlin, Kerry; Hudziak, James J.; Gelernter, Joel; Kaufman, Joan

    2014-01-01

    Objectives Determine if epigenetic markers predict dimensional ratings of depression in maltreated children. Method A Genome-wide methylation study was completed using the Illumina 450K BeadChip array in 94 maltreated and 96 non-traumatized children with saliva-derived DNA. The 450K BeadChip does not include any methylation sites in the exact location as sites in candidate genes previously examined in the literature, so a test for replication of prior research findings was not feasible. Results Methylation in three genes emerged as genomewide-significant predictors of depression: DNA-Binding Protein Inhibitor ID-3 (ID3); Glutamate Receptor, Ionotropic NMDA 1 (GRIN1); and Tubulin Polymerization Promoting Protein (TPPP) (p<5.0 × 10−7, all analyses). These genes are all biologically relevant–with ID3 involved in the stress response, GRIN1 involved in neural plasticity, and TPPP involved in neural circuitry development. Methylation in CpG sites in candidate genes were not predictors of depression at significance levels corrected for whole genome testing, but maltreated and control children did have significantly different beta values after Bonferroni correction at multiple methylation sites in these candidate genes (e.g., BDNF, NR3C1, FKBP5). Conclusion This study suggests epigenetic changes in ID3, GRIN1, and TPPP genes, in combination with experiences of maltreatment, may confer risk for depression in children. It adds to a growing body of literature supporting a role for epigenetic mechanisms in the pathophysiology of stress-related psychiatric disorders. While epigenetic changes are frequently long lasting, they are not necessarily permanent. Consequently, interventions to reverse the negative biological and behavioral sequelae associated with child maltreatment are briefly discussed. PMID:24655651

  6. Geminivirus Rep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing.

    PubMed

    Rodríguez-Negrete, Edgar; Lozano-Durán, Rosa; Piedra-Aguilera, Alvaro; Cruzado, Lucia; Bejarano, Eduardo R; Castillo, Araceli G

    2013-07-01

    Cytosine methylation is an epigenetic mark that promotes gene silencing and plays an important role in genome defence against transposons and invading DNA viruses. Previous data showed that the largest family of single-stranded DNA viruses, Geminiviridae, prevents methylation-mediated transcriptional gene silencing (TGS) by interfering with the proper functioning of the plant methylation cycle. Here, we describe a novel counter-defence strategy used by geminiviruses, which reduces the expression of the plant maintenance DNA methyltransferases, METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3), in both locally and systemically infected tissues. We demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widespread among geminivirus species. Additionally, we identified Rep (Replication associated protein) as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 down-regulation. The presence of Rep suppressed TGS of an Arabidopsis thaliana transgene and of host loci whose expression was strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for replication, displays TGS suppressor activity through a mechanism distinct from that thus far described for geminiviruses. PMID:23614786

  7. MIWI2 as an Effector of DNA Methylation and Gene Silencing in Embryonic Male Germ Cells.

    PubMed

    Kojima-Kita, Kanako; Kuramochi-Miyagawa, Satomi; Nagamori, Ippei; Ogonuki, Narumi; Ogura, Atsuo; Hasuwa, Hidetoshi; Akazawa, Takashi; Inoue, Norimitsu; Nakano, Toru

    2016-09-13

    During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon. PMID:27626653

  8. BDNF promoter I methylation correlates between post-mortem human peripheral and brain tissues.

    PubMed

    Stenz, Ludwig; Zewdie, Seblewongel; Laforge-Escarra, Térèse; Prados, Julien; La Harpe, Romano; Dayer, Alexandre; Paoloni-Giacobino, Ariane; Perroud, Nader; Aubry, Jean-Michel

    2015-02-01

    Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n = 98, Pearson, r = 0.48, p = 4.5 × 10(-7)). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n = 37, r = 0.74, p = 1.4 × 10(-7)). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood. PMID:25450314

  9. ASXL2 promotes proliferation of breast cancer cells by linking ERα to histone methylation.

    PubMed

    Park, U-H; Kang, M-R; Kim, E-J; Kwon, Y-S; Hur, W; Yoon, S K; Song, B-J; Park, J H; Hwang, J-T; Jeong, J-C; Um, S-J

    2016-07-14

    Estrogen receptor alpha (ERα) has a pivotal role in breast carcinogenesis by associating with various cellular factors. Selective expression of additional sex comb-like 2 (ASXL2) in ERα-positive breast cancer cells prompted us to investigate its role in chromatin modification required for ERα activation and breast carcinogenesis. Here, we observed that ASXL2 interacts with ligand E2-bound ERα and mediates ERα activation. Chromatin immunoprecipitation-sequencing analysis supports a positive role of ASXL2 at ERα target gene promoters. ASXL2 forms a complex with histone methylation modifiers including LSD1, UTX and MLL2, which all are recruited to the E2-responsive genes via ASXL2 and regulate methylations at histone H3 lysine 4, 9 and 27. The preferential binding of the PHD finger of ASXL2 to the dimethylated H3 lysine 4 may account for its requirement for ERα activation. On ASXL2 depletion, the proliferative potential of MCF7 cells and tumor size of xenograft mice decreased. Together with our finding on the higher ASXL2 expression in ERα-positive patients, we propose that ASXL2 could be a novel prognostic marker in breast cancer. PMID:26640146

  10. Quantitative Methylation Analysis of the PCDHB Gene Cluster.

    PubMed

    Banelli, Barbara; Romani, Massimo

    2015-01-01

    Long Range Epigenetic Silencing (LRES) is a repressed chromatin state of large chromosomal regions caused by DNA hypermethylation and histone modifications and is commonly observed in cancer. At 5q31 a LRES region of 800 kb includes three multi-gene clusters (PCDHA@, PCDHB@, and PCDHG@, respectively). Multiple experimental evidences have led to consider the PCDHB cluster as a DNA methylation marker of aggressiveness in neuroblastoma, second most common solid tumor in childhood. Because of its potential involvement not only in neuroblastoma but also in other malignancies, an easy and fast assay to screen the DNA methylation content of the PCDHB cluster might be useful for the precise stratification of the patients into risk groups and hence for choosing the most appropriate therapeutic protocol. Accordingly, we have developed a simple and cost-effective Pyrosequencing(®) assay to evaluate the methylation level of 17 genes in the protocadherin B cluster (PCDHB@). The rationale behind this Pyrosequencing assay can in principle be applied to analyze the DNA methylation level of any gene cluster with high homologies for screening purposes. PMID:26103900

  11. Racial differences in genome-wide methylation profiling and gene expression in breast tissues from healthy women.

    PubMed

    Song, Min-Ae; Brasky, Theodore M; Marian, Catalin; Weng, Daniel Y; Taslim, Cenny; Dumitrescu, Ramona G; Llanos, Adana A; Freudenheim, Jo L; Shields, Peter G

    2015-12-01

    Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer. PMID:26680018

  12. MGMT promoter methylation in serum and cerebrospinal fluid as a tumor-specific biomarker of glioma

    PubMed Central

    WANG, ZHENG; JIANG, WEI; WANG, YAHONG; GUO, YANG; CONG, ZHENG; DU, FANGFANG; SONG, BIN

    2015-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is a conventional technique to predict the prognosis or individualized treatment of glioma in tumor tissue following surgery or biopsy. However, the technique cannot be applied in those glioma patients with concomitant neurological dysfunctions or advanced age. The present study aimed to find a new minimally invasive and efficient alternative method for the detection of MGMT promoter methylation. The expression of MGMT promoter methylation was assessed in peripheral blood and cerebrospinal fluid (CSF), and compared to the corresponding tumor tissue from glioma patients. The 89 patients in the study [32 World Health Organization (WHO) grade II, 19 WHO grade III and 38 WHO grade IV) were pathologically-diagnosed glioma and received radiation therapy following sample collection. The resected glioma tumor tissue (89), corresponding serum (89) and CSF (78) samples were collected for the detection of MGMT promoter methylation using methylation-specific polymerase chain reaction. The sensitivity and specificity of detecting MGMT promoter methylation in CSF and serum were compared. Among the tumor tissue samples, 51/89 (57.3%) showed MGMT promoter methylation. The specificity of the detection in the CSF and serum samples reached 100%. The sensitivity of MGMT promoter methylation detection in CSF and serum were 26/40 (65.0%) and 19/51 (37.3%), respectively (P<0.05). In the WHO II, III and IV subgroups, the sensitivities of MGMT promoter methylation detection using CSF were 8/12 (66.7%), 11/18 (61.1%) and 7/10 (70.0%), respectively, which were significantly higher than the sensitivities using serum (7/21, 33.3%; 7/19, 36.8%; and 5/11, 45.5%, respectively P<0.05). Among patients with residual postoperative tumors, the sensitivities of detecting MGMT promoter methylation using CSF and serum were 18/25 (72.0%) and 10/24 (41.7%), respectively, both of which were significantly higher than the corresponding values

  13. DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia.

    PubMed

    Ashktorab, Hassan; Daremipouran, M; Goel, Ajay; Varma, Sudhir; Leavitt, R; Sun, Xueguang; Brim, Hassan

    2014-04-01

    The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject's colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands-in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)-were significantly hypermethylated in tumor vs. normal tissues (P<0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network-the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated

  14. DNA methylation and differential gene regulation in photoreceptor cell death

    PubMed Central

    Farinelli, P; Perera, A; Arango-Gonzalez, B; Trifunovic, D; Wagner, M; Carell, T; Biel, M; Zrenner, E; Michalakis, S; Paquet-Durand, F; Ekström, P A R

    2014-01-01

    Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP. PMID:25476906

  15. DNA methylation and differential gene regulation in photoreceptor cell death.

    PubMed

    Farinelli, P; Perera, A; Arango-Gonzalez, B; Trifunovic, D; Wagner, M; Carell, T; Biel, M; Zrenner, E; Michalakis, S; Paquet-Durand, F; Ekström, P A R

    2014-01-01

    Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP. PMID:25476906

  16. Frequent methylation of eyes absent 4 gene in Barrett's esophagus and esophageal adenocarcinoma.

    PubMed

    Zou, Hongzhi; Osborn, Neal K; Harrington, Jonathan J; Klatt, Kristie K; Molina, Julian R; Burgart, Lawrence J; Ahlquist, David A

    2005-04-01

    Most esophageal adenocarcinomas arise within Barrett's esophagus but the cause of this increasingly prevalent condition remains unknown. Early detection improves survival and discriminant screening markers for Barrett's esophagus and cancer are needed. This study was designed to explore the natural history of eyes absent 4 (EYA4) gene methylation in the neoplastic progression of Barrett's esophagus and to evaluate methylated EYA4 as a candidate marker. Aberrant promoter methylation of EYA4 was studied by methylation-specific PCR using bisulfite-treated DNA from esophageal adenocarcinomas, Barrett's esophagus, and normal epithelia, and then confirmed by sequencing. Eight cancer cell lines were treated with the demethylation agent 5-aza-2'-deoxycytidine, and EYA4 mRNA expression with and without treatment was quantified by real-time reverse-transcription PCR. EYA4 hypermethylation was detected in 83% (33 of 40) of esophageal adenocarcinomas and 77% (27 of 35) of Barrett's tissues, but only in 3% (2 of 58) of normal esophageal and gastric mucosa samples (P < 0.001). The unmethylated cancer cell lines had much higher EYA4 mRNA expression than the methylated cancer cell lines. Demethylation caused by 5-aza-2'-deoxycytidine increased the mRNA expression level by a median of 3.2-fold in methylated cells, but its effect on unmethylated cells was negligible. Results indicate that aberrant promoter methylation of EYA4 is very common during tumorigenesis in Barrett's esophagus, occurs in early metaplasia, seems to be an important mechanism of down-regulating EYA4 expression, and represents an intriguing candidate marker for Barrett's metaplasia and esophageal cancer. PMID:15824152

  17. Examining the Impact of Gene Variants on Histone Lysine Methylation

    PubMed Central

    Van Rechem, Capucine; Whetstine, Johnathan R.

    2015-01-01

    In recent years, there has been a boom in the amount of genome-wide sequencing data that has uncovered important and unappreciated links between certain genes, families of genes and enzymatic processes and diseases such as cancer. Such studies have highlighted the impact that chromatin modifying enzymes could have in cancer and other genetic diseases. In this review, we summarize characterized mutations and single nucleotide polymorphisms (SNPs) in histone lysine methyltransferases (KMTs), histone lysine demethylases (KDMs) and histones. We primarily focus on variants with strong disease correlations and discuss how they could impact histone lysine methylation dynamics and gene regulation. PMID:24859469

  18. Candidate Luminal B Breast Cancer Genes Identified by Genome, Gene Expression and DNA Methylation Profiling

    PubMed Central

    Addou-Klouche, Lynda; Finetti, Pascal; Saade, Marie-Rose; Manai, Marwa; Carbuccia, Nadine; Bekhouche, Ismahane; Letessier, Anne; Charafe-Jauffret, Emmanuelle; Jacquemier, Jocelyne; Spicuglia, Salvatore; de The, Hugues; Viens, Patrice; Bertucci, François; Birnbaum, Daniel; Chaffanet, Max

    2014-01-01

    Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype. PMID:24416132

  19. Activation of endocrine-related gene expression in placental choriocarcinoma cell lines following DNA methylation knock-down.

    PubMed

    Hogg, K; Robinson, W P; Beristain, A G

    2014-07-01

    Increasingly, placental DNA methylation is assessed as a factor in pregnancy-related complications, yet the transcriptional impact of such findings is not always clear. Using a proliferative in vitro placental model, the effect of DNA methylation loss on gene activation was evaluated at a number of genes selected for being differentially methylated in pre-eclampsia-associated placentae in vivo. We aimed to determine whether reduced DNA methylation at specific loci was associated with transcriptional changes at the corresponding gene, thus providing mechanistic underpinnings for previous clinical findings and to assess the degree of transcriptional response amongst our candidate genes. BeWo and JEG3 choriocarcinoma cells were exposed to 1 μM 5-Aza-2'-deoxycytidine (5-Aza-CdR) or vehicle control for 48 h, and re-plated and cultured for a further 72 h in normal media before cells were harvested for RNA and DNA. Bisulphite pyrosequencing confirmed that DNA methylation was reduced by ∼30-50% points at the selected loci studied in both cell lines. Gene activation, measured by qRT-PCR, was highly variable and transcript specific, indicating differential sensitivity to DNA methylation. Most notably, loss of DNA methylation at the leptin (LEP) promoter corresponded to a 200-fold and 40-fold increase in LEP expression in BeWo and JEG3 cells, respectively (P < 0.01). Transcripts of steroidogenic pathway enzymes CYP11A1 and HSD3B1 were up-regulated ∼40-fold in response to 5-Aza-CdR exposure in BeWo cells (P < 0.01). Other transcripts, including aromatase (CYP19), HSD11B2, inhibin (INHBA) and glucocorticoid receptor (NR3C1) were more moderately, although significantly, affected by loss of associated DNA methylation. These data present a mixed effect of DNA methylation changes at selected loci supporting cautionary interpretation of DNA methylation results in the absence of functional data. PMID:24623739

  20. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation.

    PubMed

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-09-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  1. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation

    PubMed Central

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-01-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  2. Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

    SciTech Connect

    Sun Hong; Zhou Xue; Chen Haobin; Li Qin; Costa, Max

    2009-06-15

    Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1.

  3. Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line

    SciTech Connect

    Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee; Oh, Seikwan; Ahn, Jung-Hyuck

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may

  4. Universal light-switchable gene promoter system

    DOEpatents

    Quail, Peter H.; Huq, Enamul; Tepperman, James; Sato, Sae

    2005-02-22

    An artificial promoter system that can be fused upstream of any desired gene enabling reversible induction or repression of the expression of the gene at will in any suitable host cell or organisms by light is described. The design of the system is such that a molecule of the plant photoreceptor phytochrome is targeted to the specific DNA binding site in the promoter by a protein domain that is fused to the phytochrome and that specifically recognizes this binding site. This bound phytochrome, upon activation by light, recruits a second fusion protein consisting of a protein that binds to phytochrome only upon light activation and a transcriptional activation domain that activates expression of the gene downstream of the promoter.

  5. Identification of Novel Gene Targets and Putative Regulators of Arsenic-Associated DNA Methylation in Human Urothelial Cells and Bladder Cancer

    PubMed Central

    Rager, Julia E.; Miller, Sloane; Tulenko, Samantha E.; Smeester, Lisa; Ray, Paul D.; Yosim, Andrew; Currier, Jenna M.; Ishida, María C.; González-Horta, Maria del Carmen; Sánchez-Ramírez, Blanca; Ballinas-Casarrubias, Lourdes; Gutiérrez-Torres, Daniela S.; Drobná, Zuzana; Del Razo, Luz M.; García-Vargas, Gonzalo G.; Kim, William Y.; Zhou, Yi-Hui; Wright, Fred A.; Stýblo, Miroslav; Fry, Rebecca C.

    2016-01-01

    There is strong epidemiologic evidence linking chronic exposure to inorganic arsenic (iAs) to a myriad of adverse health effects, including cancer of the bladder. The present study set out to identify DNA methylation patterns associated with iAs and its metabolites in exfoliated urothelial cells (EUCs) that originate primarily from the urinary bladder, one of the targets of arsenic (As)-induced carcinogenesis. Genome-wide, gene-specific promoter DNA methylation levels were assessed in EUCs from 46 residents of Chihuahua, Mexico, and the relationship was examined between promoter methylation profiles and the intracellular concentrations of total As (tAs) and As species. A set of 49 differentially methylated genes was identified with increased promoter methylation associated with EUC tAs, iAs, and/or monomethylated As (MMAs) enriched for their roles in metabolic disease and cancer. Notably, no genes had differential methylation associated with EUC dimethylated As (DMAs), suggesting that DMAs may influence DNA methylation-mediated urothelial cell responses to a lesser extent than iAs or MMAs. Further analysis showed that 22 of the 49 As-associated genes (45%) are also differentially methylated in bladder cancer tissue identified using The Cancer Genome Atlas repository. Both the As- and cancer-associated genes are enriched for the binding sites of common transcription factors known to play roles in carcinogenesis, demonstrating a novel potential mechanistic link between iAs exposure and bladder cancer. PMID:26039340

  6. Analysis of CpG methylation and genomic footprinting at the tyrosine aminotransferase gene: DNA methylation alone is not sufficient to prevent protein binding in vivo.

    PubMed Central

    Weih, F; Nitsch, D; Reik, A; Schütz, G; Becker, P B

    1991-01-01

    Specific DNA sequences from several DNase I hypersensitive sites located upstream of the tyrosine aminotransferase (TAT) gene are bound by ubiquitous nuclear factors in vitro. Genomic footprinting has shown, however, that proteins are excluded from their potential binding sites in cells where the gene is inactive and that the absence of in vivo footprints is correlated with CpG methylation and altered chromatin structures at these sites. In vitro, interactions of proteins with sequences of the TAT gene, including binding of the transcription factor CREB to the cAMP-responsive element (CRE), are prevented by a methylated CpG dinucleotide in the respective binding sites, suggesting that methylation of DNA might be sufficient to exclude proteins from their sites in vivo. To test directly whether the absence of in vivo footprints is the result of DNA methylation, we treated two different cell lines with 5-azacytidine to demethylate CpG dinucleotides. While genomic sequencing confirmed demethylation at two widely separated regions upstream of the TAT promoter, no footprints appeared in these cell lines, even though proteins capable of binding these sites in vitro were present in the nuclei. Thus, the simple model whereby protein exclusion in vivo is caused solely by DNA methylation is not appropriate in this case. The nucleosomal organization of the potential binding sites suggests that chromatin structure is a dominant determinant in maintaining the inactive state of these sites. Images PMID:1714382

  7. Integration of gene expression and DNA methylation profiles provides a molecular subtype for risk assessment in atherosclerosis.

    PubMed

    Ma, Sheng-Chao; Zhang, Hui-Ping; Kong, Fan-Qi; Zhang, Hui; Yang, Cheng; He, Yang-Yang; Wang, Yan-Hua; Yang, An-Ning; Tian, Ju; Yang, Xiao-Ling; Zhang, Ming-Hao; Xu, Hua; Jiang, Yi-Deng; Yu, Zheng

    2016-06-01

    The aim of the present study was to identify an effective method for detecting early‑phase atherosclerosis (AS), as well as to provide useful DNA methylation profiles to serve as biomarkers for the detection of AS. A total of 300 individuals (150 AS patients and 150 healthy subjects) were recruited for peripheral blood DNA methylation analyses at 12 gene promoter loci using nested methylation‑specific polymerase chain reaction in a test set. Based on the test set, the promoter methylation of TIMP metallopeptidase inhibitor 1 (TIMP1), ATP binding cassette subfamily A member 1 (ABCA1), and acetyl-CoA acetyltransferase 1 (ACAT1) were determined to be candidate biomarkers; demonstrating the highest sensitivity (88%) and specificity (90%). The biomarkers that were candidates for early AS detection were validated in an independent validation set (n=100). In the validation set, the combination of TIMP1, ABCA1 and ACAT1 methylation achieved sensitivity, specificity and coincidence rate values of 88, 70 and 79%, respectively. In the current pilot study, the patterns of DNA methylation of AS‑associated genes were observed to be significantly altered in the peripheral blood of AS patients. Thus, the AS-specific methylation of the three‑gene panel (TIMP1, ABCA1, and ACAT1) may serve as a valuable biomarker for the early detection of AS. PMID:27082829

  8. Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus.

    PubMed

    Yuan, Cong; Zhang, Yingying; Liu, Yan; Zhang, Ting; Wang, Zaizhao

    2016-09-01

    DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. PMID:27474941

  9. Placental adiponectin gene DNA methylation levels are associated with mothers' blood glucose concentration.

    PubMed

    Bouchard, Luigi; Hivert, Marie-France; Guay, Simon-Pierre; St-Pierre, Julie; Perron, Patrice; Brisson, Diane

    2012-05-01

    Growing evidence suggests that epigenetic profile changes occurring during fetal development in response to in utero environment variations could be one of the mechanisms involved in the early determinants of adult chronic diseases. In this study, we tested whether maternal glycemic status is associated with the adiponectin gene (ADIPOQ) DNA methylation profile in placenta tissue, in maternal circulating blood cells, and in cord blood cells. We found that lower DNA methylation levels in the promoter of ADIPOQ on the fetal side of the placenta were correlated with higher maternal glucose levels during the second trimester of pregnancy (2-h glucose after the oral glucose tolerance test; r(s) ≤ -0.21, P < 0.05). Lower DNA methylation levels on the maternal side of the placenta were associated with higher insulin resistance index (homeostasis model assessment of insulin resistance) during the second and third trimesters of pregnancy (r(s) ≤ -0.27, P < 0.05). Finally, lower DNA methylation levels were associated with higher maternal circulating adiponectin levels throughout pregnancy (r(s) ≤ -0.26, P < 0.05). In conclusion, the ADIPOQ DNA methylation profile was associated with maternal glucose status and with maternal circulating adiponectin concentration. Because adiponectin is suspected to have insulin-sensitizing proprieties, these epigenetic adaptations have the potential to induce sustained glucose metabolism changes in the mother and offspring later in life. PMID:22396200

  10. Promoter architectures and developmental gene regulation.

    PubMed

    Haberle, Vanja; Lenhard, Boris

    2016-09-01

    Core promoters are minimal regions sufficient to direct accurate initiation of transcription and are crucial for regulation of gene expression. They are highly diverse in terms of associated core promoter motifs, underlying sequence composition and patterns of transcription initiation. Distinctive features of promoters are also seen at the chromatin level, including nucleosome positioning patterns and presence of specific histone modifications. Recent advances in identifying and characterizing promoters using next-generation sequencing-based technologies have provided the basis for their classification into functional groups and have shed light on their modes of regulation, with important implications for transcriptional regulation in development. This review discusses the methodology and the results of genome-wide studies that provided insight into the diversity of RNA polymerase II promoter architectures in vertebrates and other Metazoa, and the association of these architectures with distinct modes of regulation in embryonic development and differentiation. PMID:26783721

  11. Reversible histone methylation regulates brain gene expression and behavior

    PubMed Central

    Xu, Jun; Andreassi, Megan

    2011-01-01

    Epigenetic chromatin remodeling, including reversible histone methylation, regulates gene transcription in brain development and synaptic plasticity. Aberrant chromatin modifications due to mutant chromatin enzymes or chemical exposures have been associated with neurological or psychiatric disorders such as mental retardation, schizophrenia, depression, and drug addiction. Some chromatin enzymes, such as histone demethylases JARID1C and UTX, are coded by X-linked genes which are not X-inactivated in females. The higher expression of JARID1C and UTX in females could contribute to sex differences in brain development and behavior. PMID:20816965

  12. Expression level and DNA methylation status of Glutathione-S-transferase genes in normal murine prostate and TRAMP tumors

    PubMed Central

    Mavis, Cory K.; Kinney, Shannon R. Morey; Foster, Barbara A.; Karpf, Adam R.

    2010-01-01

    BACKGROUND Glutathione-S-transferase (Gst) genes are down-regulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors. METHODS Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n=15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5’ CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured. RESULTS Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells. CONCLUSIONS Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression. PMID:19444856

  13. DNA methylation status is more reliable than gene expression at detecting cancer in prostate biopsy

    PubMed Central

    Paziewska, A; Dabrowska, M; Goryca, K; Antoniewicz, A; Dobruch, J; Mikula, M; Jarosz, D; Zapala, L; Borowka, A; Ostrowski, J

    2014-01-01

    Background: We analysed critically the potential usefulness of RNA- and DNA-based biomarkers in supporting conventional histological diagnostic tests for prostate carcinoma (PCa) detection. Methods: Microarray profiling of gene expression and DNA methylation was performed on 16 benign prostatic hyperplasia (BPH) and 32 cancerous and non-cancerous prostate samples extracted by radical prostatectomy. The predictive value of the selected biomarkers was validated by qPCR-based methods using tissue samples extracted from the 58 prostates and, separately, using 227 prostate core biopsies. Results: HOXC6, AMACR and PCA3 expression showed the best discrimination between PCa and BPH. All three genes were previously reported as the most promising mRNA-based markers for distinguishing cancerous lesions from benign prostate lesions; however, none were sufficiently sensitive and specific to meet the criteria for a PCa diagnostic biomarker. By contrast, DNA methylation levels of the APC, TACC2, RARB, DGKZ and HES5 promoter regions achieved high discriminating sensitivity and specificity, with area under the curve (AUCs) reaching 0.95−1.0. Only a small overlap was detected between the DNA methylation levels of PCa-positive and PCa-negative needle biopsies, with AUCs ranging between 0.854 and 0.899. Conclusions: DNA methylation-based biomarkers reflect the prostate malignancy and might be useful in supporting clinical decisions for suspected PCa following an initial negative prostate biopsy. PMID:24937670

  14. Antipsychotic drugs attenuate aberrant DNA methylation of DTNBP1 (dysbindin) promoter in saliva and post-mortem brain of patients with schizophrenia and Psychotic bipolar disorder.

    PubMed

    Abdolmaleky, Hamid M; Pajouhanfar, Sara; Faghankhani, Masoomeh; Joghataei, Mohammad Taghi; Mostafavi, Ashraf; Thiagalingam, Sam

    2015-12-01

    Due to the lack of genetic association between individual genes and schizophrenia (SCZ) pathogenesis, the current consensus is to consider both genetic and epigenetic alterations. Here, we report the examination of DNA methylation status of DTNBP1 promoter region, one of the most credible candidate genes affected in SCZ, assayed in saliva and post-mortem brain samples. The Illumina DNA methylation profiling and bisulfite sequencing of representative samples were used to identify methylation status of the DTNBP1 promoter region. Quantitative methylation specific PCR (qMSP) was employed to assess methylation of DTNBP1 promoter CpGs flanking a SP1 binding site in the saliva of SCZ patients, their first-degree relatives and control subjects (30, 15, and 30/group, respectively) as well as in post-mortem brains of patients with SCZ and bipolar disorder (BD) versus controls (35/group). qRT-PCR was used to assess DTNBP1 expression. We found DNA hypermethylation of DTNBP1 promoter in the saliva of SCZ patients (∼12.5%, P = 0.036), particularly in drug-naïve patients (∼20%, P = 0.011), and a trend toward hypermethylation in their first-degree relatives (P = 0.085) versus controls. Analysis of post-mortem brain samples revealed an inverse correlation between DTNBP1 methylation and expression, and normalization of this epigenetic change by classic antipsychotic drugs. Additionally, BD patients with psychotic depression exhibited higher degree of methylation versus other BD patients (∼80%, P = 0.025). DTNBP1 promoter DNA methylation may become a key element in a panel of biomarkers for diagnosis, prevention, or therapy in SCZ and at risk individuals pending confirmatory studies with larger sample sizes to attain a higher degree of significance. PMID:26285059

  15. [Modifications of gene expression by tumor promoters].

    PubMed

    Zhang, C; Zhao, Q; Guo, S; Zhao, M; Cheng, S

    1995-02-01

    The modifications of gene expression by tumor promoters were analyzed in vitro and in vivo. The results of slot blot hybridizations showed that tumor promoter TPA induced c-fos and c-myc expressions in mouse fibroblast cell line BALB/3T3 and rat liver, decreased the levels of Rb RNA in BALB/3T3 cell line and of alpha 1-I3 RNA in rat liver. It was also demonstrated that tumor promoter phenobarbital influenced c-fos and c-myc expressions and decreased alpha 1I3 mRNA level in rat liver during a long term experiment. Phenobarbital was found to have no effect on c-fos and c-myc expressions in rat liver during a short experiment. Tumor promoters induced the expressions of c-fos and c-myc which were positively-related to cancer formation and inhibited the expressions of Rb and alpha 1-I3 which were negatively-related to cancer formation. This implied that tumor promotion played an important role in cancer development and tumor promoters exerted their effects selectively according to the attributes of different genes. PMID:7540119

  16. Aberrant gene methylation in non-neoplastic mucosa as a predictive marker of ulcerative colitis-associated CRC

    PubMed Central

    Castagliuolo, Ignazio; Erroi, Francesca; Kotsafti, Andromachi; Basato, Silvia; Brun, Paola; D'Incà, Renata; Rugge, Massimo

    2016-01-01

    Background Promoter hypermethylation plays a major role in cancer through transcriptional silencing of critical genes. The aim of our study is to evaluate the methylation status of these genes in the colonic mucosa without dysplasia or adenocarcinoma at the different steps of sporadic and UC-related carcinogenesis and to investigate the possible role of genomic methylation as a marker of CRC. Results The expression of Dnmts 1 and 3A was significantly increased in UC-related carcinogenesis compared to non inflammatory colorectal carcinogenesis. In non-neoplastic colonic mucosa, the number of methylated genes resulted significantly higher in patients with CRC and in those with UC-related CRC compared to the HC and UC patients and patients with dysplastic lesion of the colon. The number of methylated genes in non-neoplastic colonic mucosa predicted the presence of CRC with good accuracy either in non inflammatory and inflammatory related CRC. Methods Colonic mucosal samples were collected from healthy subjects (HC) (n = 30) and from patients with ulcerative colitis (UC) (n = 29), UC and dysplasia (n = 14), UC and cancer (n = 10), dysplastic adenoma (n = 14), and colon adenocarcinoma (n = 10). DNA methyltransferases-1, -3a, -3b, mRNA expression were quantified by real time qRT-PCR. The methylation status of CDH13, APC, MLH1, MGMT1 and RUNX3 gene promoters was assessed by methylation-specific PCR. Conclusions Methylation status of APC, CDH13, MGMT, MLH1 and RUNX3 in the non-neoplastic mucosa may be used as a marker of CRC: these preliminary results could allow for the adjustment of a patient's surveillance interval and to select UC patients who should undergo intensive surveillance. PMID:26862732

  17. Alternative promoters of gene MAGE4a

    SciTech Connect

    De Plaen, E.; Naerhuyzen, B.; De Smet, C.

    1997-03-01

    Gene MAGE-4 (HGMW-approved symbol MAGE4) is expressed in several types of tumors, but not in normal tissues, except testis and placenta. The 5{prime} end of this gene contains eight homologous exons spread over a 5.8-kb region. These exons are alternatively spliced to a unique second exon and a unique third exon, which encodes a protein of 317 amino acids. The analysis of transcripts found in testis, placenta, and a sarcoma cell line showed that each of the alternative first exons is used in at least one of these tissues. Various regions of the promoter of the fifth alternative exon (1.5) were cloned in a luciferase reporter plasmid, and the constructs were transfected in a sarcoma cell line that expresses MAGE-4. Two Ets motifs located between positions -70 and -29 relative to the transcription start site were found to drive 55% of the promoter activity. A region containing an Sp1 consensus binding site located upstream of the two Ets motifs was found to be responsible for 44% of the transcriptional activity. MAGE-4a promoters 1.4 and 1.6, which also contain the Sp1 and the two Ets binding motifs, supported a level of transcription comparable to that of promoter 1.5, whereas promoter 1.1, which contains only one Ets binding site, was sixfold less active. In line with observations made with gene MAGE-1 (HGMW-approved symbol MAGE1), we found that promoter 1.5 stimulated a high level of transcription in a melanoma cell line that does not express MAGE-4. This suggests that the tumor-specific expression of MAGE genes is not determined by the presence of specific transcription factors. 26 refs., 7 figs., 2 tabs.

  18. A genome-wide search for eigenetically regulated genes in zebra finch using MethylCap-seq and RNA-seq

    PubMed Central

    Steyaert, Sandra; Diddens, Jolien; Galle, Jeroen; De Meester, Ellen; De Keulenaer, Sarah; Bakker, Antje; Sohnius-Wilhelmi, Nina; Frankl-Vilches, Carolina; Van der Linden, Annemie; Van Criekinge, Wim; Vanden Berghe, Wim; De Meyer, Tim

    2016-01-01

    Learning and memory formation are known to require dynamic CpG (de)methylation and gene expression changes. Here, we aimed at establishing a genome-wide DNA methylation map of the zebra finch genome, a model organism in neuroscience, as well as identifying putatively epigenetically regulated genes. RNA- and MethylCap-seq experiments were performed on two zebra finch cell lines in presence or absence of 5-aza-2′-deoxycytidine induced demethylation. First, the MethylCap-seq methodology was validated in zebra finch by comparison with RRBS-generated data. To assess the influence of (variable) methylation on gene expression, RNA-seq experiments were performed as well. Comparison of RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed remarkable enrichment for neurological networks. A subset of genes was validated using Exon Arrays, quantitative RT-PCR and CpG pyrosequencing on bisulfite-treated samples. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control. PMID:26864856

  19. Gene Expression and DNA Methylation Status of Glutathione S-Transferase Mu1 and Mu5 in Urothelial Carcinoma

    PubMed Central

    Wang, Shou-Chieh; Huang, Chin-Chin; Shen, Cheng-Huang; Lin, Lei-Chen; Zhao, Pei-Wen; Chen, Shih-Ying; Deng, Yu-Chiao; Liu, Yi-Wen

    2016-01-01

    Bladder cancer is highly recurrent after therapy, which has an enormous impact on the health and financial condition of the patient. It is worth developing diagnostic tools for bladder cancer. In our previous study, we found that the bladder carcinogen BBN increased urothelial global DNA CpG methylation and decreased GSTM1 protein expression in mice. Here, the correlation of BBN-decreased GSTM1 and GSTM gene CpG methylation status was analyzed in mice bladders. BBN treatment decreased the protein and mRNA expression of GSTM1, and the CpG methylation ratio of GSTM1 gene promoter was slightly increased in mice bladders. Unlike mouse GSTM1, the human GSTM1 gene tends to be deleted in bladder cancers. Among 7 human bladder cancer cell lines, GSTM1 gene is really null in 6 cell lines except one, T24 cells. The CpG methylation level of GSTM1 was 9.9% and 5-aza-dC did not significantly increase GSTM1 protein and mRNA expression in T24 cells; however, the GSTM5 gene was CpG hypermethylated (65.4%) and 5-aza-dC also did not affect the methylation ratio and mRNA expression. However, in other cell lines without GSTM1, 5-aza-dC increased GSTM5 expression and decreased its CpG DNA methylation ratio from 84.6% to 61.5% in 5637, and from 97.4% to 75% in J82 cells. In summary, two biomarkers of bladder tumor were provided. One is the GSTM1 gene which is down-regulated in mice bladder carcinogenesis and is usually deleted in human urothelial carcinoma, while the other is the GSTM5 gene, which is inactivated by DNA CpG methylation. PMID:27404495

  20. Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

    PubMed

    Navasa, Nicolás; Martin-Ruiz, Itziar; Atondo, Estíbaliz; Sutherland, James D; Angel Pascual-Itoiz, Miguel; Carreras-González, Ana; Izadi, Hooman; Tomás-Cortázar, Julen; Ayaz, Furkan; Martin-Martin, Natalia; Torres, Iviana M; Barrio, Rosa; Carracedo, Arkaitz; Olivera, Elias R; Rincón, Mercedes; Anguita, Juan

    2015-01-01

    MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions. PMID:26419808

  1. Poor survival is associated with the methylated degree of zinc-finger protein 545 (ZNF545) DNA promoter in gastric cancer.

    PubMed

    Deng, Jingyu; Liang, Han; Ying, Guoguang; Dong, Qiuping; Zhang, Rupeng; Yu, Jun; Fan, Daiming; Hao, Xishan

    2015-02-28

    Zinc-finger protein 545 (ZNF545) was identified as a gastric tumour suppressor and potentially independent prognostic factor. At the present study, we found that lower expression of ZNF545 was specific in gastric cancer (GC) tissues, and the inconsistently methylated levels of ZNF545 promoter were identified in the gastric cancer tissues. In the methylation-specific PCR (MSP) analysis cohort, we found that GC patients with hypermethylated ZNF545 promoter exhibited significantly shorter median OS than those with unmethylated ZNF545 promoter and those with hypomethylated ZNF545 promoter. In the other cohort, we also demonstrated that GC patients with three or more methylated CpG sites in the ZNF545 promoter were significantly associated with poor survival by using the bisulphite gene sequencing (BGS). The methylated degrees of five CpG sites (-232, -214, -176, -144 and -116) could also provide distinct survival discrimination of patients with GC. These findings indicated that the methylated CpG sites of the ZNF545 promoter could be used for the clinical prediction of the prognosis of GC. PMID:25714013

  2. Site-specific methylation changes in the glucocorticoid receptor exon 1F promoter in relation to life adversity: systematic review of contributing factors

    PubMed Central

    Daskalakis, Nikolaos P.; Yehuda, Rachel

    2014-01-01

    There has been recent interest in epigenetics in psychiatry since it offers a means of understanding how stressful life experiences, in interaction with the genotype, result in epigenetic changes that result in altered gene expression, ultimately affecting the risk for mental disorders. Many studies focused on methylation of the glucocorticoid receptor exon 1F promoter following an initial observation that changes in this region could be modulated by the environment. This review examines all published studies that have attempted to measure methylation in this region using different techniques, several tissue types, populations at different behavioral state and stages of development. Methodological issues have been raised with the aim of attempting to understand methylation quantification and site of action. We propose that it is useful to examine whether methylation at specific sites within the promoter region may be particularly relevant to psychiatric vulnerability to stress-related outcomes. PMID:25484853

  3. Promoter-restricted histone code, not the differentially methylated DNA regions or antisense transcripts, marks the imprinting status of IGF2R in human and mouse.

    PubMed

    Vu, Thanh H; Li, Tao; Hoffman, Andrew R

    2004-10-01

    Imprinting of the mouse Igf2r depends upon an intronic differentially methylated DNA region (DMR) and the presence of the Air antisense transcript. However, biallelic expression of mouse Igf2r in brain occurs despite the presence of Air, and biallelic expression of human IGF2R in peripheral tissues occurs despite the presence of an intronic DMR. We examined histone modifications throughout the mouse and human Igf2r/IGF2R using chromatin immuno-precipitation (ChIP) assays in combination with quantitative real time PCR. Methylation of Lys4 and Lys9 of histone H3 in the promoter regions marks the active and silenced alleles, respectively. We measured di- and tri-methyl Lys4 and Lys9 across the Igf2r and Air promoters. While both di- and tri-methyl Lys4 marked the active Igf2r and the active Air allele, tri-methyl Lys9, but not di-methyl Lys9, marked the suppressed Air allele. We show here that enrichment of parental allele-specific histone modifications in the promoter region, rather than the presence of DNA methylation or antisense transcription, correctly identifies the tissue- and species- specific imprinting status of Igf2r/IGF2R. We discuss these findings in light of recent progress in identifying specific components of the epigenetic marks in imprinted genes. PMID:15294879

  4. Age and Obesity Promote Methylation and Suppression of 5-Alpha Reductase 2–Implications for Personalized Therapy in Benign Prostatic Hyperplasia

    PubMed Central

    Bechis, Seth K.; Otsetov, Alexander G.; Ge, Rongbin; Wang, Zongwei; Vangel, Mark G.; Wu, Chin-Lee; Tabatabaei, Shahin; Olumi, Aria F.

    2016-01-01

    Purpose 5α reductase inhibitors (5ARIs) are a main modality of treatment for men suffering from symptomatic benign prostatic hyperplasia (BPH). Over 30% of men do not respond to the therapeutic effects of 5ARIs. We have found that 1/3 of adult prostate samples do not express 5AR2 secondary to epigenetic modifications. We sought to evaluate whether 5AR2 expression in BPH specimens of symptomatic men was linked to methylation of the 5AR2 gene promoter and identify associations with age, obesity, cardiac risk factors, and prostate specific antigen (PSA). Materials and Methods Prostate samples from men undergoing transurethral prostate resection were used. 5AR2 protein expression and gene promoter methylation status were determined by common assays. Clinical variables included age, body mass index (BMI), hypertension, hyperlipidemia, diabetes, PSA, and prostate volume. Univariate and multivariate statistical analyses were performed, followed by stepwise logistic regression modeling. Results BMI and age were significantly correlated with methylation of the 5AR2 gene promoter (p<0.05), whereas prostate volume, PSA, or use of BPH medication were not. Methylation was highly correlated with 5AR protein expression (p<0.0001). In a predictive model, both increasing age and BMI significantly predicted methylation status and protein expression (p<0.01). Conclusions Increasing age and BMI correlate with increased 5AR2 gene promoter methylation and decreased protein expression in men with symptomatic BPH. These results highlight the interplay between age, obesity and gene regulation. Our findings suggest the presence of an individualized epigenetic signature for symptomatic BPH, which may be important for choosing appropriate personalized treatment options. PMID:25916673

  5. The Escherichia coli regulatory protein OxyR discriminates between methylated and unmethylated states of the phage Mu mom promoter.

    PubMed Central

    Bölker, M; Kahmann, R

    1989-01-01

    Expression of the phage Mu mom gene is transcriptionally regulated by DNA methylation. Three GATC sites upstream of the mom promoter have to be methylated by the Escherichia coli deoxyadenosine methylase (Dam) to allow initiation of transcription. An E. coli dam strain was mutagenized with Tn5 in an attempt to isolate mutants which allow mom gene expression. Three independent Tn5 mutants were isolated, each mapped to a gene at 89.6 min which we designate momR. The wildtype gene was cloned and sequenced, it encodes a protein of 305 amino acids. The protein belongs to a group of related bacterial activators recently identified as the LysR family (Henikoff et al., 1988). MomR protein was overproduced and purified. Expression of momR is autoregulated; MomR binds to a 43 bp region upstream of its coding sequence. In the mom promoter MomR protects a 43 bp region containing the three GATC sites. Specific binding to these sequences was observed only with unmethylated DNA. Fortuitously, we learned that MomR is identical to OxyR, a regulatory protein responding to oxidative stress. We discuss the implications of this control for Mu development. Images PMID:2551682

  6. P01.05ZBTB18 METHYLATION PROMOTES MESENCHYMAL TRANSFORMATION IN GLIOBLASTOMA

    PubMed Central

    Fedele, V.; Dai, F.; Marinescu, V.; Kim, H.; Weyerbrock, A.; Prinz, M.; Bredel, M.; Carro, M.

    2014-01-01

    Although studies performed in the past decade have contributed to a better classification and characterization of GBM, the overall survival and outcome of GBM patients have not seen major improvement. The mesenchymal signature is described as the subtype of GBM with the worst prognosis, primarily due to inherent resistance of the tumors to contemporary therapies. The identification of molecular determinants of mesenchymal transformation could potentially allow for the discovery of new therapeutic targets. The Zing Finger And BTB Domain Containing 18 (ZBTB18; formerly ZNF238) is a transcriptional repressor with a crucial role in brain development and neuronal differentiation. Consistent with our previous study, which provided preliminary evidence of a role for ZBTB18 in a network of mesenchymal transformation in GBM, our new data have discovered epigenetic silencing, that is promoter methylation, as a mechanism to downregulate ZBTB18 in these tumors. We show that ZBTB18 functions as a master regulator of a mesenchymal gene signature in GBM and that loss of ZBTB18 contributes to the highly invasive mesenchymal phenotype. Conversely, re-expression of ZBTB18 reverses the phenotype and impairs tumor-forming ability. Overall, our results support a tumor suppressor role of ZBTB18 in the brain and identify promoter hypermethylation as a mechanism to silence ZBTB18 in the mesenchymal subtype of GBM, which provides a new mechanistic opportunity to specifically target this tumor subclass.

  7. A Longitudinal Study of BDNF Promoter Methylation and Depression in Breast Cancer

    PubMed Central

    Kang, Hee-Ju; Kim, Seon-Young; Kim, Sung-Wan; Shin, Il-Seon; Kim, Hye-Ran; Park, Min-Ho; Shin, Myung-Geun; Yoon, Jung-Han; Yoon, Jin-Sang

    2015-01-01

    Objective Brain-derived neurotrophic factor (BDNF) is investigated in depression related to medical disorders and its secretion is influenced by epigenetic factors. We investigated the association between BDNF promoter methylation and depression following mastectomy for breast cancer. Methods In total, 309 patients with breast cancer were evaluated 1 week after mastectomy, and 244 (79%) were followed up 1 year later. Depression was diagnosed (major or minor depressive disorder) according to DSM-IV criteria and depression severity was estimated by Montgomery-Asberg Depression Rating Scale (MADRS). We assessed BDNF promoter methylation using leukocyte DNA. The effects of BDNF methylation on depression diagnosis and severity were investigated using multivariate logistic and linear regression models, respectively. The two-way interaction between BDNF methylation and the val66met polymorphism on depression was also evaluated using multivariate logistic regression models. Results Higher BDNF methylation was independently associated with depression diagnosis and with more severe symptoms at both 1 week and 1 year after mastectomy. No significant methylation-genotype interactions were found. Conclusion A role for BDNF in depression related to breast cancer was supported. Indeed, the association between depression and BDNF methylation may be useful for identifying patients who are at high risk for depression and for suggesting directions for promising drug research. PMID:26508964

  8. Longitudinal associations between BDNF promoter methylation and late-life depression.

    PubMed

    Kang, Hee-Ju; Kim, Jae-Min; Bae, Kyung-Yeol; Kim, Sung-Wan; Shin, Il-Seon; Kim, Hye-Ran; Shin, Myung-Geun; Yoon, Jin-Sang

    2015-04-01

    Reduced brain-derived neurotrophic factor (BDNF) function has been suggested as a risk factor for late-life depression. BDNF secretion is influenced by epigenetic (DNA promoter methylation) and genetic (val66met polymorphism) profiles. We investigated the independent and interactive effects of BDNF methylation and val66met polymorphism on late-life depression. In total, 732 Korean community residents aged ≥ 65 years were evaluated, and 521 of them without depression at baseline were followed up 2 years later. Depression was determined using the Geriatric Mental State Schedule, and depression severity was evaluated with the Geriatric Depression Scale. Demographic and clinical covariates were obtained. The effects of BDNF methylation and polymorphism on the diagnosis of depression were investigated using a multivariate logistic regression model, and the relationships between BDNF methylation and depression severity were evaluated using partial correlation tests. Higher BDNF methylation was independently associated with the prevalence and incidence of depression and severe depressive symptoms. No significant methylation-genotype interactions were found. BDNF promoter methylation could be a proxy biomarker for depression late in life. PMID:25648279

  9. Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.

    PubMed

    Yeo, Seungeun; Jeong, Sangkyun; Kim, Janghwan; Han, Jee-Soo; Han, Yong-Mahn; Kang, Yong-Kook

    2007-08-01

    Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation. PMID:17548060

  10. DNMT3B isoforms without catalytic activity stimulate gene body methylation as accessory proteins in somatic cells

    PubMed Central

    Duymich, Christopher E.; Charlet, Jessica; Yang, Xiaojing; Jones, Peter A.; Liang, Gangning

    2016-01-01

    Promoter DNA methylation is a key epigenetic mechanism for stable gene silencing, but is correlated with expression when located in gene bodies. Maintenance and de novo DNA methylation by catalytically active DNA methyltransferases (DNMT1 and DNMT3A/B) require accessory proteins such as UHRF1 and DNMT3L. DNMT3B isoforms are widely expressed, although some do not have active catalytic domains and their expression can be altered during cell development and tumourigenesis, questioning their biological roles. Here, we show that DNMT3B isoforms stimulate gene body methylation and re-methylation after methylation-inhibitor treatment. This occurs independently of the isoforms' catalytic activity, demonstrating a similar functional role to the accessory protein DNMT3L, which is only expressed in undifferentiated cells and recruits DNMT3A to initiate DNA methylation. This unexpected role for DNMT3B suggests that it might substitute for the absent accessory protein DNMT3L to recruit DNMT3A in somatic cells. PMID:27121154

  11. Abnormal PTPN11 enhancer methylation promotes rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and joint inflammation

    PubMed Central

    Maeshima, Keisuke; Stanford, Stephanie M.; Hammaker, Deepa; Sacchetti, Cristiano; Zeng, Li-fan; Ai, Rizi; Zhang, Vida; Boyle, David L.; Aleman Muench, German R.; Feng, Gen-Sheng; Whitaker, John W.; Zhang, Zhong-Yin; Wang, Wei; Bottini, Nunzio; Firestein, Gary S.

    2016-01-01

    The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor– binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1β stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA. PMID:27275015

  12. Regional distribution of body fat in relation to DNA methylation within the LPL, ADIPOQ and PPARγ promoters in subcutaneous adipose tissue

    PubMed Central

    Drogan, D; Boeing, H; Janke, J; Schmitt, B; Zhou, Y; Walter, J; Pischon, T; Tierling, S

    2015-01-01

    Obesity may be related to differential DNA methylation and thus to differential expression of key genes in adipose tissue metabolism, such as LPL, ADIPOQ and PPARγ. Using subcutaneous adipose tissue (SAT) from 59 individuals of the European Prospective Investigation into Cancer and Nutrition–Potsdam study, we performed quantitative DNA methylation analysis within the promoters of LPL (LPL-CG1 and -CG2), ADIPOQ (ADIPOQ-CG1 and-CG2) and PPARγ (PPARγ-CG1). We then studied DNA methylation in relation to SAT gene expression, body composition measured using whole-body magnetic resonance imaging, body mass index (BMI), waist circumference (WC) and long-term changes in BMI and WC. For LPL-CG1 and LPL-CG2, higher methylation levels were associated with lower LPL expression, but with higher past WC gain. LPL-CG1 was also positively associated with BMI, WC, and visceral and subcutaneous fat mass. ADIPOQ-CG1 or -CG2 methylation exhibited no association with ADIPOQ expression or with anthropometric parameters. PPARγ-CG1 methylation was significantly higher in individuals with higher visceral fat mass. Among the investigated sites, LPL-CG1 methylation showed the strongest association with gene expression and regional body fat distribution, thereby possibly linking the degree of obesity with major metabolic processes in SAT. PMID:26148147

  13. Methylation of the ATM promoter in glioma cells alters ionizing radiation sensitivity

    SciTech Connect

    Roy, Kanaklata; Wang, Lilin; Makrigiorgos, G. Mike; Price, Brendan D. . E-mail: brendan_price@dfci.harvard.edu

    2006-06-09

    Glioblastomas are among the malignancies most resistant to radiation therapy. In contrast, cells lacking the ATM protein are highly sensitive to ionizing radiation. The relationship between ATM protein expression and radiosensitivity in 3 glioma cell lines was examined. T98G cells exhibited normal levels of ATM protein, whereas U118 and U87 cells had significantly lower levels of ATM and increased (>2-fold) sensitivity to ionizing radiation compared to T98G cells. The ATM promoter was methylated in U87 cells. Demethylation by azacytidine treatment increased ATM protein levels in the U87 cells and decreased their radiosensitivity. In contrast, the ATM promoter in U118 cells was not methylated. Further, expression of exogenous ATM did not significantly alter the radiosensitivity of U118 cells. ATM expression is therefore heterogeneous in the glioma cells examined. In conclusion, methylation of the ATM promoter may account for the variable radiosensitivity and heterogeneous ATM expression in a fraction of glioma cells.

  14. Regulated recruitment of tumor suppressor BRCA1 to the p21 gene by coactivator methylation

    PubMed Central

    Lee, Young-Ho; Bedford, Mark T.; Stallcup, Michael R.

    2011-01-01

    Tumor suppression by p53 and BRCA1 involves regulation of cell cycle, apoptosis, and DNA repair and is influenced by transcriptional coactivators and post-translational modifications. Here we show that coactivator-associated arginine methyltransferase 1 (CARM1) methylates Arg 754 in the KIX region of coactivator p300. Methylated p300 and p300 protein fragments are preferentially recognized by BRCT domains of BRCA1, identifying the BRCT domain as a novel methylarginine-binding module. CARM1 and p300 cooperate with BRCA1 and p53 to induce expression of the critical cell cycle and proliferation regulator p21WAF1/CIP1 in response to DNA damage. This induction was severely attenuated by elimination of CARM1 or its methyltransferase activity, or by mutation of Arg 754 of p300. Absence of CARM1 methyltransferase activity led to failure of cells to arrest in the G1 phase of the cell cycle in response to DNA damage. CARM1 methyltransferase activity was required for induction of some p53 target genes (p21 and Gadd45) but not others (Bax) by DNA damage. Recruitment of BRCA1 to the p53-binding region of the p21 promoter in response to DNA damage required methylation of Arg 754 of p300 by CARM1. Thus, coactivator methylation may be crucial for fine-tuning the tumor suppressor function of BRCA1 and other BRCT domain proteins. PMID:21245169

  15. Alterations in gene expression and DNA methylation during murine and human lung alveolar septation.

    PubMed

    Cuna, Alain; Halloran, Brian; Faye-Petersen, Ona; Kelly, David; Crossman, David K; Cui, Xiangqin; Pandit, Kusum; Kaminski, Naftali; Bhattacharya, Soumyaroop; Ahmad, Ausaf; Mariani, Thomas J; Ambalavanan, Namasivayam

    2015-07-01

    DNA methylation, a major epigenetic mechanism, may regulate coordinated expression of multiple genes at specific time points during alveolar septation in lung development. The objective of this study was to identify genes regulated by methylation during normal septation in mice and during disordered septation in bronchopulmonary dysplasia. In mice, newborn lungs (preseptation) and adult lungs (postseptation) were evaluated by microarray analysis of gene expression and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-Seq). In humans, microarray gene expression data were integrated with genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm and term lung. Genes with reciprocal changes in expression and methylation, suggesting regulation by DNA methylation, were identified. In mice, 95 genes with inverse correlation between expression and methylation during normal septation were identified. In addition to genes known to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its extracellular matrix (Tnc, Eln, etc.), genes involved with immune and antioxidant defense (Stat4, Sod3, Prdx6, etc.) were also observed. In humans, 23 genes were differentially methylated with reciprocal changes in expression in bronchopulmonary dysplasia compared with preterm or term lung. Genes of interest included those involved with detoxifying enzymes (Gstm3) and transforming growth factor-β signaling (bone morphogenetic protein 7 [Bmp7]). In terms of overlap, 20 genes and three pathways methylated during mouse lung development also demonstrated changes in methylation between preterm and term human lung. Changes in methylation correspond to altered expression of a number of genes associated with lung development, suggesting that DNA methylation of these genes may regulate normal and abnormal alveolar septation. PMID:25387348

  16. Alterations in Gene Expression and DNA Methylation during Murine and Human Lung Alveolar Septation

    PubMed Central

    Cuna, Alain; Halloran, Brian; Faye-Petersen, Ona; Kelly, David; Crossman, David K.; Cui, Xiangqin; Pandit, Kusum; Kaminski, Naftali; Bhattacharya, Soumyaroop; Ahmad, Ausaf; Mariani, Thomas J.

    2015-01-01

    DNA methylation, a major epigenetic mechanism, may regulate coordinated expression of multiple genes at specific time points during alveolar septation in lung development. The objective of this study was to identify genes regulated by methylation during normal septation in mice and during disordered septation in bronchopulmonary dysplasia. In mice, newborn lungs (preseptation) and adult lungs (postseptation) were evaluated by microarray analysis of gene expression and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-Seq). In humans, microarray gene expression data were integrated with genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm and term lung. Genes with reciprocal changes in expression and methylation, suggesting regulation by DNA methylation, were identified. In mice, 95 genes with inverse correlation between expression and methylation during normal septation were identified. In addition to genes known to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its extracellular matrix (Tnc, Eln, etc.), genes involved with immune and antioxidant defense (Stat4, Sod3, Prdx6, etc.) were also observed. In humans, 23 genes were differentially methylated with reciprocal changes in expression in bronchopulmonary dysplasia compared with preterm or term lung. Genes of interest included those involved with detoxifying enzymes (Gstm3) and transforming growth factor-β signaling (bone morphogenetic protein 7 [Bmp7]). In terms of overlap, 20 genes and three pathways methylated during mouse lung development also demonstrated changes in methylation between preterm and term human lung. Changes in methylation correspond to altered expression of a number of genes associated with lung development, suggesting that DNA methylation of these genes may regulate normal and abnormal alveolar septation. PMID:25387348

  17. Methylation of the PTEN promoter defines low-grade gliomas and secondary glioblastoma

    PubMed Central

    Wiencke, John K.; Zheng, Shichun; Jelluma, Nanette; Tihan, Tarik; Vandenberg, Scott; Tamgüney, Tanja; Baumber, Rachel; Parsons, Ramon; Lamborn, Kathleen R.; Berger, Mitchel S.; Wrensch, Margaret R.; Haas-Kogan, Daphne Adele; Stokoe, David

    2007-01-01

    Glioblastoma multiforme (GBM) can present as either de novo or secondary tumors arising from previously diagnosed low-grade gliomas. Although these tumor types are phenotypically indistinguishable, de novo and secondary GBMs are associated with distinct genetic characteristics. PTEN mutations, which result in activation of the phosphoinositide 3-kinase (PI3K) signal transduction pathway, are frequent in de novo but not in secondary GBMs or their antecedent low-grade tumors. Results we present here show that grade II astrocytomas, oligodendrogliomas, and oligoastrocytomas commonly display methylation of the PTEN promoter, a finding that is absent in nontumor brain specimens and rare in de novo GBMs. Methylation of the PTEN promoter correlates with protein kinase B (PKB/Akt) phosphorylation, reflecting functional activation of the PI3K pathway. Our results also demonstrate frequent methylation of the PTEN promoter in grade III astrocytomas and secondary GBMs, consistent with the hypothesis that these tumors arise from lower grade precursors. PTEN methylation is rare in de novo GBMs and is mutually exclusive with PTEN mutations. We conclude that methylation of the PTEN promoter may represent an alternate mechanism by which PI3K signaling is increased in grade II and III gliomas as well as secondary GBMs, a finding that offers new therapeutic approaches in these patients. PMID:17504928

  18. Methylation of the PTEN promoter defines low-grade gliomas and secondary glioblastoma.

    PubMed

    Wiencke, John K; Zheng, Shichun; Jelluma, Nanette; Tihan, Tarik; Vandenberg, Scott; Tamgüney, Tanja; Baumber, Rachel; Parsons, Ramon; Lamborn, Kathleen R; Berger, Mitchel S; Wrensch, Margaret R; Haas-Kogan, Daphne Adele; Stokoe, David

    2007-07-01

    Glioblastoma multiforme (GBM) can present as either de novo or secondary tumors arising from previously diagnosed low-grade gliomas. Although these tumor types are phenotypically indistinguishable, de novo and secondary GBMs are associated with distinct genetic characteristics. PTEN mutations, which result in activation of the phosphoinositide 3-kinase (PI3K) signal transduction pathway, are frequent in de novo but not in secondary GBMs or their antecedent low-grade tumors. Results we present here show that grade II astrocytomas, oligodendrogliomas, and oligoastrocytomas commonly display methylation of the PTEN promoter, a finding that is absent in nontumor brain specimens and rare in de novo GBMs. Methylation of the PTEN promoter correlates with protein kinase B (PKB/Akt) phosphorylation, reflecting functional activation of the PI3K pathway. Our results also demonstrate frequent methylation of the PTEN promoter in grade III astrocytomas and secondary GBMs, consistent with the hypothesis that these tumors arise from lower grade precursors. PTEN methylation is rare in de novo GBMs and is mutually exclusive with PTEN mutations. We conclude that methylation of the PTEN promoter may represent an alternate mechanism by which PI3K signaling is increased in grade II and III gliomas as well as secondary GBMs, a finding that offers new therapeutic approaches in these patients. PMID:17504928

  19. Preeclampsia is Associated with Decreased Methylation of the GNA12 Promoter.

    PubMed

    Ye, Weiping; Shen, Lu; Xiong, Ying; Zhou, Yun; Gu, Hang; Yang, Zujing

    2016-01-01

    Preeclampsia, characterized by high blood pressure, albuminuria and other systemic disorders, is a serious complication during pregnancy. It has been reported that GNA12 is overexpressed during preeclampsia. In this study, we investigated the potential association between the methylation of the GNA12 promoter and preeclampsia. The methylation level at eight CpG sites of the GNA12 promoter was analyzed by MassARRAY in placenta and peripheral blood DNA samples from 50 preeclampsia patients and 50 normal pregnant women. In the placenta DNA samples, the methylation level at three CpG sites of the GNA12 promoter was significantly lower in the preeclampsia patients than in the controls. The difference was also significant at two of the three CpG sites in the peripheral blood DNA samples. The mRNA expression level of GNA12 in placenta was analyzed by real-time quantitative PCR in 20 cases and 20 controls. Consistent with the decreased methylation level, the mRNA expression level of GNA12 was higher in preeclampsia patients than in controls. Our results showed that preeclampsia is associated with decreased methylation of the GNA12 promoter, which can be detected in both the placenta and the peripheral blood of the pregnant women. PMID:26767593

  20. Corruption of the Intra-Gene DNA Methylation Architecture Is a Hallmark of Cancer

    PubMed Central

    Bartlett, Thomas E.; Zaikin, Alexey; Olhede, Sofia C.; West, James; Teschendorff, Andrew E.; Widschwendter, Martin

    2013-01-01

    Epigenetic processes - including DNA methylation - are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers. PMID:23874574

  1. Corruption of the intra-gene DNA methylation architecture is a hallmark of cancer.

    PubMed

    Bartlett, Thomas E; Zaikin, Alexey; Olhede, Sofia C; West, James; Teschendorff, Andrew E; Widschwendter, Martin

    2013-01-01

    Epigenetic processes--including DNA methylation--are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test [Formula: see text] in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers. PMID:23874574

  2. Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13 S) functions.

    PubMed

    Weisshaar, B; Langner, K D; Jüttermann, R; Müller, U; Zock, C; Klimkait, T; Doerfler, W

    1988-07-20

    The inactivating effect of sequence-specific promoter methylations was extensively studied by using the late E2A promoter of adenovirus type 2 (Ad2) DNA. The modification of the three 5' CCGG 3' sequences at nucleotides +24, +6 and -215, relative to the cap site in this promoter, sufficed to silence the gene in transient expression either in Xenopus laevis oocytes or in mammalian cells, and after the fixation of the E2A promoter-chloramphenicol-acetyltransferase (CAT) gene construct in the genome of hamster cells. It will now be demonstrated that the inactivation of the late promoter of Ad2 DNA can be reversed by transactivating functions that are encoded in the 13S messenger RNA of the E1A region of Ad2 DNA. The reactivation of a methylation-inactivated eukaryotic promoter by transactivating functions has general significance in that the value of a regulatory signal can be fully realized only by its controlled reversibility. It was demonstrated in transient expression experiments that the 5' CCGG 3'-methylated late E2A promoter was at least partly reactivated in cell lines constitutively expressing the E1 region of Ad2 or of adenovirus type 5 (Ad5) DNA. The reactivation led to transcriptional initiation at the authentic cap sites of the late E2A promoter and was not associated with promoter demethylation, at least not in both DNA complements. Reactivation of the methylation-inactivated E2A promoter could also be demonstrated in two BHK21 cell lines (mc14 and mc20), which carried the late E2A promoter-CAT gene assembly in an integrated form. In these cell lines the late E2A promoter was methylated and the CAT gene was not expressed. By transfection of cell lines mc14 and mc20, the reactivating functions were shown to reside in the pAd2E1A-13 S cDNA clone of Ad2 DNA. The pAd2E1A-12 S cDNA clone or the pAd2E1B clone showed no reactivating function. These findings implicated the E1A 289 amino acid residue protein of Ad2, a well-known transactivator, as the

  3. Relationships among folate, alcohol consumption, gene variants in one-carbon metabolism and p16INK4a methylation and expression in healthy breast tissues.

    PubMed

    Llanos, Adana A; Dumitrescu, Ramona G; Brasky, Theodore M; Liu, Zhenhua; Mason, Joel B; Marian, Catalin; Makambi, Kepher H; Spear, Scott L; Kallakury, Bhaskar V S; Freudenheim, Jo L; Shields, Peter G

    2015-01-01

    p16(INK4a) is a tumor suppressor gene, frequently hypermethylated in breast cancer; this epigenetic silencing of p16(INK4a) occurs early in carcinogenesis. The risk factors and functional consequences of p16(INK4a) methylation are unknown. Alcohol consumption, a breast cancer risk factor, impedes folate metabolism and may thereby alter gene methylation since folate plays a pivotal role in DNA methylation. In a cross-sectional study of 138 women with no history of breast cancer who underwent reduction mammoplasty, we studied breast cancer risk factors, plasma and breast folate concentrations, variation in one-carbon metabolism genes, p16(INK4a) promoter methylation and P16 protein expression. Logistic regression was used to estimate multivariable-adjusted odds ratios (OR) and 95% confidence intervals (CI). p16(INK4a) methylation was negatively correlated with P16 expression (r = -0.28; P = 0.002). Alcohol consumption was associated with lower breast folate (P = 0.03), higher p16(INK4a) promoter methylation (P = 0.007) and less P16 expression (P = 0.002). Higher breast folate concentrations were associated with lower p16(INK4a) promoter methylation (P = 0.06). Genetic variation in MTRR (rs1801394) and MTHFD1 (rs1950902) was associated with higher p16 (INK4a) promoter methylation (OR = 2.66, 95% CI: 1.11-6.42 and OR = 2.72, 95% CI: 1.12-6.66, respectively), whereas variation in TYMS (rs502396) was associated with less P16 protein expression (OR = 0.22, 95% CI: 0.05-0.99). Given that this is the first study to indicate that alcohol consumption, breast folate and variation in one-carbon metabolism genes are associated with p16(INK4a) promoter methylation and P16 protein expression in healthy tissues; these findings require replication. PMID:25344837

  4. Methylation-Associated Gene Silencing of RARB in Areca Carcinogens Induced Mouse Oral Squamous Cell Carcinoma

    PubMed Central

    Tsou, Yung-An; Fan, Shin-Ru; Tsai, Ming-Hsui; Chen, Hsiao-Ling; Chang, Nai-Wen; Cheng, Ju-Chien

    2014-01-01

    Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis. PMID:25197641

  5. Relationship of DNA Methylation and Gene Expression in Idiopathic Pulmonary Fibrosis

    PubMed Central

    Pedersen, Brent S.; Rabinovich, Einat; Hennessy, Corinne E.; Davidson, Elizabeth J.; Murphy, Elissa; Guardela, Brenda Juan; Tedrow, John R.; Zhang, Yingze; Singh, Mandal K.; Correll, Mick; Schwarz, Marvin I.; Geraci, Mark; Sciurba, Frank C.; Quackenbush, John; Spira, Avrum; Kaminski, Naftali; Schwartz, David A.

    2014-01-01

    Rationale: Idiopathic pulmonary fibrosis (IPF) is an untreatable and often fatal lung disease that is increasing in prevalence and is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control gene expression and are likely to regulate the IPF transcriptome. Objectives: To identify methylation marks that modify gene expression in IPF lung. Methods: We assessed DNA methylation (comprehensive high-throughput arrays for relative methylation arrays [CHARM]) and gene expression (Agilent gene expression arrays) in 94 patients with IPF and 67 control subjects, and performed integrative genomic analyses to define methylation–gene expression relationships in IPF lung. We validated methylation changes by a targeted analysis (Epityper), and performed functional validation of one of the genes identified by our analysis. Measurements and Main Results: We identified 2,130 differentially methylated regions (DMRs; <5% false discovery rate), of which 738 are associated with significant changes in gene expression and enriched for expected inverse relationship between methylation and expression (P < 2.2 × 10−16). We validated 13/15 DMRs by targeted analysis of methylation. Methylation–expression quantitative trait loci (methyl-eQTL) identified methylation marks that control cis and trans gene expression, with an enrichment for cis relationships (P < 2.2 × 10−16). We found five trans methyl-eQTLs where a methylation change at a single DMR is associated with transcriptional changes in a substantial number of genes; four of these DMRs are near transcription factors (castor zinc finger 1 [CASZ1], FOXC1, MXD4, and ZDHHC4). We studied the in vitro effects of change in CASZ1 expression and validated its role in regulation of target genes in the methyl-eQTL. Conclusions: These results suggest that DNA methylation may be involved in the pathogenesis of IPF. PMID:25333685

  6. Differential Methylation during Maize Leaf Growth Targets Developmentally Regulated Genes1[C][W][OPEN

    PubMed Central

    Candaele, Jasper; Demuynck, Kirin; Mosoti, Douglas; Beemster, Gerrit T.S.; Inzé, Dirk; Nelissen, Hilde

    2014-01-01

    DNA methylation is an important and widespread epigenetic modification in plant genomes, mediated by DNA methyltransferases (DMTs). DNA methylation is known to play a role in genome protection, regulation of gene expression, and splicing and was previously associated with major developmental reprogramming in plants, such as vernalization and transition to flowering. Here, we show that DNA methylation also controls the growth processes of cell division and cell expansion within a growing organ. The maize (Zea mays) leaf offers a great tool to study growth processes, as the cells progressively move through the spatial gradient encompassing the division zone, transition zone, elongation zone, and mature zone. Opposite to de novo DMTs, the maintenance DMTs were transcriptionally regulated throughout the growth zone of the maize leaf, concomitant with differential CCGG methylation levels in the four zones. Surprisingly, the majority of differentially methylated sequences mapped on or close to gene bodies and not to repeat-rich loci. Moreover, especially the 5′ and 3′ regions of genes, which show overall low methylation levels, underwent differential methylation in a developmental context. Genes involved in processes such as chromatin remodeling, cell cycle progression, and growth regulation, were differentially methylated. The presence of differential methylation located upstream of the gene anticorrelated with transcript expression, while gene body differential methylation was unrelated to the expression level. These data indicate that DNA methylation is correlated with the decision to exit mitotic cell division and to enter cell expansion, which adds a new epigenetic level to the regulation of growth processes. PMID:24488968

  7. DNA methylation in transcriptional repression of two differentially expressed X-linked genes, GPC3 and SYBL1

    PubMed Central

    Huber, Reid; Hansen, R. Scott; Strazzullo, Maria; Pengue, Gina; Mazzarella, Richard; D’Urso, Michele; Schlessinger, David; Pilia, Giuseppe; Gartler, Stanley M.; D’Esposito, Maurizio

    1999-01-01

    Methylation of CpG islands is an established transcriptional repressive mechanism and is a feature of silencing in X chromosome inactivation. Housekeeping genes that are subject to X inactivation exhibit differential methylation of their CpG islands such that the inactive alleles are hypermethylated. In this report, we examine two contrasting X-linked genes with CpG islands for regulation by DNA methylation: SYBL1, a housekeeping gene in the Xq pseudoautosomal region, and GPC3, a tissue-specific gene in Xq26 that is implicated in the etiology of the Simpson–Golabi–Behmel overgrowth syndrome. We observed that in vitro methylation of either the SYBL1 or the GPC3 promoter resulted in repression of reporter constructs. In normal contexts, we found that both the Y and inactive X alleles of SYBL1 are repressed and hypermethylated, whereas the active X allele is expressed and unmethylated. Furthermore, the Y and inactive X alleles of SYBL1 were derepressed by treatment with the demethylating agent azadeoxycytidine. GPC3 is also subject to X inactivation, and the active X allele is unmethylated in nonexpressing leukocytes as well as in an expressing cell line, suggesting that methylation is not involved in the tissue-specific repression of this allele. The inactive X allele, however, is hypermethylated in leukocytes, presumably reflecting early X inactivation events that become important for gene dosage in expressing lineages. These and other data suggest that all CpG islands on Xq, including the pseudoautosomal region, are subject to X inactivation-induced methylation. Additionally, methylation of SYBL1 on Yq may derive from a process related to X inactivation that targets large chromatin domains for transcriptional repression. PMID:9892682

  8. System-Wide Associations between DNA-Methylation, Gene Expression, and Humoral Immune Response to Influenza Vaccination

    PubMed Central

    Zimmermann, Michael T.; Oberg, Ann L.; Grill, Diane E.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; Kennedy, Richard B.; Poland, Gregory A.

    2016-01-01

    Failure to achieve a protected state after influenza vaccination is poorly understood but occurs commonly among aged populations experiencing greater immunosenescence. In order to better understand immune response in the elderly, we studied epigenetic and transcriptomic profiles and humoral immune response outcomes in 50–74 year old healthy participants. Associations between DNA methylation and gene expression reveal a system-wide regulation of immune-relevant functions, likely playing a role in regulating a participant’s propensity to respond to vaccination. Our findings show that sites of methylation regulation associated with humoral response to vaccination impact known cellular differentiation signaling and antigen presentation pathways. We performed our analysis using per-site and regionally average methylation levels, in addition to continuous or dichotomized outcome measures. The genes and molecular functions implicated by each analysis were compared, highlighting different aspects of the biologic mechanisms of immune response affected by differential methylation. Both cis-acting (within the gene or promoter) and trans-acting (enhancers and transcription factor binding sites) sites show significant associations with measures of humoral immunity. Specifically, we identified a group of CpGs that, when coordinately hypo-methylated, are associated with lower humoral immune response, and methylated with higher response. Additionally, CpGs that individually predict humoral immune responses are enriched for polycomb-group and FOXP2 transcription factor binding sites. The most robust associations implicate differential methylation affecting gene expression levels of genes with known roles in immunity (e.g. HLA-B and HLA-DQB2) and immunosenescence. We believe our data and analysis strategy highlight new and interesting epigenetic trends affecting humoral response to vaccination against influenza; one of the most common and impactful viral pathogens. PMID:27031986

  9. Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

    PubMed Central

    Römer, Winfried; Sonnleitner, Alois

    2015-01-01

    Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5), a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex) to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs) in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type. PMID:25767807

  10. c-Myc inhibits TP53INP1 expression via promoter methylation in esophageal carcinoma

    SciTech Connect

    Weng, Wenhao; Yang, Qinyuan; Huang, Miaolong; Qiao, Yongxia; Xie, Yuan; Yu, Yongchun; Jing, An; Li, Zhi

    2011-02-11

    Research highlights: {yields} TP53INP1 expression is down-regulated in esophageal carcinoma and is associated with CGI-131 methylation. {yields} Inhibition of CGI-131 methylation upregulates TP53INP1 expression in ESCC cell lines. {yields} Ectopic expression of TP53INP1 inhibits growth of ESCC cells by inducing apoptosis and inhibiting cell cycle progression. {yields} c-Myc binds to the promoter of TP53INP1 in vivo and vitro and recruits DNMT3A to TP53INP1 promoter for CGI-131 methylation. -- Abstract: Tumor protein p53-induced nuclear protein 1 (TP53INP1) is a well known stress-induced protein that plays a role in both cell cycle arrest and p53-mediated apoptosis. Loss of TP53INP1 expression has been reported in human melanoma, breast carcinoma, and gastric cancer. However, TP53INP1 expression and its regulatory mechanism in esophageal squamous cell carcinoma (ESCC) remain unclear. Our findings are in agreement with previous reports in that the expression of TP53INP1 was downregulated in 28% (10/36 cases) of ESCC lesions, and this was accompanied by significant promoter methylation. Overexpression of TP53INP1 induced G1 cell cycle arrest and increased apoptosis in ESCC cell lines (EC-1, EC-109, EC-9706). Furthermore, our study showed that the oncoprotein c-Myc bound to the core promoter of TP53INP1 and recruited DNA methyltransferase 3A to methylate the local promoter region, leading to the inhibition of TP53INP1 expression. Our findings revealed that TP53INP1 is a tumor suppressor in ESCC and that c-Myc-mediated DNA methylation-associated silencing of TP53INP1 contributed to the pathogenesis of human ESCC.

  11. CHST11 gene expression and DNA methylation in breast cancer

    PubMed Central

    HERMAN, DAMIR; LEAKEY, TATIANA I.; BEHRENS, ALICE; YAO-BORENGASSER, AIWEI; COONEY, CRAIG A.; JOUSHEGHANY, FARIBA; PHANAVANH, BOUNLEUT; SIEGEL, ERIC R.; SAFAR, A. MAZIN; KOROURIAN, SOHEILA; KIEBER-EMMONS, THOMAS; MONZAVI-KARBASSI, BEHJATOLAH

    2015-01-01

    methylation status of this gene also has potential as a prognostic biomarker. PMID:25586191

  12. Promoter DNA demethylation of Keap1 gene in diabetic cardiomyopathy

    PubMed Central

    Liu, Zhong-Zhi; Zhao, Xiang-Zhi; Zhang, Xue-Song; Zhang, Mei

    2014-01-01

    Researches have shown that the onset of diabetes is closely associated with oxidative stress and the chronic exposure leads to the development of complications such as diabetic cardiomyopathy. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. The aim of the present study was to investigate the status of Nrf2 mediated antioxidant system in myocardial biopsies of non-diabetic (NDM) and type-2 diabetic (DM-T2) cardiomyopathy patients. The western blot analysis of antioxidant proteins, real-time PCR analysis of Nrf2/Keap1 gene and bisulphate DNA sequencing analysis to study the methylation status of the CpG islands of Keap1 promoter DNA were performed. The immunoblot analysis showed the decreased level of antioxidant proteins other than Keap1 in the diabetic cardiopathy patients. Similarly, mRNA levels of Keap1 showed 5-fold increase in diabetic patients. Further analysis on promoter region of Keap1 gene revealed 80% demethylation in diabetic patients. Altogether, our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. Decreased Nrf2 activity represses the transcription of many antioxidant enzyme genes and alters the redox-balance up on diabetes. Thus, our study clearly demonstrates the failure of Nrf2 mediated antioxidant system revealed in biopsies of diabetic cardiomyopathy. PMID:25674242

  13. Methylation Profiling of Multiple Tumor Suppressor Genes in Hepatocellular Carcinoma and the Epigenetic Mechanism of 3OST2 Regulation

    PubMed Central

    Chen, Haiyan; Zhang, Tingguo; Sheng, Yan; Zhang, Cheng; Peng, Yunfei; Wang, Xiao; Zhang, Cuijuan

    2015-01-01

    DNA methylation is considered as a significant mechanism that silences tumor suppressor genes (TSGs) and could be used in the early diagnosis of cancer. Histone modifications often work together with DNA methylation; however, how these epigenetic alterations regulate TSGs remains unclear. Here, we determined the methylation status of ten TSGs (3OST2, ppENK, CHFR, LKB1, THBS1, HIC1, SLIT2, EDNRB, COX2, and CLDN7) in hepatocellular carcinoma (HCC) and corresponding noncancerous tissues. Methylation profiling revealed that four genes had very high frequencies of methylation in HCCs, but interestingly, similar high frequencies were also detected in corresponding noncancerous tissues (97.9% vs 95.8% for SLIT2, 93.8% vs 81.3% for EDNRB, 66.7% vs 85.4% for HIC1, and 56.3% vs 56.3% for ppENK, P > 0.05). Only the 3OST2 gene was frequently methylated in HCCs and there was significant difference between HCCs and corresponding noncancerous tissues (68.8% vs 37.5%, P < 0.05). 5-aza-2'-deoxycytidine (5-Aza-CdR) or trichostatin A (TSA) alone could partially reverse 3OST2 methylation, and their combination resulted in complete reversal. UHRF1 and histone H3R8me2s were both enriched on the hypermethylated 3OST2 promoter, but H3R8me2a was not. After 5-Aza-CdR or TSA treatment, the UHRF1 and H3R8me2s enrichment was decreased, while H3R8me2a enrichment increased. We demonstrated that 3OST2 methylation may play a critical role in the earliest steps of hepatocarcinogenesis and is directly regulated by UHRF1. Furthermore, H3R8me2s acted as a repressive mark, while H3R8me2a was correlated with 3OST2 transcriptional activity. PMID:26185536

  14. Developmental patterns of chromatin structure and DNA methylation responsible for epigenetic expression of a maize regulatory gene.

    PubMed Central

    Hoekenga, O A; Muszynski, M G; Cone, K C

    2000-01-01

    Epigenetic regulatory mechanisms heritably alter patterns of gene expression without changes in DNA sequence. Epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure. Pl-Blotched is a stable epigenetic allele of the maize anthocyanin regulatory gene, purple plant1(pl). Pl-Blotched plants display a variegated pattern of pigmentation that contrasts sharply with the uniformly dark purple pigmentation of plants carrying the dominant Pl-Rhoades allele. Previously, we showed that the lower level of pigmentation in Pl-Blotched is correlated with lower pl mRNA levels and increased DNA methylation at some sites. To explore how DNA methylation, chromatin structure, and developmental stage might contribute to the expression of Pl-Blotched, we used methylation-sensitive restriction enzymes and DNaseI sensitivity assays to compare the methylation status and chromatin structure of Pl-Blotched and Pl-Rhoades at different stages in development. Both alleles exhibit developmentally sensitive changes in methylation. In Pl-Blotched, methylation of two diagnostic HpaII/MspI sites increases progressively, coincident with the juvenile-to-adult transition in growth. In seedlings, the chromatin encompassing the coding region of the gene is less sensitive to DNaseI digestion in Pl-Blotched than in Pl-Rhoades. Developmental maturation from seedling to adult is accompanied by expansion of this closed chromatin domain to include the promoter and downstream flanking sequences. We provide evidence to show that chromatin structure, rather than DNA methylation, is the primary epigenetic determinant for the phenotypic differences between Pl-Blotched and Pl-Rhoades. PMID:10924483

  15. Fibrillarin methylates H2A in RNA polymerase I trans-active promoters in Brassica oleracea

    PubMed Central

    Loza-Muller, Lloyd; Rodríguez-Corona, Ulises; Sobol, Margarita; Rodríguez-Zapata, Luis C.; Hozak, Pavel; Castano, Enrique

    2015-01-01

    Fibrillarin is a well conserved methyltransferase involved in several if not all of the more than 100 methylations sites in rRNA which are essential for proper ribosome function. It is mainly localized in the nucleoli and Cajal bodies inside the cell nucleus where it exerts most of its functions. In plants, fibrillarin binds directly the guide RNA together with Nop56, Nop58, and 15.5ka proteins to form a snoRNP complex that selects the sites to be methylated in pre-processing of ribosomal RNA. Recently, the yeast counterpart NOP1 was found to methylate histone H2A in the nucleolar regions. Here we show that plant fibrillarin can also methylate histone H2A. In Brassica floral meristem cells the methylated histone H2A is mainly localized in the nucleolus but unlike yeast or human cells it also localize in the periphery of the nucleus. In specialized transport cells the pattern is altered and it exhibits a more diffuse staining in the nucleus for methylated histone H2A as well as for fibrillarin. Here we also show that plant fibrillarin is capable of interacting with H2A and carry out its methylation in the rDNA promoter. PMID:26594224

  16. Serotonin 1B Receptor Gene (HTR1B) Methylation as a Risk Factor for Callous-Unemotional Traits in Antisocial Boys.

    PubMed

    Moul, Caroline; Dobson-Stone, Carol; Brennan, John; Hawes, David J; Dadds, Mark R

    2015-01-01

    The serotonin system is thought to play a role in the aetiology of callous-unemotional (CU) traits in children. Previous research identified a functional single nucleotide polymorphism (SNP) from the promoter region of the serotonin 1B receptor gene as being associated with CU traits in boys with antisocial behaviour problems. This research tested the hypothesis that CU traits are associated with reduced methylation of the promoter region of the serotonin 1B receptor gene due to the influence of methylation on gene expression. Participants (N = 117) were boys with antisocial behaviour problems aged 3-16 years referred to University of New South Wales Child Behaviour Research Clinics. Participants volunteered a saliva sample from which the genotype of a SNP from the promoter region of the serotonin 1B receptor gene and the methylation levels of 30 CpG sites from 3 CpG regions surrounding the location of this polymorphism were assayed. Lower levels of serotonin 1B receptor gene methylation were associated with higher levels of CU traits. This relationship, however, was found to be moderated by genotype and carried exclusively by two CpG sites for which levels of methylation were negatively associated with overall methylation levels in this region of the gene. Results provide support to the emerging literature that argues for a genetically-driven system-wide alteration in serotonin function in the aetiology of CU traits. Furthermore, the results suggest that there may be two pathways to CU traits that involve methylation of the serotonin 1B receptor gene; one that is driven by a genotypic risk and another that is associated with risk for generally increased levels of methylation. Future research that aims to replicate and further investigate these results is required. PMID:25993020

  17. Methylation and mRNA expression levels of P15, death-associated protein kinase, and suppressor of cytokine signaling-1 genes in multiple myeloma

    PubMed Central

    Liu, Lin; Tan, Lin; He, Zhenxin

    2016-01-01

    Objective(s): The aim of this study was to investigate the methylation status and mRNA expression levels of P15, death-associated protein kinase (DAPK), and suppressor of cytokine signaling-1 (SOCS1) genes in multiple myeloma (MM). Materials and Methods: The bone marrow samples of 54 MM patients were collected and the methylation status of the P15, DAPK, and SOCS1 gene promoter regions was determined by methylation-specific polymerase chain reaction. Automated sequencing technology was used to sequence the amplified products in order to analyze the base methylation sites. mRNA expression levels were determined using real-time fluorescent quantitative polymerase chain reaction. Results: Among the 54 MM patients, the positive methylation rates of the P15, DAPK, and SOCS1 genes were 27.78%, 18.52%, and 16.67%, respectively. The methylation results were confirmed by sequencing. The positive methylation rates of the P15, DAPK, and SOCS1 genes showed no correlation with patient gender, age, typing, staging, and grouping (P>0.05). There was no significant difference in the mRNA expression levels of the P15, DAPK, and SOCS1 genes between the MM patient group and the control group (P>0.05). Conclusions: Aberrant methylation of the P15, DAPK, and SOCS1 genes exists in MM, and these genes may play certain roles in pathogenesis of MM. There was no significant difference in mRNA expression levels between the methylated group and the non-methylated group, suggesting that these genes are regulated by other mechanisms during their transcription.

  18. Identification and characterization of methylation-dependent/independent DNA regulatory elements in the human SLC9B1 gene

    PubMed Central

    Kumar, Priya L.; James, Paul F.

    2015-01-01

    The human NHEDC1 (hNHEDC1) protein is thought to be essential for sperm motility and fertility however the mechanisms regulating its gene expression are largely unknown. In this study we have identified multiple DNA regulatory elements in the 5′ end of the gene encoding hNHEDC1 (SLC9B1) and have explored the role that DNA methylation at these elements plays in the regulation of its expression. We first show that the full-length hNHEDC1 protein is testis-specific for the tissues that we tested and that it localizes to the cells of the seminiferous tubules. In silico analysis of the SLC9B1 gene locus identified two putative promoters (P1 and P2) and two CpG islands - CpGI (overlapping with P1) and CpGII (intragenic) - at the 5′ end of the gene. By deletion analysis of P1, we show that the region from −23bp to +200bp relative to the transcription start site (TSS) is sufficient for optimal promoter activity in a germ cell line. Additionally, in vitro methylation of the P1 (the −500bp to +200bp region relative to the TSS) abolishes its activity in germ cells and somatic cells strongly suggesting that DNA methylation at this promoter could regulate SLC9B1 expression. Furthermore, bisulfite-sequencing analysis of the P1/CpGI uncovered reduced methylation in the testis vs. lung whereas CpGII displayed no differences in methylation between these two tissues. Additionally, treatment of HEK 293 cells with 5-Aza2-Deoxycytidine led to upregulation of NHEDC1 transcript and reduced methylation in the promoter CpGI. Finally, we have uncovered both enhancer and silencer functions of the intragenic SLC9B1 CpGII. In all, our data suggests that SLC9B1 gene expression could be regulated via a concerted action of DNA methylation-dependent and independent mechanisms mediated by these multiple DNA regulatory elements. PMID:25701605

  19. Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing

    PubMed Central

    Kenyon, Jonathan; Nickel-Meester, Gabrielle; Qing, Yulan; Santos-Guasch, Gabriela; Drake, Ellen; PingfuFu; Sun, Shuying; Bai, Xiaodong; Wald, David; Arts, Eric; Gerson, Stanton L.

    2016-01-01

    Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1, an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from −938 to −337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34+ selected hematopoietic stem and progenitor cells. PMID:27570841

  20. Regional DNA methylation differences between humans and chimpanzees are associated with genetic changes, transcriptional divergence and disease genes.

    PubMed

    Fukuda, Kei; Ichiyanagi, Kenji; Yamada, Yoichi; Go, Yasuhiro; Udono, Toshifumi; Wada, Seitaro; Maeda, Toshiyuki; Soejima, Hidenobu; Saitou, Naruya; Ito, Takashi; Sasaki, Hiroyuki

    2013-07-01

    Changes in gene expression have been proposed to have an important role in the evolutionary changes in phenotypes. Interspecific changes in gene expression can result not only from genetic changes in regulatory regions but also from epigenetic changes in such regions. Here we report the identification of genomic regions showing differences in DNA methylation between humans and chimpanzees (termed S-DMRs for species-specific differentially methylated regions) on chromosomes 21 and 22. These regional methylation differences are frequently associated with genes, including those relevant to a disease, such as Alzheimer's disease, diabetes mellitus or cancer. Methylation differences are often correlated with changes in promoter activity or alternative splicing. Comparative studies including other great ape species provide evidence for the contribution of genetic changes to some of these S-DMRs. Genetic changes responsible for the S-DMRs include gain or loss of CTCF-binding site and changes in CpG density in microsatellite repeats. Our results suggest that DNA methylation changes, often caused by small sequence changes, contribute to transcriptional and phenotypic diversification in hominid evolution. PMID:23739127

  1. ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter.

    PubMed

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

    2014-08-01

    One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance. PMID:25089713

  2. ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter

    PubMed Central

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

    2014-01-01

    One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance. PMID:25089713

  3. Methylation of multiple genes in hepatitis C virus associated hepatocellular carcinoma.

    PubMed

    Zekri, Abdel-Rahman N; Bahnasy, Abeer A; Shoeab, Fatma Elzahraa M; Mohamed, Waleed S; El-Dahshan, Dina H; Ali, Fahmey T; Sabry, Gilane M; Dasgupta, Nairajana; Daoud, Sayed S

    2014-01-01

    We studied promoter methylation (PM) of 11 genes in Peripheral Blood Lymphocytes (PBLs) and tissues of hepatitis C virus (HCV) associated hepatocellular carcinoma (HCC) and chronic hepatitis (CH) Egyptian patients. The present study included 31 HCC with their ANT, 38 CH and 13 normal hepatic tissue (NHT) samples. In all groups, PM of APC, FHIT, p15, p73, p14, p16, DAPK1, CDH1, RARβ, RASSF1A, O(6)MGMT was assessed by methylation-specific PCR (MSP). APC and O6-MGMT protein expression was assessed by immunohistochemistry (IHC) in the studied HCC and CH (20 samples each) as well as in a different HCC and CH set for confirmation of MSP results. PM was associated with progression from CH to HCC. Most genes showed high methylation frequency (MF) and the methylation index (MI) increased with disease progression. MF of p14, p73, RASSF1A, CDH1 and O(6)MGMT was significantly higher in HCC and their ANT. MF of APC was higher in CH. We reported high concordance between MF in HCC and their ANT, MF in PBL and CH tissues as well as between PM and protein expression of APC and O(6)MGMT. A panel of 4 genes (APC, p73, p14, O(6)MGMT) classifies the cases independently into HCC and CH with high accuracy (89.9%), sensitivity (83.9%) and specificity (94.7%). HCV infection may contribute to hepatocarcinogenesis through enhancing PM of multiple genes. PM of APC occurs early in the cascade while PM of p14, p73, RASSF1A, RARB, CDH1 and O(6)MGMT are late changes. A panel of APC, p73, p14, O6-MGMT could be used in monitoring CH patients for early detection of HCC. Also, we found that, the methylation status is not significantly affected by whether the tissue was from the liver or PBL, indicating the possibility of use PBL as indicator to genetic profile instead of liver tissue regardless the stage of disease. PMID:25685469

  4. MGMT-B gene promoter hypermethylation in patients with inflammatory bowel disease - a novel finding.

    PubMed

    Mokarram, Pooneh; Kavousipour, Soudabeh; Sarabi, Mostafa Moradi; Mehrabani, Golnosh; Fahmidehkar, Mohammad Ali; Shamsdin, Seyedeh Azra; Alipour, Abbas; Naini, Mahvash Alizade

    2015-01-01

    Inflammatory bowel disease (IBD) is a disease strongly associated with colorectal cancer (CRC) as a well-known precancerous condition. Alterations in DNA methylation and mutation in K-ras are believed to play an early etiopathogenic role in CRC and may also an initiating event through deregulation of molecular signaling. Epigenetic silencing of APC and SFRP2 in the WNT signaling pathway may also be involved in IBD-CRC. The role of aberrant DNA methylation in precancerous state of colorectal cancer (CRC) is under intensive investigation worldwide. The aim of this study was to investigate the status of promoter methylation of MGMT-B, APC1A and SFRP2 genes, in inflamed and normal colon tissues of patients with IBD compared with control normal tissues. A total of 52 IBD tissues as well as corresponding normal tissues and 30 samples from healthy participants were obtained. We determined promoter methylation status of MGMT-B, SFRP2 and APC1A genes by chemical treatment with sodium bisulfite and subsequent MSP. The most frequently methylated locus was MGMT-B (71%; 34 of 48), followed by SFRP2 (66.6 %; 32 of 48), and APC1A (43.7%; 21 of 48). Our study demonstrated for the first time that hypermethylation of the MGMT-B and the SFRP2 gene promoter regions might be involved in IBD development. Methylation of MGMT-B and SFRP2 in IBD patients may provide a method for early detection of IBD-associated neoplasia. PMID:25773792

  5. MGMT promoter methylation is associated with temozolomide response and prolonged progression-free survival in disseminated cutaneous melanoma.

    PubMed

    Tuominen, Rainer; Jewell, Rosalyn; van den Oord, Joost J; Wolter, Pascal; Stierner, Ulrika; Lindholm, Christer; Hertzman Johansson, Carolina; Lindén, Diana; Johansson, Hemming; Frostvik Stolt, Marianne; Walker, Christy; Snowden, Helen; Newton-Bishop, Julia; Hansson, Johan; Egyházi Brage, Suzanne

    2015-06-15

    To investigate the predictive and prognostic value of O(6) -methylguanine DNA methyltransferase (MGMT) inactivation by analyses of promoter methylation in pretreatment tumor biopsies from patients with cutaneous melanoma treated with dacarbazine (DTIC) or temozolomide (TMZ) were performed. The patient cohorts consisted of Belgian and Swedish disseminated melanoma patients. Patients were subdivided into those receiving single-agent treatment with DTIC/TMZ (cohort S, n = 74) and those treated with combination chemotherapy including DTIC/TMZ (cohort C, n = 79). Median follow-up was 248 and 336 days for cohort S and cohort C, respectively. MGMT promoter methylation was assessed by three methods. The methylation-related transcriptional silencing of MGMT mRNA expression was assessed by real-time RT-PCR. Response to chemotherapy and progression-free survival (PFS) and overall survival were correlated to MGMT promoter methylation status. MGMT promoter methylation was detected in tumor biopsies from 21.5 % of the patients. MGMT mRNA was found to be significantly lower in tumors positive for MGMT promoter methylation compared to tumors without methylation in both treatment cohorts (p < 0.005). DTIC/TMZ therapy response rate was found to be significantly associated with MGMT promoter methylation in cohort S (p = 0.0005), but did not reach significance in cohort C (p = 0.16). Significantly longer PFS was observed among patients with MGMT promoter-methylated tumors (p = 0.002). Multivariate Cox regression analysis identified presence of MGMT promoter methylation as an independent variable associated with longer PFS. Together, this implies that MGMT promoter methylation is associated with response to single-agent DTIC/TMZ and longer PFS in disseminated cutaneous melanoma. PMID:25400033

  6. Gene structure, DNA methylation, and imprinted expression of the human SNRPN gene

    SciTech Connect

    Glenn, C.C.; Jong, T.C.; Filbrandt, M.M.

    1996-02-01

    The human SNRPN (small nuclear ribonucleoprotein polypeptide N) gene is one of a gene family that encode proteins involved in pre-mRNA splicing and maps to the smallest deletion region involved in the Prader-Willi syndrome (PWS) within chromosome 15q11-q13. Paternal only expression of SNRPN has previously been demonstrated by use of cell lines from PWS patients (maternal allele only) and Angelman syndrome (AS) patients (paternal allele only). We have characterized two previously unidentified 5{prime} exons of the SNRPN gene and demonstrate that exons -1 and 0 are included in the full-length transcript. This gene is expressed in a wide range of somatic tissues and at high, approximately equal levels in all regions of the brain. Both the first exon of SNRPN (exon -1) and the putative transcription start site are embedded within a CpG island. This CpG island is extensively methylated on the repressed maternal allele and is unmethylated on the expressed paternal allele, in a wide range of fetal and adult somatic cells. This provides a quick and highly reliable diagnostic assay for PWS and AS, which is based on DNA-methylation analysis that has been tested on >100 patients in a variety of tissues. Conversely, several CpG sites {approximately}22 kb downstream of the transcription start site in intron 5 are preferentially methylated on the expressed paternal allele in somatic tissues and male germ cells, whereas these same sites are unmethylated in fetal oocytes. These findings are consistent with a key role for DNA methylation in the imprinted inheritance and subsequent gene expression of the human SNRPN gene. 59 refs., 9 figs., 1 tab.

  7. Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression

    PubMed Central

    Gordon, Katrina; Clouaire, Thomas; Bao, Xun X.; Kemp, Sadie E.; Xenophontos, Maria; de Las Heras, Jose Ignacio; Stancheva, Irina

    2014-01-01

    Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells. PMID:24371281

  8. Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells

    PubMed Central

    Reiner, Andrew H.; Coll, Mar; Verhulst, Stefaan; Mannaerts, Inge; Øie, Cristina I.; Smedsrød, Bård; Najimi, Mustapha; Sokal, Etienne; Luttun, Aernout; Sancho-Bru, Pau; Collas, Philippe; van Grunsven, Leo A.

    2015-01-01

    Background & Aims Liver fibrogenesis – scarring of the liver that can lead to cirrhosis and liver cancer – is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs. Material and methods Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR. Results We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiation-associated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis. Conclusions Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation. PMID:26353929

  9. Ageing, chronic alcohol consumption and folate are determinants of genomic DNA methylation, p16 promoter methylation and the expression of p16 in the mouse colon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elder age and chronic alcohol consumption are important risk factors for the development of colon cancer. Each factor can alter genomic and gene-specific DNA methylation. This study examined the effects of aging and chronic alcohol consumption on genomic and p16-specific methylation, and p16 express...

  10. APC promoter is frequently methylated in pancreatic juice of patients with pancreatic carcinomas or periampullary tumors

    PubMed Central

    Ginesta, Mireia M.; Diaz-Riascos, Zamira Vanessa; Busquets, Juli; Pelaez, Núria; Serrano, Teresa; Peinado, Miquel Àngel; Jorba, Rosa; García-Borobia, Francisco Javier; Capella, Gabriel; Fabregat, Joan

    2016-01-01

    Early detection of pancreatic and periampullary neoplasms is critical to improve their clinical outcome. The present authors previously demonstrated that DNA hypermethylation of adenomatous polyposis coli (APC), histamine receptor H2 (HRH2), cadherin 13 (CDH13), secreted protein acidic and cysteine rich (SPARC) and engrailed-1 (EN-1) promoters is frequently detected in pancreatic tumor cells. The aim of the present study was to assess their prevalence in pancreatic juice of carcinomas of the pancreas and periampullary area. A total of 135 pancreatic juices obtained from 85 pancreatic cancer (PC), 26 ampullary carcinoma (AC), 10 intraductal papillary mucinous neoplasm (IPMN) and 14 chronic pancreatitis (CP) patients were analyzed. The methylation status of the APC, HRH2, CDH13, SPARC and EN-1 promoters was analyzed using methylation specific-melting curve analysis (MS-MCA). Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were also tested with allele-specific quantitative polymerase chain reaction amplification. Out of the 5 promoters analyzed, APC (71%) and HRH2 (65%) were the most frequently methylated in PC juice. APC methylation was also detected at a high frequency in AC (76%) and IPMN (80%), but only occasionally observed in CP (7%). APC methylation had a high sensitivity (71–80%) for all types of cancer analyzed. The panel (where a sample scored as positive when ≥2 markers were methylated) did not outperform APC as a single marker. Finally, KRAS detection in pancreatic juice offered a lower sensitivity (50%) and specificity (71%) for detection of any cancer. APC hypermethylation in pancreatic juice, as assessed by MS-MCA, is a frequent event of potential clinical usefulness in the diagnosis of pancreatic and periampullary neoplasms. PMID:27602165

  11. Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

    PubMed

    Schroeder, Diane I; Jayashankar, Kartika; Douglas, Kory C; Thirkill, Twanda L; York, Daniel; Dickinson, Pete J; Williams, Lawrence E; Samollow, Paul B; Ross, Pablo J; Bannasch, Danika L; Douglas, Gordon C; LaSalle, Janine M

    2015-08-01

    Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo. PMID:26241857

  12. Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas

    PubMed Central

    Schroeder, Diane I.; Jayashankar, Kartika; Douglas, Kory C.; Thirkill, Twanda L.; York, Daniel; Dickinson, Pete J.; Williams, Lawrence E.; Samollow, Paul B.; Ross, Pablo J.; Bannasch, Danika L.; Douglas, Gordon C.; LaSalle, Janine M.

    2015-01-01

    Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo. PMID:26241857

  13. DNA Methylation and Gene Expression Profiling of Ewing Sarcoma Primary Tumors Reveal Genes That Are Potential Targets of Epigenetic Inactivation

    PubMed Central

    Patel, Nikul; Black, Jennifer; Chen, Xi; Marcondes, A. Mario; Grady, William M.; Lawlor, Elizabeth R.; Borinstein, Scott C.

    2012-01-01

    The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs) using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA)-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1,) and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis. PMID:23024594

  14. Methylation of Epstein-Barr virus Rta promoter in EBV primary infection, reactivation and lymphoproliferation.

    PubMed

    Germi, Raphaële; Guigue, Nicolas; Lupo, Julien; Semenova, Touyana; Grossi, Laurence; Vermeulen, Odile; Epaulard, Olivier; de Fraipont, Florence; Morand, Patrice

    2016-10-01

    During Epstein-Barr virus (EBV) latency, the EBV genome is largely silenced by methylation. This silencing is overturned during the switch to the lytic cycle. A key event is the production of the viral protein Zta which binds to three Zta-response elements (ZRE) from the Rta promoter (Rp), two of which (ZRE2 and ZRE3) include three CpG motifs methylated in the latent genome. The bisulphite pyrosequencing reaction was used to quantify the methylation of ZRE2, ZRE3a, and ZRE3b in EBV-positive cell lines and in ex vivo samples of EBV-related diseases, in order to assess whether the level of methylation in these ZREs could provide additional information to viral DNA load and serology in the characterization of EBV-associated diseases. In PBMC from two patients with infectious mononucleosis, over time Rp became increasingly methylated whereas EBV load decreased. In tonsil from patients with chronic tonsillitis, the methylation was less than in EBV-associated tumors, regardless of the viral load. This was even more striking when only the ZRE3a and ZRE3b were considered since some samples presented unbalanced profiles on ZRE2. EBV reactivation in cell culture showed that the reduction in the overall level of methylation was closely related to the production of unmethylated virions. Thus, an assessment of the level of methylation may help to better characterize EBV replication in PBMC and in biopsies with high EBV load, during infectious mononucleosis and EBV-associated cancers. J. Med. Virol. 88:1814-1820, 2016. © 2016 Wiley Periodicals, Inc. PMID:26990870

  15. DNA methylation analysis of secreted frizzled-related protein 2 gene for the early detection of colorectal cancer in fecal DNA

    PubMed Central

    Babaei, Hadi; Mohammadi, Mohsen; Salehi, Rasoul

    2016-01-01

    Background: The early detection of colorectal cancer (CRC) with high sensitivity screening is essential for the reduction of cancer-specific mortality. Abnormally methylated genes that are responsible for the pathogenesis of cancers can be used as biomarkers for the detection of CRC. The methylation status of the secreted frizzled-related protein 2 (SFRP2) gene was evaluated for their use as a marker in the noninvasive detection of CRC. Materials and Methods: Methylation-specific polymerase chain reaction was performed to analyze the promoter CpG methylation of SFRP2 in the fecal DNA of 25 patients with CRC and 25 individuals exhibiting normal colonoscopy results. Results: Promoter methylation levels of SFRP2 in CRC patients and in healthy controls were 60% and 8%, respectively. Methylation of the SFRP2 promoter in fecal DNA is associated with the presence of colorectal tumors. Conclusion: Hence, the detection of aberrantly methylated DNA in fecal samples may present a promising, noninvasive screening method for CRC.

  16. Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40

    PubMed Central

    Ayadi, Wajdi; Allaya, Nesrine; Frikha, Hanèn; Trigui, Emna; Khabir, Abdelmajid; Ghorbel, Abdelmonem; Daoud, Jamel; Frikha, Mounir; Mokdad-Gargouri, Raja

    2014-01-01

    To further explore the epigenetic changes in nasopharyngeal carcinoma (NPC), methylation-sensitive arbitrarily primed PCR was performed on NPC biopsies and nontumor nasopharyngeal samples. We have shown mainly two DNA fragments that appeared to be differentially methylated in NPCs versus nontumors. The first, defined as hypermethylated, corresponds to a CpG island at the 5′-end of the tetratricopeptide repeat domain 40 (TTC40) gene, whereas the second, defined as hypo-methylated, is located on repetitive sequences at chromosomes 16p11.1 and 13.1. Thereafter, the epigenetic alteration on the 5′-TTC40 gene was confirmed by methylation-specific PCR, showing a significant aberrant methylation in NPCs, compared to nontumors. In addition, the bisulfite sequencing analysis has shown a very high density of methylated cytosines in C15, C17, and X666 NPC xenografts. To assess whether TTC40 gene is silenced by aberrant methylation, we examined the gene expression by reverse transcription-PCR. Our analysis showed that the mRNA expression was significantly lower in tumors than in nontumors, which is associated with 5′-TTC40 gene hypermethylation. In conclusion, we found that the 5′-TTC40 gene is frequently methylated and is associated with the loss of mRNA expression in NPCs. Hypermethylation of 5′-TTC40 gene might play a role in NPC development; nevertheless, other studies are needed. PMID:25101295

  17. Reduction of TIP30 in esophageal squamous cell carcinoma cells involves promoter methylation and microRNA-10b

    SciTech Connect

    Dong, Wenjie; Shen, Ruizhe; Cheng, Shidan

    2014-10-31

    Highlights: • TIP30 expression is frequently suppressed in ESCC. • TIP30 was hypermethylated in ESCC. • Reduction of TIP30 was significantly correlated with LN metastasis. • miR-10b is a direct regulator of TIP30. - Abstract: TIP30 is a putative tumor suppressor that can promote apoptosis and inhibit angiogenesis. However, the role of TIP30 in esophageal squamous cell carcinoma (ESCC) biology has not been investigated. Immunohistochemistry was used to investigate the expression of TIP30 in 70 ESCC. Hypermethylation of TIP30 was evaluated by the methylation specific PCR (MSP) method in ESCC (tumor and paired adjacent non-tumor tissues). Lost expression of TIP30 was observed in 50 of 70 (71.4%) ESCC. 61.4% (43 of 70) of primary tumors analyzed displayed TIP30 hypermethylation, indicating that this aberrant characteristic is common in ESCC. Moreover, a statistically significant inverse association was found between TIP30 methylation status and expression of the TIP30 protein in tumor tissues (p = 0.001). We also found that microRNA-10b (miR-10b) targets a homologous DNA region in the 3′untranslated region of the TIP30 gene and represses its expression at the transcriptional level. Reporter assay with 3′UTR of TIP30 cloned downstream of the luciferase gene showed reduced luciferase activity in the presence of miR-10b, providing strong evidence that miR-10b is a direct regulator of TIP30. These results suggest that TIP30 expression is regulated by promoter methylation and miR-10b in ESCC.

  18. Prognostic value of aberrant promoter hypermethylation of tumor-related genes in early-stage head and neck cancer.

    PubMed

    Misawa, Kiyoshi; Mochizuki, Daiki; Imai, Atsushi; Endo, Shiori; Mima, Masato; Misawa, Yuki; Kanazawa, Takeharu; Carey, Thomas E; Mineta, Hiroyuki

    2016-05-01

    Staging and pathological grading are useful, but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Accordingly, molecular biomarkers that predict the risk of recurrence are necessary to improve clinical outcomes. The methylation statuses of the promoters of 11 tumor-related genes (p16, RASSF1A, E-cadherin, H-cadherin, MGMT, DAPK, DCC, COL1A2, TAC1, SST, and GALR1) were analyzed in 133 HNSCC cases using quantitative methylation-specific PCR. We detected frequent methylation of p16 (44%), RASSF1A (18%), E-cadherin (53%), H-cadherin (35%), MGMT (35%), DAPK (53%), DCC (42%), COL1A2 (44%), TAC1 (61%), SST (64%), and GALR1 (44%) in HNSCC. Disease-free survival was lower in patients with 6-11 methylated genes than in those with 0-5 methylated genes (log-rank test, P = 0.001). In a multivariate Cox proportional hazards analysis, the methylation of E-cadherin, COL1A2, TAC1, and GALR1 was associated with poor survival, with hazard ratios of 4.474 (95% CI, 1.241-16.124). In a joint analysis of these four genes, patients with 2-4 methylated genes had a significantly lower survival rate than those with 0-1 methylated genes in early-stage HNSCC. Importantly, the methylation of some genes was closely related to poor prognosis in early-stage HNSCC, providing strong evidence that these hypermethylated genes are valuable biomarkers for prognostic evaluation. PMID:27027429

  19. Differential DNA methylation and expression of inflammatory and zinc transporter genes defines subgroups of osteoarthritic hip patients

    PubMed Central

    Rushton, Michael D; Young, David A; Loughlin, John; Reynard, Louise N

    2015-01-01

    Objectives We have previously shown that the cartilage DNA methylome delineates two clusters of osteoarthritic (OA) hip patients, characterised by differential methylation of inflammatory genes, while others have demonstrated a link between zinc homeostasis and inflammation in OA. We aimed to investigate these effects at the methylation and gene expression level. Methods We used our previously generated methylation data while quantitative PCR was used to measure gene expression using RNA from the hip cartilage of members of both clusters and from control individuals without hip OA. Results One of the OA clusters is characterised by the promoter hypomethylation and increased expression of inflammation-associated genes including IL1A and TNF. Furthermore, we show that the increase in expression of these genes is accompanied by increased expression of several zinc transporter genes. In addition, the zinc responsive transcription factor MTF1 is also upregulated, which is accompanied by an increase in the expression of its targets the metalloproteinases MMP13 and ADAMTS5. Conclusions We have identified a subgroup of OA hip patients that are epigenetically and transcriptiomically characterised by a cartilage inflammatory phenotype with concurrent differential regulation of zinc regulators. The identification of subgroups enhances stratified phenotyping of OA patients and has important implications for future therapeutic applications. PMID:25854584

  20. Trichloroethylene-Induced Gene Expression and DNA Methylation Changes in B6C3F1 Mouse Liver

    PubMed Central

    Tong, Jian; Chen, Tao

    2014-01-01

    Trichloroethylene (TCE), widely used as an organic solvent in the industry, is a common contaminant in air, soil, and water. Chronic TCE exposure induced hepatocellular carcinoma in mice, and occupational exposure in humans was suggested to be associated with liver cancer. To understand the role of non-genotoxic mechanism(s) for TCE action, we examined the gene expression and DNA methylation changes in the liver of B6C3F1 mice orally administered with TCE (0, 100, 500 and 1000 mg/kg b.w. per day) for 5 days. After 5 days TCE treatment at a dose level of 1000 mg/kg b.w., a total of 431 differentially expressed genes were identified in mouse liver by microarray, of which 291 were up-regulated and 140 down-regulated. The expression changed genes were involved in key signal pathways including PPAR, proliferation, apoptosis and homologous recombination. Notably, the expression level of a number of vital genes involved in the regulation of DNA methylation, such as Utrf1, Tet2, DNMT1, DNMT3a and DNMT3b, were dysregulated. Although global DNA methylation change was not detected in the liver of mice exposed to TCE, the promoter regions of Cdkn1a and Ihh were found to be hypo- and hypermethylated respectively, which correlated negatively with their mRNA expression changes. Furthermore, the gene expression and DNA methylation changes induced by TCE were dose dependent. The overall data indicate that TCE exposure leads to aberrant DNA methylation changes, which might alter the expression of genes involved in the TCE-induced liver tumorgenesis. PMID:25549359

  1. Methylation of miRNA genes in the response to temperature stress in Populus simonii.

    PubMed

    Ci, Dong; Song, Yuepeng; Tian, Min; Zhang, Deqiang

    2015-01-01

    DNA methylation and miRNAs provide crucial regulation of the transcriptional and post-transcriptional responses to abiotic stress. In this study, we used methylation-sensitive amplification polymorphisms to identify 1066 sites that were differentially methylated in response to temperature stress in Populus simonii. Among these loci, BLAST searches of miRBase identified seven miRNA genes. Expression analysis by quantitative real-time PCR suggested that the methylation pattern of these miRNA genes probably influences their expression. Annotation of these miRNA genes in the sequenced genome of Populus trichocarpa found three target genes (Potri.007G090400, Potri.014G042200, and Potri.010G176000) for the miRNAs produced from five genes (Ptc-MIR396e and g, Ptc-MIR156i and j, and Ptc-MIR390c) respectively. The products of these target genes function in lipid metabolism to deplete lipid peroxide. We also constructed a network based on the interactions between DNA methylation and miRNAs, miRNAs and target genes, and the products of target genes and the metabolic factors that they affect, including H2O2, malondialdehyde, catalase (CAT), and superoxide dismutase. Our results suggested that DNA methylation probably regulates the expression of miRNA genes, thus affecting expression of their target genes, likely through the gene-silencing function of miRNAs, to maintain cell survival under abiotic stress conditions. PMID:26579167

  2. Methylation of miRNA genes in the response to temperature stress in Populus simonii

    PubMed Central

    Ci, Dong; Song, Yuepeng; Tian, Min; Zhang, Deqiang

    2015-01-01

    DNA methylation and miRNAs provide crucial regulation of the transcriptional and post-transcriptional responses to abiotic stress. In this study, we used methylation-sensitive amplification polymorphisms to identify 1066 sites that were differentially methylated in response to temperature stress in Populus simonii. Among these loci, BLAST searches of miRBase identified seven miRNA genes. Expression analysis by quantitative real-time PCR suggested that the methylation pattern of these miRNA genes probably influences their expression. Annotation of these miRNA genes in the sequenced genome of Populus trichocarpa found three target genes (Potri.007G090400, Potri.014G042200, and Potri.010G176000) for the miRNAs produced from five genes (Ptc-MIR396e and g, Ptc-MIR156i and j, and Ptc-MIR390c) respectively. The products of these target genes function in lipid metabolism to deplete lipid peroxide. We also constructed a network based on the interactions between DNA methylation and miRNAs, miRNAs and target genes, and the products of target genes and the metabolic factors that they affect, including H2O2, malondialdehyde, catalase (CAT), and superoxide dismutase. Our results suggested that DNA methylation probably regulates the expression of miRNA genes, thus affecting expression of their target genes, likely through the gene-silencing function of miRNAs, to maintain cell survival under abiotic stress conditions. PMID:26579167

  3. DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line

    PubMed Central

    Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

    2010-01-01

    Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2′-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5′-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator TaxBLV decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267LTaxSN 5′-LTR compared with the L267 5′-LTR. Interestingly, DNA methylation inhibitors and TaxBLV synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the −154 or −129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at −129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5′-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency. PMID:20413592

  4. Methylation changes in the TFAP2E promoter region are associated with BRAF mutation and poorer overall & disease free survival in colorectal cancer

    PubMed Central

    Beggs, Andrew D.; Dilworth, Mark P.; Domingo, Enric; Midgley, Rachel; Kerr, David; Tomlinson, Ian P.M.; Middleton, Gary W.

    2015-01-01

    Introduction BRAF mutant colorectal cancer carries a poor prognosis which is thought to be related to poor response to conventional chemotherapy. BRAF mutation is associated with the serrated tumour phenotype. We hypothesised that one of the mechanisms by which BRAF mutant colorectal cancer demonstrate poor outcomes with chemotherapy is abnormal gene methylation Methods The Cancer Genome Atlas (TCGA) methylation data was analysed using a linear regression model with BRAF mutation as an independent variable. Expression datasets were also obtained to correlate functional changes. Top differentially methylated probes were taken forward for validation by methylation pyrosequencing. These probes were analysed on a cohort of patients enriched for BRAF mutations taken from the VICTOR and QUASAR2 studies. Results In an analysis of 91 tumours (9 BRAF mutant, 82 wild type), the Illumina probe cg11835197 was the probe identified as top differentially methylated (p = 2.56×10-7, Bayes Factor (BF) =6.54). This probe covered a region −413bp from the promoter region of TFAP2E. We found a complex pattern of CpG specific methylation of this region which was associated with both overall (p=0.044) and disease free (p=0.046) survival. Discussion BRAF mutant tumours may attain part of their chemoresistance from abnormal TFAP2E methylation, which has not previously been described. PMID:26097884

  5. V2 of tomato yellow leaf curl virus can suppress methylation-mediated transcriptional gene silencing in plants.

    PubMed

    Wang, Bi; Li, Fangfang; Huang, Changjun; Yang, Xiuling; Qian, Yajuan; Xie, Yan; Zhou, Xueping

    2014-01-01

    Tomato yellow leaf curl virus (TYLCV) is a DNA virus belonging to the genus Begomovirus. TYLCV replicates using double-stranded DNA intermediates that can become the target of plant transcriptional gene silencing (TGS). Here, we show that the V2 protein of TYLCV can suppress TGS of a transcriptionally silenced green fluorescent protein (GFP) transgene in Nicotiana benthamiana line 16-TGS. Through bisulfite sequencing and chop-PCR, we demonstrated that the TYLCV V2 can reverse GFP transgene silencing by reducing the methylation levels in the 35S promoter sequence. Both AtSN1 and MEA-ISR loci in Arabidopsis thaliana were previously reported to be strongly methylated, and we show that the methylation status of both loci was significantly reduced in TYLCV V2 transgenic Arabidopsis plants. We conclude that TYLCV can efficiently suppress TGS when it infects plants, and its V2 protein is responsible for the TGS suppression activity. PMID:24187017

  6. TEMOZOLOMIDE FOR RECURRENT INTRACRANIAL EPENDYMOMA OF THE ADULT: PATTERNS OF RESPONSE, SURVIVAL AND CORRELATIONS WITH MGMT PROMOTER METHYLATION

    PubMed Central

    Soffietti, Riccardo; Bosa, Chiara; Bertero, Luca; Trevisan, Elisa; Cassoni, Paola; Morra, Isabella; Rudà, Roberta

    2014-01-01

    BACKGROUND: A variety of agents have been investigated with modest results in recurrent grade II and III ependymomas failing surgery and/or radiotherapy. Few data are available on the role of temozolomide (TMZ). We investigated patterns of response, outcome and correlations with MGMT promoter methylation in a cohort of patients with recurrent ependymomas of the adult receiving temozolomide as salvage therapy. METHODS: We retrospectively studied all patients aged ≥18 years with recurrent intracranial ependymoma, who received as part of their treatment standard temozolomide between 1999 and 2011. Clinical information were retrieved from the database and follow-up visits, while MRI images were reviewd by an investigator blind to patients' outcome. Response to TMZ on MRI was evaluated according to Macdonald Criteria. An analysis of MGMT gene promoter methylation by PCR was performed. RESULTS: We found 18 evaluable patients of whom 12 were males and 6 females, and 10 (56%) were of grade III and 8 (44%) of grade II. Tumor location at initial surgery was supratentorial in 11 (61%) patients and infratentorial in 7 (39%), and type of progression before TMZ was local in 10 (56%), local and spinal in 6 (33%) and spinal alone in 2 (11%). Median age was 42 years (18-61) and median KPS 70 (60-90). Previous treatments consisted of radiotherapy (either adjuvant or at relapse) in 17/18 (94%) patients, and chemotherapy (cisplatin + VP16, PCV, BCNU) in 6/18 (33%). A median of 8 cycles of TMZ (1-24) were administered. Best response to TMZ was as follows: CR 1/18 (5%) and PR 3/18 (17%), with an overall RR of 22%; SD 7/18 (39%) and PD 7/18 (39%). Maximum reponse in 3 out of 4 patients was observed after 10, 14 and 15 cycles, respectively. All 4 responding patients were chemotherapy-naive. Responses occurred in both anaplastic (2) and low grade (2) tumors. Median PFS was 9 months (1 month-13 years), while PFS 6 and 12 were 72% and 39%, respectively. Median OS was 31 months (3 months-14

  7. CDKN2B, SLC19A3 and DLEC1 promoter methylation alterations in the bone marrow of patients with acute myeloid leukemia during chemotherapy

    PubMed Central

    HONG, QINGXIAO; LI, YIRUN; CHEN, XIAOYING; YE, HUADAN; TANG, LINLIN; ZHOU, ANNAN; HU, YAN; GAO, YUTING; CHEN, RONGRONG; XIA, YONGMING; DUAN, SHIWEI

    2016-01-01

    Previous studies have demonstrated that promoter hypermethylation of tumor suppressor genes contributes to the occurrence and development of acute myeloid leukemia (AML). However, the association of DNA methylation with chemotherapeutic outcomes remains unknown. In the present study, 15 patients with AML were recruited, and the promoter methylation status of cyclin-dependent kinase inhibitor 2B (CDKN2B), solute carrier family 19 member 3 (SLC19A3) and deleted in lung and esophageal cancer 1 (DLEC1) genes was examined prior to and following various chemotherapeutic regimens in order to identify any alterations. The results suggested that chemotherapy-induced hypermethylation of CDKN2B and DLEC1 may be specific to males and females, respectively, and that there were no alterations in SLC19A3 methylation following chemotherapy. These results may provide an improved understanding of gene methylation to guide the development of an individualized chemotherapy for AML. Due to the complexity of AML and the wide range of treatment types, future studies with a larger sample size are required in order to verify the results of the present investigation. PMID:27168825

  8. An Innovative Fluorescent Semi-quantitative Methylation-specific PCR Method for the Determination of MGMT Promoter Methylation is Reflecting Intra-tumor Heterogeneity.

    PubMed

    Nguyen, Aurelia; Legrain, Michele; Noel, Georges; Coca, Andres; Meyer Ea, Nicolos; Schott, Roland; Lasthaus, Christelle; Chenard, Marie Pierrette; Gaub, Marie Pierre; Lessinger, Jean Marc; Guenot, Dominique; Entz-Werle, Natacha

    2015-01-01

    High grade gliomas (HGG) are usually associated with a very dismal prognosis, which was moderately improving in the last decade with the introduction of the alkylating agent temozolomide in their treatment. The methylation status of MGMT (O6 methylguanine DNA-methyltransferase) promoter is one of the strongest predictive and prognostic factors for the patient chemoresponse. For instance, the molecular method of assessment for MGMT promoter status is not standardized. In this background, we developed a fluorescent capillary gel electrophoresis-based methylation specific-PCR. This technique allowed a semi-quantitative estimate of the relative ratio between methylated and unmethylated alleles. The efficacy and accuracy of the technique was assessed in a retrospective cohort of 178 newly diagnosed adult HGGs, who were homogeneously treated. First, we analyzed the impact on survival of different cut-off points in the MGMT promoter methylation and, to go further, we correlated these different rates to other well-known prognostic molecular factors involved in adult HGGs. This strategy allowed to validate our technique as a very sensitive technique (detection of a low methylation percentage, < 5%), which was feasible in fresh-frozen as well as in FFPE samples and had the propensity to detect intra-tumor heterogeneity. This technique identified a new sub-group of anaplastic oligodendrogliomas or oligoastrocytomas defined by a minor methylation and a worse outcome and, therefore, will help to substratify accurately into more homogeneous subgroups of methylated tumors. PMID:26118907

  9. The role of PAQR3 gene promoter hypermethylation in breast cancer and prognosis.

    PubMed

    Chen, Jinpeng; Wang, Feiran; Xu, Junfei; He, Zhixian; Lu, Yuhua; Wang, Zhiwei

    2016-09-01

    PAQR3 is a tumor suppressor in breast cancer and its expression regulation mechanism has not been well elucidated. In this study, we found that PAQR3 expression was downregulated in breast cancer tissues, and the downregulation of PAQR3 expression was found to be significantly associated with aberrant methylation of the gene promoter. Methylation‑specific PCR showed that hypermethylation of the PAQR3 gene was observed in 71.8% of the breast cancers, whereas it was found in only 28.2% of the corresponding non‑tumor tissues. Moreover, we found that the PAQR3 promoter methylation status was related to lymph node metastasis (P=0.01). In addition, overexpression of PAQR3 inhibited breast cancer cell invasion and growth. Furthermore, PAQR3 expression was restored in MCF‑7 cells after treatment with the demethylating agent, 5-aza-2'-deoxycytidine, and the effect of demethylation induced invasion and proliferation suppression of MCF‑7 cells. Collectively, our results suggested that the aberrant methylation of PAQR3 underlies its downregulation in breast cancer and our data indicated that epigenetic silencing of PAQR3 gene expression by promoter hypermethylation may play an important role in breast cancer. PMID:27461225

  10. Expression and promoter methylation of Wnt inhibitory factor-1 in the development of oral submucous fibrosis.

    PubMed

    Zhou, Shanghui; Chen, Ling; Mashrah, Mubarak; Zhu, Yun; He, Zhijing; Hu, Yuhua; Xiang, Tingxiu; Yao, Zhigang; Guo, Feng; Zhang, Chenping

    2015-11-01

    Oral squamous cell carcinoma (OSCC) is a type of head and neck malignancy with a high mortality rate. Oral submucous fibrosis (OSF) is the pre-cancerous lesion of OSCC, whose molecular mechanisms in OSCC tumorigenesis remain largely unclear. Activation of the Wnt/β-catenin signaling pathway plays an important role in oral mucous carcinogenesis, although rare mutations of Wnt signaling molecules are found in OSCC, suggesting an epigenetic mechanism mediating aberrant Wnt/β‑catenin signaling in OSCC. Wnt inhibitory factor-1 (WIF1) is an Wnt antagonist, and its downregulation and methylation have been reported in a number of malignancies. However, the expression and methylation of WIF1 in the development of OSF have yet to be reported. In the present study, we investigated the WIF1 expression level by immuno-histochemical staining and semi‑quantitative RT-PCR in normal oral, OSF and OSCC tissues, as well as the methylation status by methylation-specific PCR and bisulfite genomic sequencing. The results showed that WIF1 was readily expressed in normal oral mucous tissues, but decreased gradually in OSF early, moderately advanced and advanced tissues, and was less expressed in OSCC tissues. Moreover, WIF1 was able to translocate from the nuclear to cytoplasm in OSF and OSCC tissues. Furthermore, WIF1 was frequently methylated in OSCC cases with betel quid chewing habit, but not in normal oral mucous and different stages of OSF tissues, suggesting WIF1 methylation is tumor-specific in the development of OSF. Thus, the results demonstrated that WIF1 is frequently downregulated or silenced by promoter methylation in the carcinogenesis of OSF, which serves as a potential epigenetic biomarker for the early detection of OSCC. PMID:26352791

  11. Genome-wide methylation analysis in vestibular schwannomas shows putative mechanisms of gene expression modulation and global hypomethylation at the HOX gene cluster.

    PubMed

    Torres-Martín, Miguel; Lassaletta, Luis; de Campos, Jose M; Isla, Alberto; Pinto, Giovanny R; Burbano, Rommel R; Melendez, Bárbara; Castresana, Javier S; Rey, Juan A

    2015-04-01

    Schwannomas are tumors that develop from Schwann cells in the peripheral nerves and commonly arise from the vestibular nerve. Vestibular schwannomas can present unilaterally and sporadically or bilaterally when the tumor is associated with neurofibromatosis Type 2 (NF2) syndrome. The molecular hallmark of the disease is biallelic inactivation of the NF2 gene. The epigenetic signature of schwannomas remains poorly understood and is mostly limited to DNA methylation of the NF2 gene, whose altered expression due to epigenetic factors in this tumor is controversial. In this study, we tested the genomewide DNA methylation pattern of schwannomas to shed light on this epigenetic alteration in these particular tumors. The methodology used includes Infinium Human Methylation 450K BeadChip microarrays in a series of 36 vestibular schwannomas, 4 nonvestibular schwannomas, and 5 healthy nerves. Our results show a trend toward hypomethylation in schwannomas. Furthermore, homeobox (HOX) genes, located at four clusters in the genome, displayed hypomethylation in several CpG sites in the vestibular schwannomas but not in the nonvestibular schwannomas. Several microRNA (miRNA) and protein-coding genes were also found to be hypomethylated at promoter regions and were confirmed as upregulated by expression analysis; including miRNA-21, Met Proto-Oncogene (MET), and PMEPA1. We also detected methylation patterns that might be involved in alternative transcripts of several genes such as NRXN1 or MBP, which would increase the complexity of the methylation and expression patterns. Overall, our results show specific epigenetic signatures in several coding genes and miRNAs that could potentially be used as therapeutic targets. PMID:25533176

  12. Association of weight regain with specific methylation levels in the NPY and POMC promoters in leukocytes of obese men: a translational study.

    PubMed

    Crujeiras, Ana B; Campion, Javier; Díaz-Lagares, Angel; Milagro, Fermin I; Goyenechea, Estíbaliz; Abete, Itziar; Casanueva, Felipe F; Martínez, J Alfredo

    2013-09-10

    Specific methylation of appetite-related genes in leukocytes could serve as a useful biomarker to predict weight regain after an energy restriction program. We aimed to evaluate whether the pre-intervention DNA methylation patterns involved in the epigenetic control of appetite-regulatory genes in leukocytes are associated with the weight regain process. Eighteen men who lost ≥5% of body weight after an 8-week nutritional intervention were categorized as "regainers" (≥10% weight regain) and "non-regainers" (<10% weight regain) 32weeks after stopping dieting. At baseline, leukocytes were isolated and DNA was analyzed for epigenetic methylation patterns of appetite-related gene promoters by MALDI-TOF mass spectrometry. Regainers showed higher methylation levels than non-regainers in proopiomelanocortin (POMC) CpG sites +136bp and +138bp (fold change from non-regainers=26%; p=0.020) and lower methylation of the whole analyzed region of neuropeptide Y (NPY; fold change from non-regainers=-22%; p=0.033), as well as of several individual NPY-promoter CpG sites. Importantly, total baseline NPY methylation was associated with weight-loss regain (r=-0.76; p<0.001), baseline plasma ghrelin levels (r=0.60; p=0.011) and leptin/ghrelin ratio (r=-0.52; p=0.046). Lower methylation levels of POMC CpG sites +136bp and +138bp were associated with success in weight-loss maintenance (odds ratio=0.042 [95% CI 0.01-0.57]; p=0.018), whereas lower total methylation levels in NPY promoter were associated with higher risk of weight regain (odds ratio=14.0 [95% CI 1.13-172]; p=0.039). Therefore, the study of leukocyte methylation levels reflects a putative epigenetic regulation of NPY and POMC, which might be implicated in the weight regain process and be used as biomarkers for predicting weight regain after dieting. PMID:23831408

  13. Maternal and paternal chromosomes 7 show differential methylation of many genes in lymphoblast DNA.

    PubMed

    Hannula, K; Lipsanen-Nyman, M; Scherer, S W; Holmberg, C; Höglund, P; Kere, J

    2001-04-01

    Genomic imprinting, the differential expression of paternal and maternal alleles, involves many chromosomal regions and plays a role in development and growth. Differential methylation of maternal and paternal alleles is a hallmark of imprinted genes, and thus methylation assays are widely used to support the identification of novel imprinted genes. Either blood or lymphoblast DNAs are most often used in these assays, even though methylation levels may change in cell culture. We undertook a systematic survey of parent-of-origin-specific methylation of chromosome 7 genes and ESTs by comparing DNA samples from cases of maternal and paternal uniparental disomy for chromosome 7 using DNA from fresh blood and lymphoblast cell lines. Our results revealed that up to 41% of genes and ESTs show parent-of-origin-specific methylation differences in lymphoblast DNA after only a short time in culture, whereas methylation differences were not seen in blood DNA. The methylation changes occurred most commonly on paternal chromosome 7, whereas alterations on maternal chromosome 7 were more infrequent and weaker. These findings indicate that methylation patterns may change significantly during cell culture in a parent-of-origin-dependent manner and suggest that methylation is maintained differently on maternal and paternal chromosomes 7. PMID:11352560

  14. NNMT promotes epigenetic remodeling in cancer by creating a metabolic methylation sink

    PubMed Central

    Ulanovskaya, Olesya A.; Zuhl, Andrea M.; Cravatt, Benjamin F.

    2013-01-01

    Nicotinamide N-methyltransferase (NNMT) is overexpressed in a variety of human cancers, where it contributes to tumorigenesis by a still poorly understood mechanism. Here, we show using metabolomics that NNMT impairs the methylation potential of cancer cells by consuming methyl units from S-adenosyl methionine to create the stable metabolic product 1-methylnicotinamide. As a result, NNMT-expressing cancer cells possess an altered epigenetic state that includes hypomethylated histones and other cancer-related proteins combined with heightened expression of pro-tumorigenic gene products. Our findings thus point to a direct mechanistic link between the deregulation of a metabolic enzyme and widespread changes in the methylation landscape of cancer cells. PMID:23455543

  15. PTHrP in differentiating human mesenchymal stem cells: transcript isoform expression, promoter methylation, and protein accumulation.

    PubMed

    Longo, Alessandra; Librizzi, Mariangela; Naselli, Flores; Caradonna, Fabio; Tobiasch, Edda; Luparello, Claudio

    2013-10-01

    Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5' and 3' ends and the existence of three different transcriptional promoters (P1, P2 and P3), two of which (P2 and P3) contain CpG islands. It is known that the expression of PTHrP isoforms may be differentially regulated in a developmental stage- and tissue-specific manner. To search for novel molecular markers of stemness/differentiation, here we have examined isoform expression in fat-derived mesenchymal stem cells both maintained in stem conditions and induced toward adipo- and osteogenesis. In addition, the expression of the splicing isoforms derived from P2 and P3 promoters was correlated to the state of methylation of the latter. Moreover, we also performed a quantitative evaluation of intracellular and secreted PTHrP protein product in undifferentiated stem cells and in parallel cultures at various differentiation stages. The data obtained indicate that from the stemness condition to that of osteo- and adipo-genic differentiated cells, the expression of isoforms becomes increasingly selective, thereby being a potential gene signature for the monitoring of cell stem or committed/differentiating state and that the switching-off of PTHrP isoform expression is mostly promoter methylation-dependent. Moreover, PTHrP intracellular retention is down-regulated in osteo-differentiating cells whereas the secretion of the protein in the extracellular medium is up-regulated with respect to stem cells, thereby suggesting that these variations of the intracellular and extracellular levels of PTHrP could potentially be enclosed in the list of the available protein signature of osteogenic differentiation. PMID:23810909

  16. MGMT Promoter Methylation and BRAF V600E Mutations Are Helpful Markers to Discriminate Pleomorphic Xanthoastrocytoma from Giant Cell Glioblastoma.

    PubMed

    Lohkamp, Laura-Nanna; Schinz, Maren; Gehlhaar, Claire; Guse, Katrin; Thomale, Ulrich-Wilhelm; Vajkoczy, Pet