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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. 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

  3. 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

  4. 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...

  5. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    PubMed Central

    Uno, Miyuki; Oba-Shinjo, Sueli Mieko; Camargo, Anamaria Aranha; Moura, Ricardo Pereira; de Aguiar, Paulo Henrique; Cabrera, Hector Navarro; Begnami, Marcos; Rosemberg, Sérgio; Teixeira, Manoel Jacobsen; Marie, Suely Kazue Nagahashi

    2011-01-01

    OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue. PMID:22012047

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. Methylation loss at H19 imprinted gene correlates with methylenetetrahydrofolate reductase gene promoter hypermethylation in semen samples from infertile males.

    PubMed

    Rotondo, John C; Selvatici, Rita; Di Domenico, Maura; Marci, Roberto; Vesce, Fortunato; Tognon, Mauro; Martini, Fernanda

    2013-09-01

    Aberrant methylation at the H19 paternal imprinted gene has been identified in different cohorts of infertile males. The causes of H19 methylation errors are poorly understood. In this study, we investigated the methylation status of the H19 gene in semen DNA samples from infertile males affected by MTHFR gene promoter hypermethylation. DNA from normal and abnormal semen samples harbouring MTHFR gene promoter hypermethylated, hmMTHFR-nor and hmMTHFR-abn, and without MTHFR methylation, MTHFR-nor and MTHFR-abn, were investigated for methylation status in the H19 locus using bisulfite-treated DNA PCR, followed by cloning and sequencing. The prevalence of H19 hypomethylated clones was 20% in hmMTHFR-nor and 0% in MTHFR-nor semen samples (p<0.05), and 28% in hmMTHFR-abn compared with 16% in MTHFR-abn semen samples (p>0.05). These results underscore the association between H19 methylation defects and hypermethylation of the MTHFR gene promoter in normal semen samples and suggest that aberrant methylation at H19 may occur in the normal sperm of infertile males affected by MTHFR gene dysfunction. These findings provide new insights into the mechanisms causing abnormal methylation in imprinted genes and, in turn, male infertility.

  12. Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

    PubMed Central

    Durr, Megan L.; Mydlarz, Wojciech K.; Shao, Chunbo; Zahurak, Marianna L.; Chuang, Alice Y.; Hoque, Mohammad O.; Westra, William H.; Liegeois, Nanette J.; Califano, Joseph A.; Sidransky, David; Ha, Patrick K.

    2010-01-01

    Background Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected. Methodology DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results. Principal Findings The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3. Conclusions/Significance Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors. PMID:20520817

  13. 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.

  14. 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

  15. Effect of cigarette smoke condensate on gene promoter methylation in human lung cells

    PubMed Central

    2014-01-01

    Background In lung cancer, an association between tobacco smoking and promoter DNA hypermethylation has been demonstrated for several genes. However, underlying mechanisms for promoter hypermethylation in tobacco-induced cancer are yet to be fully established. Methods Promoter methylation was evaluated in control and cigarette smoke condensate (CSC) exposed human lung cells using the Methyl-Profiler DNA Methylation PCR System. PSAE cells were exposed to 0.3 or 1.0 μg/ml CSC for 72 hours and longer term for 14 and 30 days. NL-20 cells were exposed for 30 days to 10 or 100 μg/ml CSC. Results Promoters of several genes, including hsa-let-7a-3, CHD1, CXCL12, PAX5, RASSF2, and TCF21, were highly methylated (>90%); hsa-let-7a-3 was affected in both cell lines and under all exposure conditions. Level of methylation tended to increase with CSC concentration and exposure duration (statistical differences were not determined). Percentage methylation of TCF21, which was >98% at exposures of 10 or 100 μg/ml CSC, was found to be reduced to 28% and 42%, respectively, in the presence of the dietary agent genistein. Conclusions Using array techniques, several tumor suppressor genes in human lung cells were identified that undergo promoter hypermethylation, providing further evidence of their potential involvement in tobacco smoke-induced lung carcinogenesis and their use as potential biomarkers of harm in tobacco smoke exposure. Results from the study also demonstrated the potential of a dietary agent to exert chemopreventive activity in human tissue against tobacco smoke related diseases through modulation of DNA methylation. Additional studies are needed to confirm these findings. PMID:25214829

  16. Differential methylation of the promoter and first exon of the RASSF1A gene in hepatocarcinogenesis

    PubMed Central

    Jain, Surbhi; Xie, Lijia; Boldbaatar, Batbold; Lin, Selena Y.; Hamilton, James P.; Meltzer, Stephen J.; Chen, Shun-Hua; Hu, Chi-Tan; Block, Timothy M.; Song, Wei; Su, Ying-Hsiu

    2015-01-01

    Aim Aberrant methylation of the promoter, P2, and the first exon, E1, regions of the tumor suppressor gene RASSF1A, have been associated with hepatocellular carcinoma (HCC), albeit with poor specificity. This study analyzed the methylation profiles of P1, P2 and E1 regions of the gene to identify the region of which methylation most specifically corresponds to HCC and to evaluate the potential of this methylated region as a biomarker in urine for HCC screening. Methods Bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays were performed to compare methylation of the 56 CpG sites in regions P1, P2 and E1 in DNA isolated from normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC liver tissue and urine samples for the characterization of hypermethylation of the RASSF1A gene as a biomarker for HCC screening. Results In tissue, comparing HCC (n = 120) with cirrhosis and hepatitis together (n = 70), methylation of P1 had an area under the receiver operating characteristics curve (AUROC) of 0.90, whereas methylation of E1 and P2 had AUROC of 0.84 and 0.72, respectively. At 90% sensitivity, specificity for P1 methylation was 72.9% versus 38.6% for E1 and 27.1% for P2. Methylated P1 DNA was detected in urine in association with cirrhosis and HCC. It had a sensitivity of 81.8% for α-fetoprotein negative HCC. Conclusion Among the three regions analyzed, methylation of P1 is the most specific for HCC and holds great promise as a DNA marker in urine for screening of cirrhosis and HCC. PMID:25382672

  17. Promoter methylation confers kidney-specific expression of the Klotho gene.

    PubMed

    Azuma, Masahiro; Koyama, Daisuke; Kikuchi, Jiro; Yoshizawa, Hiromichi; Thasinas, Dissayabutra; Shiizaki, Kazuhiro; Kuro-o, Makoto; Furukawa, Yusuke; Kusano, Eiji

    2012-10-01

    The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant; however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to -1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho down-regulation.

  18. Promoter methylation and downregulated expression of the TBX15 gene in ovarian carcinoma

    PubMed Central

    Gozzi, Gaia; Chelbi, Sonia T.; Manni, Paola; Alberti, Loredana; Fonda, Sergio; Saponaro, Sara; Fabbiani, Luca; Rivasi, Francesco; Benhattar, Jean; Losi, Lorena

    2016-01-01

    TBX15 is a gene involved in the development of mesodermal derivatives. As the ovaries and the female reproductive system are of mesodermal origin, the aim of the present study was to determine the methylation status of the TBX15 gene promoter and the expression levels of TBX15 in ovarian carcinoma, which is the most lethal and aggressive type of gynecological tumor, in order to determine the role of TBX15 in the pathogenesis of ovarian carcinoma. This alteration could be used to predict tumor development, progression, recurrence and therapeutic effects. The study was conducted on 80 epithelial ovarian carcinoma and 17 control cases (normal ovarian and tubal tissues). TBX15 promoter methylation was first determined by pyrosequencing following bisulfite modification, then by cloning and sequencing, in order to obtain information about the epigenetic haplotype. Immunohistochemical analysis was performed to evaluate the correlation between the methylation and protein expression levels. Data revealed a statistically significant increase of the TBX15 promoter region methylation in 82% of the tumor samples and in various histological subtypes. Immunohistochemistry showed an inverse correlation between methylation levels and the expression of the TBX15 protein. Furthermore, numerous tumor samples displayed varying degrees of intratumor heterogeneity. Thus, the present study determined that ovarian carcinoma typically expresses low levels of TBX15 protein, predominantly due to an epigenetic mechanism. This may have a role in the pathogenesis of ovarian carcinoma independent of the histological subtype.

  19. Promoter methylation and downregulated expression of the TBX15 gene in ovarian carcinoma

    PubMed Central

    Gozzi, Gaia; Chelbi, Sonia T.; Manni, Paola; Alberti, Loredana; Fonda, Sergio; Saponaro, Sara; Fabbiani, Luca; Rivasi, Francesco; Benhattar, Jean; Losi, Lorena

    2016-01-01

    TBX15 is a gene involved in the development of mesodermal derivatives. As the ovaries and the female reproductive system are of mesodermal origin, the aim of the present study was to determine the methylation status of the TBX15 gene promoter and the expression levels of TBX15 in ovarian carcinoma, which is the most lethal and aggressive type of gynecological tumor, in order to determine the role of TBX15 in the pathogenesis of ovarian carcinoma. This alteration could be used to predict tumor development, progression, recurrence and therapeutic effects. The study was conducted on 80 epithelial ovarian carcinoma and 17 control cases (normal ovarian and tubal tissues). TBX15 promoter methylation was first determined by pyrosequencing following bisulfite modification, then by cloning and sequencing, in order to obtain information about the epigenetic haplotype. Immunohistochemical analysis was performed to evaluate the correlation between the methylation and protein expression levels. Data revealed a statistically significant increase of the TBX15 promoter region methylation in 82% of the tumor samples and in various histological subtypes. Immunohistochemistry showed an inverse correlation between methylation levels and the expression of the TBX15 protein. Furthermore, numerous tumor samples displayed varying degrees of intratumor heterogeneity. Thus, the present study determined that ovarian carcinoma typically expresses low levels of TBX15 protein, predominantly due to an epigenetic mechanism. This may have a role in the pathogenesis of ovarian carcinoma independent of the histological subtype. PMID:27698863

  20. 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

  1. Promoter Methylation of Glucocorticoid Receptor Gene Is Associated with Subclinical Atherosclerosis: a Monozygotic Twin Study

    PubMed Central

    Zhao, Jinying; An, Qiang; Goldberg, Jack; Quyyumi, Arshed A.; Vaccarino, Viola

    2015-01-01

    Objective Endothelial dysfunction assessed by brachial artery flow-mediated dilation (FMD) is a marker of early atherosclerosis. Glucocorticoid receptor gene (NR3C1) regulates many biological processes, including stress response, behavioral, cardiometabolic and immunologic functions. Genetic variants in NR3C1 have been associated with atherosclerosis and related risk factors. This study investigated the association of NR3C1 promoter methylation with FMD, independent of genetic and family-level environmental factors. Methods We studied 84 middle-aged, male-male monozygotic twin pairs recruited from the Vietnam Era Twin Registry. Brachial artery FMD was measured by ultrasound. DNA methylation levels at 22 CpG residues in the NR3C1 exon 1F promoter region were quantified by bisulfite pyrosequencing in genomic DNA isolated from peripheral blood leukocytes. Co-twin control analyses were conducted to examine the association of methylation variation with FMD, adjusting for smoking, physical activity, body mass index, lipids, blood pressure, fasting glucose, and depressive symptoms. Multiple testing was corrected using the false discovery rate. Results Mean methylation level across the 22 studied CpG sites was 2.02%. Methylation alterations at 12 out of the 22 CpG residues were significantly associated with FMD. On average, a 1% increase in the intra-pair difference in mean DNA methylation was associated with 2.83% increase in the intra-pair difference in FMD (95% CI: 1.46-4.20; P <0.0001) after adjusting for risk factors and multiple testing. Conclusion Methylation variation in NR3C1 exon 1F promoter significantly influences subclinical atherosclerosis, independent of genetic, early family environmental and other risk factors. PMID:26186654

  2. 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

  3. Epigenetic DNA methylation in the promoters of the Igf1 receptor and insulin receptor genes in db/db mice.

    PubMed

    Nikoshkov, Andrej; Sunkari, Vivekananda; Savu, Octavian; Forsberg, Elisabete; Catrina, Sergiu-Bogdan; Brismar, Kerstin

    2011-04-01

    We have investigated promoter methylation of the Insr, Igf1 and Igf1r genes in skeletal and cardiac muscles of normal and diabetic db/db mice. No differences in Insr promoter methylation were found in the heart and skeletal muscles and no methylation was detected in the Igf1 promoter in skeletal muscle. In skeletal muscle, db/db males exhibited a 7.4-fold increase in Igf1r promoter methylation, which was accompanied by a 1.8-fold decrease in Igf1r mRNA levels, compared with controls. More than 50% of the detected methylation events were concentrated within an 18 bp sequence that includes one of the Sp1 binding sites. We conclude that the methylation level and pattern of the Igf1r promoter in skeletal muscle is related to gender and the diabetic state. PMID:21474992

  4. 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

  5. Interplay between promoter methylation and chromosomal loss in gene silencing at 3p11-p14 in cervical cancer

    PubMed Central

    Lando, Malin; Fjeldbo, Christina S; Wilting, Saskia M; Snoek, Barbara C; Aarnes, Eva-Katrine; Forsberg, Malin F; Kristensen, Gunnar B; Steenbergen, Renske DM; Lyng, Heidi

    2015-01-01

    Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n = 149, n = 121), using Illumina 450K methylation arrays. The aim was to investigate whether hyperm-ethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof 6 genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified 3 regulation patterns encompassing 8 hypermethylated genes; a methylation driven pattern (C3orf14, GPR27, ZNF717), a gene dosage driven pattern (THOC7, PSMD6), and a combined methylation and gene dosage driven pattern (FHIT, ADAMTS9, LRIG1). In survival analysis, patients with both hypermethylation and loss of LRIG1 had a worse outcome compared to those harboring only hypermethylation or none of the events. C3orf14 emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved

  6. Expression of human histo-blood group ABO genes is dependent upon DNA methylation of the promoter region.

    PubMed

    Kominato, Y; Hata, Y; Takizawa, H; Tsuchiya, T; Tsukada, J; Yamamoto, F

    1999-12-24

    We have investigated the regulatory role of DNA methylation in the expression of the human histo-blood group ABO genes. The ABO gene promoter region contains a CpG island whose methylation status correlates well with gene expression in the cell lines tested. The CpG island was found hypomethylated in some cell lines that expressed ABO genes, whereas the other cell lines that did not express ABO genes were hypermethylated. Whereas constitutive transcriptional activity of the ABO gene promoter was demonstrated in both expressor and nonexpressor cell lines by transient transfection of reporter constructs containing the ABO gene promoter sequence, HhaI methylase-catalyzed in vitro methylation of the promoter region prior to DNA transfection suppressed the promoter activity when introduced into the expressor gastric cancer cell line KATOIII cells. On the other hand, in the nonexpressor gastric cancer cell line MKN28 cells, treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in demethylation of the ABO gene promoter and appearance of A-transferase messages, as well as A-antigens synthesized by A-transferase. Taken together, these studies suggest that DNA methylation of the ABO gene promoter may play an important role in the regulation of ABO gene expression. PMID:10601288

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Methylation status and chromatin structure of the myostatin gene promoter region in the sea perch Lateolabrax japonicus (Perciformes).

    PubMed

    Abbas, E M; Takayanagi, A; Shimizu, N; Kato, M

    2011-01-01

    Myostatin is a negative regulator of the growth and development of skeletal muscle mass. In fish, myostatin is expressed in several organs in addition to skeletal muscle. To understand the mechanisms regulating myostatin gene expression in the sea perch, Lateolabrax japonicus, we examined the methylation status of the myostatin gene promoter region in several tissues (liver, eye, kidney, brain, and heart) isolated from adult specimens. The frequency of methylated cytosines was very low in all tissues, regardless of the level of myostatin expression, suggesting that DNA methylation is not involved in the tissue-specific regulation of myostatin expression. Southern blot analysis of genomic DNA obtained from micrococcal nuclease-treated nuclei showed that chromatin digestion occurs in tissues where the myostatin gene is actively transcribed and that the myostatin gene is protected from micrococcal nuclease in tissues where myostatin is not expressed. The chromatin structure in the myostatin gene region appears to regulate its expression without DNA methylation. PMID:22183947

  15. [Methylation of FHIT gene promoter region in DNA from plasma of patients with myelodysplastic syndromes and demethylating effect of decitabine].

    PubMed

    Deng, Yin-Fen; Zhang, Lei; Zhang, Xiu-Qun; Hu, Ming-Qiu; Dai, Dan; Zhang, Xue-Zhong; Xu, Yan-Li

    2012-10-01

    This study was aimed to detect the methylation status of FHIT gene promoter region in the DNA from plasma of patients with myelodysplastic syndrome (MDS), and to investigate the demethylating effect of decitabine. Methylation-specific PCR method was used to detect the methylation status of FHIT gene promoter region in the DNA from plasma of 4 patients with MDS before and after treatment with decitabine plus semis CAG therapy (among them, 1 case of newly diagnosed MDS, 3 cases progressed into acute leukemia). The results indicated that 3 cases were found to have an increased methylation in the promoter region. After treatment with decitabine plus semis CAG, increased methylation was reversed in 2 cases. In 4 cases, 2 cases displayed clinical response. It is concluded that FHIT gene hypermethylation is associated with MDS pathogenesis. Decitabine has demethylating effect on the FHIT gene hypermethylation of plasma from MDS patients. Detecting the methylation status of FHIT gene in DNA from plasma may play a role in MDS auxiliary diagnosis or prognosis.

  16. Global indiscriminate methylation in cell-specific gene promoters following reprogramming into human induced pluripotent stem cells.

    PubMed

    Nissenbaum, Jonathan; Bar-Nur, Ori; Ben-David, Eyal; Benvenisty, Nissim

    2013-01-01

    Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm, and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global nonspecific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells. PMID:24371806

  17. Global indiscriminate methylation in cell-specific gene promoters following reprogramming into human induced pluripotent stem cells.

    PubMed

    Nissenbaum, Jonathan; Bar-Nur, Ori; Ben-David, Eyal; Benvenisty, Nissim

    2013-01-01

    Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm, and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global nonspecific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells.

  18. Global Indiscriminate Methylation in Cell-Specific Gene Promoters following Reprogramming into Human Induced Pluripotent Stem Cells

    PubMed Central

    Nissenbaum, Jonathan; Bar-Nur, Ori; Ben-David, Eyal; Benvenisty, Nissim

    2013-01-01

    Summary Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm, and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global nonspecific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells. PMID:24371806

  19. Methylation of promoters of microRNAs and their host genes in myelodysplastic syndromes

    PubMed Central

    Erdogan, Begum; Bosompem, Amma; Peng, Dunfa; Han, Leng; Smith, Emily; Kennedy, Mija E.; Alford, Catherine E.; Wu, Huiyun; Zhao, Zhongming; Mosse, Claudio A.; El-Rifai, Wael; Kim, Annette S.

    2014-01-01

    Myelodysplastic syndromes (MDS) are a group of hematopoietic malignancies characterized by ineffective hematopoiesis. Recently, we identified MDS-associated microRNAs (miRNAs) that are down-regulated in MDS. This study examines possible explanations for that observed down-regulation of miRNA expression in MDS. Since genomic losses are insufficient to explain the down-regulation of all our MDS-associated miRNAs, we explored other avenues. We demonstrate that these miRNAs are predominantly intragenic, and that, in many cases, they and their host genes are expressed in a similar pattern during myeloid maturation, suggesting their co-regulation. This co-regulation is further supported by the down-regulation of several of the host genes in MDS and increased methylation of the shared promoters of several miRNAs and their respective host genes. These studies identify a role of hypermethylation of miRNA promoters in the down-regulation of MDS-associated miRNAs, unifying research on miRNAs in MDS and epigenetic regulation in MDS into a common pathway. PMID:23547841

  20. Effect of vitrification on promoter methylation and the expression of pluripotency and differentiation genes in mouse blastocysts.

    PubMed

    Zhao, Xue-Ming; Du, Wei-Hua; Hao, Hai-Sheng; Wang, Dong; Qin, Tong; Liu, Yan; Zhu, Hua-Bin

    2012-07-01

    The present study was designed to determine the effects of vitrification on promoter methylation and the expression levels of pluripotency and differentiation genes in mouse blastocysts. Promoter region CpG methylation patterns and the expression levels of octamer-binding transcription factor (Oct4), Nanog homeobox (Nanog), caudal-type homeobox 2 (Cdx2), and heart and neural crest derivatives-expressed transcript 1 (Hand1) were analyzed in fresh and vitrified mouse blastocysts. Methylation was measured by bisulphate mutagenesis and sequencing; gene expression was determined by real-time reverse transcription-PCR. The results showed that vitrification significantly reduced the methylation levels of the Oct4 (85% vs. 62.5%), Nanog (77.5% vs. 55%), and Cdx2 promoters (4.6% vs. 1.4%; P < 0.05) in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog in vitrified blastocysts. Hand1 promoter methylation was not significantly different in the fresh (17.9%) versus vitrification group (21.4%; P > 0.05). The expression levels of Cdx2 and Hand1 were not significantly different in fresh and vitrified blastocysts. In conclusion, vitrification significantly decreased Oct4, Nanog, and Cdx2 promoter methylation in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog.

  1. 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…

  2. 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

  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.

  4. Exploring the potential relationship between Notch pathway genes expression and their promoter methylation in mice hippocampal neurogenesis.

    PubMed

    Zhang, Zhen; Gao, Feng; Kang, Xiaokui; Li, Jia; Zhang, Litong; Dong, Wentao; Jin, Zhangning; Li, Fan; Gao, Nannan; Cai, Xinwang; Yang, Shuyuan; Zhang, Jianning; Ren, Xinliang; Yang, Xinyu

    2015-04-01

    The Notch pathway is a highly conserved pathway that regulates hippocampal neurogenesis during embryonic development and adulthood. It has become apparent that intracellular epigenetic modification including DNA methylation is deeply involved in fate specification of neural stem cells (NSCs). However, it is still unclear whether the Notch pathway regulates hippocampal neurogenesis by changing the Notch genes' DNA methylation status. Here, we present the evidence from DNA methylation profiling of Notch1, Hes1 and Ngn2 promoters during neurogenesis in the dentate gyrus (DG) of postnatal, adult and traumatic brains. We observed the expression of Notch1, Hes1 and Ngn2 in hippocampal DG with qPCR, Western blot and immunofluorescence staining. In addition, we investigated the methylation status of Notch pathway genes using the bisulfite sequencing PCR (BSP) method. The number of Notch1 or Hes1 (+) and BrdU (+) cells decreased in the subgranular zone (SGZ) of the DG in the hippocampus following TBI. Nevertheless, the number of Ngn2-positive cells in the DG of injured mice was markedly higher than in the DG of non-TBI mice. Accordingly, the DNA methylation level of the three gene promoters changed with their expression in the DG. These findings suggest that the strict spatio-temporal expression of Notch effector genes plays an important role during hippocampal neurogenesis and suggests the possibility that Notch1, Hes1 and Ngn2 were regulated by changing some specific CpG sites of their promoters to further orchestrate neurogenesis in vivo.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. Methylation pattern of CDH1 promoter and its association with CDH1 gene expression in cytological cervical specimens

    PubMed Central

    Holubeková, Veronika; Mendelová, Andrea; Grendár, Marián; Meršaková, Sandra; Kapustová, Ivana; Jašek, Karin; Vaňochová, Andrea; Danko, Jan; Lasabová, Zora

    2016-01-01

    Cervical cancer is the fourth leading cause of cancer mortality in females worldwide. Infection with high-risk human papillomavirus (HPV) is essential but insufficient to cause cervical cancer, and the clearance of HPV infection is mediated by the immune system. The deficit of molecules responsible for adhesion may play a role in the development of cervical cancer. E-cadherin is encoded by the cadherin 1 (CDH1) gene, and is involved in cell adhesion by forming adherens junctions. The aim of present study was to investigate the methylation pattern of the CDH1 promoter and to identify the association between CDH1 promoter hypermethylation, CDH1 gene expression and HPV infection in cervical specimens obtained from 93 patients with low-grade squamous intraepithelial lesions (SILs), high-grade SILs or squamous cell carcinomas, and from 47 patients with normal cervical cytology (HPV-negative). The methylation pattern of the CDH1 promoter was investigated by methylation-specific polymerase chain reaction and quantitative pyrosequencing. CDH1 gene expression was measured by relative quantification. CDH1 methylation was significantly higher in both types of lesions and in cervical cancer than in normal samples, and CDH1 gene expression was significantly reduced during SIL progression (P=0.0162). However, the influence of HPV infection or HPV E6 expression on the methylation pattern of the CDH1 gene or its gene expression levels could not be confirmed. The present results support that the methylation of the CDH1 gene is age-related in patients with cervical lesions (P=0.01085), and therefore, older patients could be more susceptible to cancer than younger patients. The important methylation of the CDH1 promoter occurred near the transcription factor binding sites on nucleotides −13 and +103, which are close to the translational start codon. These results suggest that methylation at these sites may be an important event in the transcriptional regulation of E-cadherin, and

  10. 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

  11. 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

  12. 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.

  13. Promoter Methylation Status Modulate the Expression of Tumor Suppressor (RbL2/p130) Gene in Breast Cancer

    PubMed Central

    Ullah, Farman; Khan, Taimoor; Ali, Nawab; Malik, Faraz Arshad; Kayani, Mahmood Akhtar; Shah, Syed Tahir Abbas; Saeed, Muhammad

    2015-01-01

    Background Aberrant expression of tumor suppressor genes may correspond to the abnormal cell development and tumorigenesis. Rbl2/p130, a member of retinoblastoma family of proteins, has growth suppressive properties. Numerous studies reported de-regulation of Rbl2/p130 in various types of cancer as a consequence of a number of genetic alterations. However, role of epigenetic mechanisms like DNA methylation in Rbl2/p130 expression remains elusive. Methods In the current study, 76 breast cancer tumors along with normal tissues (n = 76), blood (n = 76) of respective individuals and control blood (n = 50) were analyzed. Rbl2/p130 expression was analyzed by quantitative real time PCR (syber green method). Promoter methylation status was studied through methylation specific PCR of bisulfite converted genomic DNA. Data was analyzed using various statistical tests. Results We report significantly reduced Rbl2/p130 expression (P = 0.001) in tumors tissues as compared to control samples. Similarly, Rbl2/p130 expression varies with age and disease stages (P = 0.022), which suggest its involvement in tumor progression. Aberrant promoter methylation (Δmeth) was found in almost all the diseased samples and that was significantly different (P<0.001) with control samples. Similarly, methylation status varies significantly with tumor progression stages (P = 0.022). Hyper-methylation was observed at -1, +3, +15 and +75 of Rbl2/p130 promoter flanking around the TSS. Statistical analysis revealed that Rbl2/p130 expression negatively correlates to its promoter methylation (r = -0.412) in tumor tissues. Our results reflect an epigenetic regulation of Rbl2/p130 expression in breast cancer. This highlights the importance of Rbl2/p130 promoter methylation in breast cancer pathogenesis. PMID:26271034

  14. 5-Aza-CdR can reverse gefitinib resistance caused by DAPK gene promoter methylation in lung adenocarcinoma cells.

    PubMed

    Yang, Bo; Yang, Zhi-Guang; Gao, Bao; Shao, Guo-Guang; Li, Guang-Hu

    2015-01-01

    To explore the relationship between death associated protein kinase (DAPK) gene promoter methylation and gefitinib resistance in Lung adenocarcinoma cell lines. EGFR-mutation lung adenocarcinoma cell lines PC9 and the gefitinib-resistant with T790M Mutation cell lines PC9/GR were chosen as cell models, and PC9/GR were treated with 5-aza-CdR (1 μmol/L). The experiments were divided into three groups: PC9 group, PC9/GR group and PC9/GR with 5-Aza-CdR pretreatment group. Treat three groups cell with different concentrations gefitinib, the cell proliferation was determined by MTT assay. The apoptotic rates were detected by flow cytometry. The methylation of DAPK gene promoter region was examined by methylation-specific PCR (MSP). The expressions of DAPK protein were detected by Western blot. MTT results showed that the half maximal inhibitory concentration (IC50) of PC9 and PC9/GR cell lines increase from 0.12 μmol/L to 8.52 μmol/L. But after treated with 5-aza-CdR, the IC50 of PC9/GR cell lines decrease to 4.35 μmol/L, and the resistance index (RI) decrease from 71 to 36 (P<0.05). Flow cytometry results showed that the apoptosis rate were 24.80% ± 0.28%, 12.70% ± 0.31%, 19.8% ± 0.15% respectively. MSP results showed that DAPK gene promoter region was un-methylated in PC9 cells and methylated in PC9/GR cells, when treated with 5-aza-CdR, DAPK gene promoter region was partly methylated in PC9/GR cells (P<0.05). Western blot results showed that the levels of DAPK protein were reduced significantly in PC9/GR cell lines compared with PC9, and after treated with 5-aza-CdR, the expression levels of DAPK protein in PC9/GR were increased (P<0.05). In conclusion, DAPK gene promoter methylation may contribute to the downregulation of DAPK gene and protein, and consequently affect the sensitivity of gefitinib in lung adenocarcinoma lines, induced gefitinib resistance. But 5-Aza-CdR can reverse gefitinib resistance by demethylation of DAPK gene promoter.

  15. The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats.

    PubMed

    Amaral, Cátia Lira Do; Bueno, Rafaela de Barros E Lima; Burim, Regislaine Valéria; Queiroz, Regina Helena Costa; Bianchi, Maria de Lourdes Pires; Antunes, Lusânia Maria Greggi

    2011-05-18

    Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet's effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet's effects on genomic stability and DNA methylation.

  16. 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.

  17. Long-term pancreatic beta cell exposure to high levels of glucose but not palmitate induces DNA methylation within the insulin gene promoter and represses transcriptional activity.

    PubMed

    Ishikawa, Kota; Tsunekawa, Shin; Ikeniwa, Makoto; Izumoto, Takako; Iida, Atsushi; Ogata, Hidetada; Uenishi, Eita; Seino, Yusuke; Ozaki, Nobuaki; Sugimura, Yoshihisa; Hamada, Yoji; Kuroda, Akio; Shinjo, Keiko; Kondo, Yutaka; Oiso, Yutaka

    2015-01-01

    Recent studies have implicated epigenetics in the pathophysiology of diabetes. Furthermore, DNA methylation, which irreversibly deactivates gene transcription, of the insulin promoter, particularly the cAMP response element, is increased in diabetes patients. However, the underlying mechanism remains unclear. We aimed to investigate insulin promoter DNA methylation in an over-nutrition state. INS-1 cells, the rat pancreatic beta cell line, were cultured under normal-culture-glucose (11.2 mmol/l) or experimental-high-glucose (22.4 mmol/l) conditions for 14 days, with or without 0.4 mmol/l palmitate. DNA methylation of the rat insulin 1 gene (Ins1) promoter was investigated using bisulfite sequencing and pyrosequencing analysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and increased DNA methylation at all five CpG sites within the Ins1 promoter, including the cAMP response element, in a time-dependent and glucose concentration-dependent manner. DNA methylation under experimental-high-glucose conditions was unique to the Ins1 promoter; however, palmitate did not affect DNA methylation. Artificial methylation of Ins1 promoter significantly suppressed promoter-driven luciferase activity, and a DNA methylation inhibitor significantly improved insulin mRNA suppression by experimental-high-glucose conditions. Experimental-high-glucose conditions significantly increased DNA methyltransferase activity and decreased ten-eleven-translocation methylcytosine dioxygenase activity. Oxidative stress and endoplasmic reticulum stress did not affect DNA methylation of the Ins1 promoter. High glucose but not palmitate increased ectopic triacylglycerol accumulation parallel to DNA methylation. Metformin upregulated insulin gene expression and suppressed DNA methylation and ectopic triacylglycerol accumulation. Finally, DNA methylation of the Ins1 promoter increased in isolated islets from Zucker diabetic fatty rats. This study helps to clarify the

  18. COX-2 gene promoter DNA methylation status in eutopic and ectopic endometrium of Egyptian women with endometriosis.

    PubMed

    Zidan, Haidy E; Rezk, Noha A; Alnemr, Amr Abd Almohsen; Abd El Ghany, Amany M

    2015-11-01

    The pathophysiology of COX-2 expression in endometriosis is a matter of debate. The aim was to investigate the role of DNA methylation of the NF-IL6 site within the promoter of COX-2 gene in the pathogenesis of endometriosis. The endometrial tissues (ectopic and eutopic) were collected from 60 women with endometriosis and 30 women without endometriosis (control group). The methylation status of COX-2 was examined by methylation-specific PCR. Quantitative real-time PCR (RT-PCR) was performed to measure COX-2 mRNA levels in endometrial tissues. We found significantly higher levels of COX-2 in ectopic endometriotic tissue compared with eutopic tissue. Also, we found that the frequencies of methylation status of the NF-IL6 site within the COX-2 promoter in the eutopic and ectopic endometrial tissues of endometriosis groups were significantly decreased in comparison to controls (P=0.002, P=0.000 respectively). Our study demonstrated that DNA hypomethylation of the NF-IL6 site within the promoter of COX-2 gene could be a key mechanism for its elevated expression in the eutopic and ectopic tissues of endometriosis.

  19. 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

  20. 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

  1. Association of NDRG1 gene promoter methylation with reduced NDRG1 expression in gastric cancer cells and tissue specimens.

    PubMed

    Chang, Xiaojing; Zhang, Shuanglong; Ma, Jinguo; Li, Zhenhua; Zhi, Yu; Chen, Jing; Lu, Yao; Dai, Dongqiu

    2013-05-01

    NDRG1 (N-myc downstream-regulated gene 1) plays a role in cell differentiation and suppression of tumor metastasis. This study aims to determine the expression of NDRG1 mRNA and protein in gastric cancer cell lines and tissue specimens and then assess the possible cause of its aberrant expression. Six gastric cancer cell lines and 20 pairs of normal and gastric cancer tissue samples were used to assess NDRG1 expression using Real-time PCR and Western blot. High-resolution melting analysis (HRM) and methylation-specific PCR (MSP) were performed to detect gene mutation and methylation, respectively, in cell lines and tissues samples. Expression of NDRG1 mRNA and protein was downregulated in gastric cancer cell lines and tissues. Specifically, expression of NDRG1 mRNA and protein was lower in all six gastric cancer cell lines than that of normal gastric cells, while 15 out of 20 cases of gastric cancer tissues had the reduced levels of NDRG1 mRNA and protein. HRM data showed that there was no mutation in NDRG1 gene, but MSP data showed high levels of NDRG1 gene promoter methylation in the CpG islands in both cell lines and tissue samples. Moreover, treatment with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine upregulated NDRG1 expression in gastric cancer HGC27 cells, but not in the histone deacetylase inhibitor trichostatin A-treated HGC27 cells. In conclusion, this study has shown that expression of NDRG1 mRNA and protein was reduced in gastric cancer cell lines and tissues, which is due to methylation of NDRG1 gene promoter. Further study will unearth the clinical significance of the reduced NDRG1 protein in gastric cancer.

  2. Association of the CpG methylation pattern of the proximal insulin gene promoter with type 1 diabetes.

    PubMed

    Fradin, Delphine; Le Fur, Sophie; Mille, Clémence; Naoui, Nadia; Groves, Chris; Zelenika, Diana; McCarthy, Mark I; Lathrop, Mark; Bougnères, Pierre

    2012-01-01

    The insulin (INS) region is the second most important locus associated with Type 1 Diabetes (T1D). The study of the DNA methylation pattern of the 7 CpGs proximal to the TSS in the INS gene promoter revealed that T1D patients have a lower level of methylation of CpG -19, -135 and -234 (p = 2.10(-16)) and a higher methylation of CpG -180 than controls, while methylation was comparable for CpG -69, -102, -206. The magnitude of the hypomethylation relative to a control population was 8-15% of the corresponding levels in controls and was correlated in CpGs -19 and -135 (r = 0.77) and CpG -135 and -234 (r = 0.65). 70/485 (14%) of T1D patients had a simultaneous decrease in methylation of CpG -19, -135, -234 versus none in 317 controls. CpG methylation did not correlate with glycated hemoglobin or with T1D duration. The methylation of CpG -69, -102, -180, -206, but not CpG -19, -135, -234 was strongly influenced by the cis-genotype at rs689, a SNP known to show a strong association with T1D. We hypothesize that part of this genetic association could in fact be mediated at the statistical and functional level by the underlying changes in neighboring CpG methylation. Our observation of a CpG-specific, locus-specific methylation pattern, although it can provide an epigenetic biomarker of a multifactorial disease, does not indicate whether the reported epigenetic pattern preexists or follows the establishment of T1D. To explore the effect of chronic hyperglycemia on CpG methylation, we studied non obese patients with type 2 diabetes (T2D) who were found to have decreased CpG-19 methylation versus age-matched controls, similar to T1D (p = 2.10(-6)) but increased CpG-234 methylation (p = 5.10(-8)), the opposite of T1D. The causality and natural history of the different epigenetic changes associated with T1D or T2D remain to be determined. PMID:22567146

  3. 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

  4. 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

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

    PubMed Central

    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

  6. Gene promoter DNA methylation patterns have a limited role in orchestrating transcriptional changes in the fetal liver in response to maternal folate depletion during pregnancy

    PubMed Central

    Adriaens, Michiel; Evelo, Chris T.; Ford, Dianne; Mathers, John C.

    2016-01-01

    Scope Early‐life exposures are critical in fetal programming and may influence function and health in later life. Adequate maternal folate consumption during pregnancy is essential for healthy fetal development and long‐term offspring health. The mechanisms underlying fetal programming are poorly understood, but are likely to involve gene regulation. Epigenetic marks, including DNA methylation, regulate gene expression and are modifiable by folate supply. We observed transcriptional changes in fetal liver in response to maternal folate depletion and hypothesized that these changes are concomitant with altered gene promoter methylation. Methods and results Female C57BL/6J mice were fed diets containing 2 or 0.4 mg folic acid/kg for 4 wk before mating and throughout pregnancy. At 17.5‐day gestation, genome‐wide gene expression and promoter methylation were measured by microarray analysis in male fetal livers. While 989 genes were differentially expressed, 333 promoters had altered methylation (247 hypermethylated, 86 hypomethylated) in response to maternal folate depletion. Only 16 genes had both expression and methylation changes. However, most methylation changes occurred in genomic regions neighboring expression changes. Conclusion In response to maternal folate depletion, altered expression at the mRNA level was not associated with altered promoter methylation of the same gene in fetal liver. PMID:27133805

  7. 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

  8. Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway.

    PubMed

    Ginis, Olivia; Courdavault, Vincent; Melin, Céline; Lanoue, Arnaud; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Courtois, Martine; Oudin, Audrey

    2012-05-01

    The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.

  9. 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

  10. Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls.

    PubMed

    Guo, F; Yang, B; Ju, Z H; Wang, X G; Qi, C; Zhang, Y; Wang, C F; Liu, H D; Feng, M Y; Chen, Y; Xu, Y X; Zhong, J F; Huang, J M

    2014-02-01

    The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

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

    PubMed Central

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

    2015-01-01

    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

  12. 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

    2015-12-01

    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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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...

  18. Tissue-specific promoter methylation coincides with Cyp19 gene expression in buffalo (Bubalus bubalis) placenta of different stages of gestation.

    PubMed

    Ghai, Sandeep; Monga, Rachna; Mohanty, T K; Chauhan, M S; Singh, Dheer

    2010-11-01

    Aromatase is the key enzyme for estrogen biosynthesis and is encoded by Cyp19 gene. Placental cotyledons are the main site of Cyp19 gene expression during pregnancy. The present study was aimed to investigate if DNA methylation and thus epigenetic mechanisms play a potential role in stage-specific regulation of Cyp19 expression in placental cotyledons of pregnant water buffaloes (Bubalus bubalis). Significantly higher expression of Cyp19 gene (p<0.05) in placental cotyledons of early gestation period and post parturition period was found in comparison to mid-gestation placenta. Tissue-specific promoter driven transcript analyses showed that the change in expression was mainly due to change in the relative abundance of transcripts from exon I.1 while the transcripts from exon II showed comparatively less variation. Methylation analysis of 5 CpG dinucleotides of placenta-specific promoter I.1 and proximal promoter, PII showed hypo-methylation of PI.1 in early and term placenta while hyper-methylation in mid-placenta. However, PII was found to be hypomethylated in all the three tissues. In conclusion, result of the present study demonstrated that stage-specific methylation status of PI.1, the major promoter responsible for aromatase expression in buffalo placental cotyledons, coincides with the change in expression of Cyp19 gene in different stages of pregnancy.

  19. 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.

  20. 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

  1. 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

  2. Cell-specific transcriptional regulation and reactivation of galectin-1 gene expression are controlled by DNA methylation of the promoter region.

    PubMed Central

    Benvenuto, G; Carpentieri, M L; Salvatore, P; Cindolo, L; Bruni, C B; Chiariotti, L

    1996-01-01

    The galectin-1 gene is developmentally regulated gene whose activity is strongly modulated during cell differentiation and transformation. We have previously shown that galectin-1 promoter constructs are highly active when transiently transfected in cells both expressing and not expressing the endogenous gene and that the basal activity is determined by a small region encompassing the transcription start site (from positions -50 to +50). We have now investigated the role of DNA methylation in galectin-1 gene expression. Southern blot analysis with HpaII and MspI endonucleases and sodium bisulfite analysis of genomic DNA from expressing and nonexpressing cell lines and cell hybrids showed a close correlation between gene activity and demethylation of the 5' region of the galectin-1 gene. We found that the galectin-1 promoter region is fully methylated, at every CpG site on both strands, in nonexpressing differentiated rat liver (FAO) and thyroid (PC C13) cells and unmethylated in the expressing undifferentiated liver (BRL3A) and thyroid transformed (PC myc/raf) cell lines. In addition, reactivation of the silent FAO alleles in FAO-human osteosarcoma (143tk-) hybrid cells is accompanied by a complete demethylation of the promoter region. Finally, when galectin-1 chloramphenicol acetyltransferase (CAT) promoter constructs were methylated in vitro by SssI methylase at every cytosine residue of the CpG doublets and transfected into mouse fibroblasts, the transcription of the CAT reporter gene was strongly inhibited. PMID:8649381

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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...

  8. [Estimation of the methylation status of the promoter region of the cell cycle gene P14ARF in placental tissues of spontaneous abortuses with chromosomal mosaicism].

    PubMed

    Kashevarova, A A; Tolmacheva, E N; Sukhanova, N N; Sazhenova, E A; Lebedev, I N

    2009-06-01

    The methylation status of the promoter region of the cell cycle gene P14ARF was studied in the extraembryonic mesoderm and in the chorion cytotrophoblast of 46 human spontaneous abortuses with chromosomal mosaicism. Aberrant methylation of alleles of this gene was revealed for the first time in placental tissues of 9% of embryos. The identified epimutations were found to be characteristic of embryos with aneuploid cell clones of postzygotic origin. It is suggested that epigenetic inactivation of loci responsible for the regulation of cell division and for segregation of chromosomes is associated with the occurrence of mosaic forms of the karyotype at early stages of human embryonic development. PMID:19639877

  9. Aberrant gene promoter methylation of p16, FHIT, CRBP1, WWOX, and DLC-1 in Epstein-Barr virus-associated gastric carcinomas.

    PubMed

    He, Dan; Zhang, Yi-wang; Zhang, Na-na; Zhou, Lu; Chen, Jian-ning; Jiang, Ye; Shao, Chun-kui

    2015-04-01

    Alterations in global DNA methylation and specific regulatory gene methylation are frequently found in cancer, but the significance of these epigenetic changes in EBV-associated gastric carcinoma (EBVaGC) remains unclear. We evaluated global DNA methylation status in 49 EBVaGC and 45 EBV-negative gastric carcinoma (EBVnGC) tissue samples and cell lines by 5-methylcytosine immunohistochemical staining and methylation quantification. We determined promoter methylation status and protein expression for the p16, FHIT, CRBP1, WWOX, and DLC-1 genes in tissues and studied the correlation between CpG island methylator phenotype (CIMP) class and clinicopathological characteristics. Changes in gene methylation and mRNA expression in EBVaGC cell line SNU-719 and in EBVnGC cell lines SGC-7901, BGC-823, and AGS were assessed after treatment with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA), or a combination of both, by methylation-specific PCR and quantitative real-time RT-PCR. Global genomic DNA hypomethylation was more pronounced in EBVnGC than in EBVaGC. Promoter methylation of all five genes was more frequent in EBVaGC than in EBVnGC (p < 0.05). p16 and FHIT methylation was reversely correlated with protein expression in EBVaGC. Most (41/49) EBVaGC exhibited CIMP-high (CIMP-H), and the prognosis of CIMP-H patients was significantly worse than that of CIMP-low (p = 0.027) and CIMP-none (p = 0.003) patients. Treatment with 5-aza-dC and/or TSA induced upregulation of RNA expression of all five genes in SNU-719; meanwhile, individual gene expression increased in EBVnGC cell lines. In summary, EBV-induced hypermethylation of p16, FHIT, CRBP1, WWOX, and DLC-1 may contribute to EBVaGC development. Demethylation therapy may represent a novel therapeutic strategy for EBVaGC.

  10. 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

  11. 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

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

    PubMed

    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.

  13. 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

  14. Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain

    PubMed Central

    2014-01-01

    Background The analgesic potency of opioids is reduced in neuropathic pain. However, the molecular mechanism is not well understood. Results The present study demonstrated that increased methylation of the Mu opioid receptor (MOR) gene proximal promoter (PP) in dorsal root ganglion (DRG) plays a crucial role in the decreased morphine analgesia. Subcutaneous (s.c.), intrathecal (i.t.) and intraplantar (i.pl.), not intracerebroventricular (i.c.v.) injection of morphine, the potency of morphine analgesia was significantly reduced in nerve-injured mice compared with control sham-operated mice. After peripheral nerve injury, we observed a decreased expression of MOR protein and mRNA, accompanied by an increased methylation status of MOR gene PP, in DRG. However, peripheral nerve injury could not induce a decreased expression of MOR mRNA in the spinal cord. Treatment with 5-aza-2′-deoxycytidine (5-aza-dC), inhibited the increased methylation of MOR gene PP and prevented the decreased expression of MOR in DRG, thereby improved systemic, spinal and periphery morphine analgesia. Conclusions Altogether, our results demonstrate that increased methylation of the MOR gene PP in DRG is required for the decreased morphine analgesia in neuropathic pain. PMID:25118039

  15. Environmental Stress Affects DNA Methylation of a CpG Rich Promoter Region of Serotonin Transporter Gene in a Nurse Cohort

    PubMed Central

    Alasaari, Jukka S.; Lagus, Markus; Ollila, Hanna M.; Toivola, Auli; Kivimäki, Mika; Vahtera, Jussi; Kronholm, Erkki; Härmä, Mikko; Puttonen, Sampsa; Paunio, Tiina

    2012-01-01

    Background Shift-working nurses are exposed to a stressful work environment, which puts them at an increased risk for burnout and depression. We explored the effect of environmental stress on serotonin transporter gene (SLC6A4) promoter methylation among nurses from high and low work stress environments. Methodology Using bisulfite sequencing, we investigated the methylation status of five CpG residues of a CpG-rich region in the promoter of SLC6A4 by comparing female shift working nurses from a high work stress environment (n = 24) to low work stress environment (n = 25). We also analyzed the association of 5-HTTLPR polymorphism at 5′ end of SLC6A4. Work stress was assessed by the Karasek’s Model and possible signs of burnout or depression were measured by the Maslach Burnout Index General Survey and Beck Depression Index. Methylation levels were assessed by bisulfite sequencing of DNA extracted from peripheral blood leucocytes. Restriction enzyme treatment followed by standard PCR was used to identify 5-HTTLPR genotypes. Principal Findings We found that nurses in the high stress environment had significantly lower promoter methylation levels at all five CpG residues compared to nurses in the low stress environment (p<0.01). There was no significant interaction of 5-HTTLPR genotype and work stress with methylation (p = 0.58). In unadjusted (bivariate) analysis, burnout was not significantly associated to methylation levels. However, when mutually adjusted for both, burnout and work stress were significant contributors (p = 0.038 and p<0.0001 respectively) to methylation levels. Conclusions Our findings show that environmental stress is concurrent with decreased methylation of the SLC6A4 promoter. This may lead to increased transcriptional activity of the gene, increased reuptake of serotonin from synaptic clefts, and termination of the activity of serotonin. This could present a possible coping mechanism for environmental stress in humans that

  16. Double Strand Breaks Can Initiate Gene Silencing and SIRT1-Dependent Onset of DNA Methylation in an Exogenous Promoter CpG Island

    PubMed Central

    O'Hagan, Heather M.; Mohammad, Helai P.; Baylin, Stephen B.

    2008-01-01

    Chronic exposure to inducers of DNA base oxidation and single and double strand breaks contribute to tumorigenesis. In addition to the genetic changes caused by this DNA damage, such tumors often contain epigenetically silenced genes with aberrant promoter region CpG island DNA hypermethylation. We herein explore the relationships between such DNA damage and epigenetic gene silencing using an experimental model in which we induce a defined double strand break in an exogenous promoter construct of the E-cadherin CpG island, which is frequently aberrantly DNA hypermethylated in epithelial cancers. Following the onset of repair of the break, we observe recruitment to the site of damage of key proteins involved in establishing and maintaining transcriptional repression, namely SIRT1, EZH2, DNMT1, and DNMT3B, and the appearance of the silencing histone modifications, hypoacetyl H4K16, H3K9me2 and me3, and H3K27me3. Although in most cells selected after the break, DNA repair occurs faithfully with preservation of activity of the promoter, a small percentage of the plated cells demonstrate induction of heritable silencing. The chromatin around the break site in such a silent clone is enriched for most of the above silent chromatin proteins and histone marks, and the region harbors the appearance of increasing DNA methylation in the CpG island of the promoter. During the acute break, SIRT1 appears to be required for the transient recruitment of DNMT3B and subsequent methylation of the promoter in the silent clones. Taken together, our data suggest that normal repair of a DNA break can occasionally cause heritable silencing of a CpG island–containing promoter by recruitment of proteins involved in silencing. Furthermore, with contribution of the stress-related protein SIRT1, the break can lead to the onset of aberrant CpG island DNA methylation, which is frequently associated with tight gene silencing in cancer. PMID:18704159

  17. 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

  18. 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

  19. 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

  20. 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.

  1. PGC−1α Promoter Methylation in Parkinson’s Disease

    PubMed Central

    Su, Xiaomin; Chu, Yaping; Kordower, Jeffrey H.; Li, Bin; Cao, Hong; Huang, Liang; Nishida, Maki; Song, Lei; Wang, Difei; Federoff, Howard J.

    2015-01-01

    The etiopathogenesis of sporadic Parkinson’s disease (PD) remains elusive although mitochondrial dysfunction has long been implicated. Recent evidence revealed reduced expression of peroxisome proliferator-activated receptor gamma coactivator−1 α (PGC−1α) and downstream regulated nuclear encoded respiratory complex genes in affected brain tissue from PD patients. We sought to determine whether epigenetic modification of the PGC−1α gene could account for diminished expression. In substantia nigra from PD patients but not control subjects, we show significant promoter-proximal non-canonical cytosine methylation of the PGC−1α gene but not an adjacent gene. As neuroinflammation is a prominent feature of PD and a mediator of epigenetic change, we evaluated whether the pro-inflammatory fatty acid, palmitate, would stimulate PGC−1α promoter methylation in different cell types from the CNS. Indeed, in mouse primary cortical neurons, microglia and astrocytes, palmitate causes PGC−1α gene promoter non-canonical cytosine methylation, reduced expression of the gene and reduced mitochondrial content. Moreover, intracerebroventricular (ICV) injection of palmitate to transgenic human α−synuclein mutant mice resulted in increased PGC−1α promoter methylation, decreased PGC−1α expression and reduced mitochondrial content in substantia nigra. Finally we provide evidence that dysregulation of ER stress and inflammatory signaling is associated with PGC−1α promoter methylation. Together, these data strengthen the connection between saturated fatty acids, neuroflammation, ER stress, epigenetic alteration and bioenergetic compromise in PD. PMID:26317511

  2. Early-life stress changes expression of GnRH and kisspeptin genes and DNA methylation of GnRH3 promoter in the adult zebrafish brain.

    PubMed

    Khor, Yee Min; Soga, Tomoko; Parhar, Ishwar S

    2016-02-01

    Early-life stress can cause long-term effects in the adulthood such as alterations in behaviour, brain functions and reproduction. DNA methylation is a mechanism of epigenetic change caused by early-life stress. Dexamethasone (DEX) was administered to zebrafish larvae to study its effect on reproductive dysfunction. The level of GnRH2, GnRH3, Kiss1 and Kiss2 mRNAs were measured between different doses of DEX treatment groups in adult zebrafish. Kiss1 and GnRH2 expression were increased in the 200mg/L DEX treated while Kiss2 and GnRH3 mRNA levels were up-regulated in the 2mg/L DEX-treated zebrafish. The up-regulation may be related to programming effect of DEX in the zebrafish larvae, causing overcompensation mechanism to increase the mRNA levels. Furthermore, DEX treatment caused negative impact on the development and maturation of the testes, in particular spermatogenesis. Therefore, immature gonadal development may cause positive feedback by increasing GnRH and Kiss. This indicates that DEX can alter the regulation of GnRH2, GnRH3, Kiss1 and Kiss2 in adult zebrafish, which affects maturation of gonads. Computer analysis of 1.5 kb region upstream of the 5' UTR of Kiss1, Kiss2, GnRH2 and GnRH3 promoter showed that there are putative binding sites of glucocorticoid response element and transcription factors involved in stress response. GnRH3 promoter analysed from pre-optic area, ventral telencephalon and ventral olfactory bulb showed higher methylation at CpG residues located on -1410, -1377 and -1355 between control and 2mg/L DEX-treated groups. Hence, early-life DEX treatment can alter methylation of GnRH3 gene promoter, which subsequently affects gene regulation and reproductive functions.

  3. 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

  4. 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

  5. DNA Methylation Profile at the DNMT3L Promoter

    PubMed Central

    Gokul, Gopinathan; Gautami, Bhimana; Malathi, Surapaneni; Sowjanya, A. Pavani; Poli, Usha Rani; Jain, Meenakshi; Ramakrishna, Gayatri; Khosla, Sanjeev

    2007-01-01

    Epigenetic events play a prominent role during cancer development. This is evident from the fact that almost all cancer types show aberrant DNA methylation. These abnormal DNA methylation levels are not restricted to just a few genes but affect the whole genome. Previous studies have shown genome-wide DNA hypomethylation and gene-specific hypermethylation to be a hallmark of most cancers. Molecules like DNA methyltransferase act as effectors of epigenetic reprogramming. In the present study we have examined the possibility that the reprogramming genes themselves undergo epigenetic modifications reflecting their changed transcriptional status during cancer development. Comparison of DNA methylation status between the normal and cervical cancer samples was carried out at the promoters of a few reprogramming molecules. Our study revealed statistically significant DNA methylation differences within the promoter of DNMT3L. A regulator of de novo DNA methyltransferases DNMT3A and DNMT3B, DNMT3L promoter was found to have lost DNA methylation to varying levels in 14 out of 15 cancer cervix samples analysed. The present study highlights the importance of DNA methylation profile at DNMT3L promoter not only as a promising biomarker for cervical cancer, which is the second most common cancer among women worldwide, but also provides insight into the possible role of DNMT3L in cancer development. PMID:17965599

  6. Solar-simulated ultraviolet radiation induces histone 3 methylation changes in the gene promoters of matrix metalloproteinases 1 and 3 in primary human dermal fibroblasts.

    PubMed

    Gesumaria, Lisa; Matsui, Mary S; Kluz, Thomas; Costa, Max

    2015-05-01

    Molecular signalling pathways delineating the induction of matrix metalloproteinases (MMPs) by ultraviolet radiation (UVR) are currently well-defined; however, the effects of UVR on epigenetic mechanisms of MMP induction are not as well understood. In this study, we examined solar-simulated UVR (ssUVR)-induced gene expression changes and alterations to histone methylation in the promoters of MMP1 and MMP3 in primary human dermal fibroblasts (HDF). Gene expression changes, including the increased expression of MMP1 and MMP3, were observed using Affymetrix GeneChip arrays and confirmed by qRT-PCR. Using ChIP-PCR, we showed for the first time that in HDF irradiated with 12 J/cm(2) ssUVR, the H3K4me3 transcriptional activating mark increased and the H3K9me2 transcriptional silencing mark decreased in abundance in promoters, correlating with the observed elevation of MMP1 and MMP3 mRNA levels following ssUVR exposure. Changes in mRNA levels due to a single exposure were transient and decreased 5 days after exposure. PMID:25707437

  7. Solar-simulated ultraviolet radiation induces histone 3 methylation changes in the gene promoters of matrix metalloproteinases 1 and 3 in primary human dermal fibroblasts.

    PubMed

    Gesumaria, Lisa; Matsui, Mary S; Kluz, Thomas; Costa, Max

    2015-05-01

    Molecular signalling pathways delineating the induction of matrix metalloproteinases (MMPs) by ultraviolet radiation (UVR) are currently well-defined; however, the effects of UVR on epigenetic mechanisms of MMP induction are not as well understood. In this study, we examined solar-simulated UVR (ssUVR)-induced gene expression changes and alterations to histone methylation in the promoters of MMP1 and MMP3 in primary human dermal fibroblasts (HDF). Gene expression changes, including the increased expression of MMP1 and MMP3, were observed using Affymetrix GeneChip arrays and confirmed by qRT-PCR. Using ChIP-PCR, we showed for the first time that in HDF irradiated with 12 J/cm(2) ssUVR, the H3K4me3 transcriptional activating mark increased and the H3K9me2 transcriptional silencing mark decreased in abundance in promoters, correlating with the observed elevation of MMP1 and MMP3 mRNA levels following ssUVR exposure. Changes in mRNA levels due to a single exposure were transient and decreased 5 days after exposure.

  8. 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...

  9. Methylated DNA Binding Domain Protein 2 (MBD2) Coordinately Silences Gene Expression through Activation of the MicroRNA hsa-mir-496 Promoter in Breast Cancer Cell Line

    PubMed Central

    Alvarado, Sebastian; Wyglinski, Joanne; Suderman, Matthew; Andrews, Stephen A.; Szyf, Moshe

    2013-01-01

    Methylated DNA binding protein 2 (MBD2) binds methylated promoters and suppresses transcription in cis through recruitment of a chromatin modification repressor complex. We show here a new mechanism of action for MBD2: suppression of gene expression indirectly through activation of microRNA hsa-mir-496. Overexpression of MBD2 in breast epithelial cell line MCF-10A results in induced expression and demethylation of hsa-mir-496 while depletion of MBD2 in a human breast cancer cell lines MCF-7 and MDA-MB231 results in suppression of hsa-mir-496. Activation of hsa-mir-496 by MBD2 is associated with silencing of several of its target genes while depletion of MBD2 leads to induction of hsa-mir-496 target genes. Depletion of hsa-mir-496 by locked nucleic acid (LNA) antisense oligonucleotide leads to activation of these target genes in MBD2 overexpressing cells supporting that hsa-mir-496 is mediating in part the effects of MBD2 on gene expression. We demonstrate that MBD2 binds the promoter of hsa-mir-496 in MCF-10A, MCF-7 and MDA-MB-231 cells and that it activates an in vitro methylated hsa-mir-496 promoter driving a CG-less luciferase reporter in a transient transfection assay. The activation of hsa-mir-496 is associated with reduced methylation of the promoter. Taken together these results describe a novel cascade for gene regulation by DNA methylation whereby activation of a methylated microRNA by MBD2 that is associated with loss of methylation triggers repression of downstream targets. PMID:24204564

  10. Analysis of the transcriptional regulation of cancer-related genes by aberrant DNA methylation of the cis-regulation sites in the promoter region during hepatocyte carcinogenesis caused by arsenic

    PubMed Central

    Miao, Zhuang; Wu, Lin; Lu, Ming; Meng, Xianzhi; Gao, Bo; Qiao, Xin; Zhang, Weihui; Xue, Dongbo

    2015-01-01

    Liver is the major organ for arsenic methylation metabolism and may be the potential target of arsenic-induced cancer. In this study, normal human liver cell was treated with arsenic trioxide, and detected using DNA methylation microarray. Some oncogenes, tumor suppressor genes, transcription factors (TF), and tumor-associated genes (TAG) that have aberrant DNA methylation have been identified. However, simple functional studies of genes adjacent to aberrant methylation sites cannot well reflect the regulatory relationship between DNA methylation and gene transcription during the pathogenesis of arsenic-induced liver cancer, whereas a further analysis of the cis-regulatory elements and their trans-acting factors adjacent to DNA methylation can more precisely reflect the relationship between them. MYC and MAX (MYC associated factor X) were found to participating cell cycle through a bioinformatics analysis. Additionally, it was found that the hypomethylation of cis-regulatory sites in the MYC promoter region and the hypermethylation of cis-regulatory sites in the MAX promoter region result in the up-regulation of MYC mRNA expression and the down-regulation of MAX mRNA, which increased the hepatocyte carcinogenesis tendency. PMID:26046465

  11. Prognostic significance of aberrant gene methylation in gastric cancer.

    PubMed

    Shi, Jing; Zhang, Guanjun; Yao, Demao; Liu, Wei; Wang, Na; Ji, Meiju; He, Nongyue; Shi, Bingyin; Hou, Peng

    2012-01-01

    Promoter methylation acts as an important alternative to genetic alterations for gene inactivation in gastric carcinogenesis. Although a number of gastric cancer-associated genes have been found to be methylated in gastric cancer, valuable methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. In the present study, we used methylation-specific PCR (MSP) to analyze promoter methylation of 9 gastric cancer-associated genes, including MLF1, MGMT, p16, RASSF2, hMLH1, HAND1, HRASLS, TM, and FLNc, and their association with clinicopathological characteristics and clinical outcome in a large cohort of gastric cancers. Our data showed that all of these genes were aberrantly methylated in gastric cancer, ranging from 8% to 51%. Moreover, gene methylation was strongly associated with certain clinicopathological characteristics, such as tumor differentiation, lymph node metastasis, and cancer-related death. Of interest, methylation of MGMT, p16, RASSF2, hMLH1, HAND1, and FLNc was closely associated with poor survival in gastric cancer, particularly MGMT, p16, RASSF2 and FLNc. Thus, our findings suggested these epigenetic events may contribute to the initiation and progression of gastric cancer. Importantly, methylation of some genes were closely relevant to poor prognosis in gastric cancer, providing the strong evidences that these hypermethylated genes may be served as valuable biomarkers for prognostic evaluation in this cancer.

  12. 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

  13. Evaluation of MYB Promoter Methylation in Salivary Adenoid Cystic Carcinoma

    PubMed Central

    Shao, Chunbo; Bai, Weiliang; Junn, Jacqueline C.; Uemura, Mamoru; Hennessey, Patrick T.; Zaboli, David; Sidransky, David; Califano, Joseph A.; Ha, Patrick K.

    2011-01-01

    Summary The transcription factor MYB was recently proposed to be a promising oncogene candidate in salivary gland adenoid cystic carcinoma (ACC). However, the up-regulation of MYB in ACC could not be explained solely by deletion of its 3′ end. It is widely accepted that the promoter methylation status can regulate the transcription of genes, especially in human cancers. Therefore, it is important to know whether MYB promoter demethylation could explain the over-expression of MYB in ACC. By using the Methprimer program, we identified nine CpG islands in the promoter of MYB. All of these CpG islands were located within the −864 to +2,082 nt region relative to the transcription start site of MYB. We then used bisulfite genomic sequencing to evaluate the methylation levels of the CpG islands of MYB in 18 primary ACC tumors, 13 normal salivary gland tissues and nine cancer cell lines. Using cell lines, we also determined the relative MYB expression levels and correlated these with the methylation levels. With bisulfite genomic sequencing, we found no detectable methylation in the CpG islands of MYB in either ACC or normal salivary gland tissues. There was a variable degree of MYB expression in the cell lines tested, but none of these cell lines demonstrated promoter methylation. Promoter hypomethylation does not appear to explain the differential expression of MYB in ACC. An alternative mechanism needs to be proposed for the transcriptional control of MYB in ACC. PMID:21324728

  14. 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

  15. 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

  16. Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation

    PubMed Central

    Leng, Shuguang; Stidley, Christine A.; Willink, Randy; Bernauer, Amanda; Do, Kieu; Picchi, Maria A.; Sheng, Xin; Frasco, Melissa, A.; Berg, David Van Den; Gilliland, Frank D.; Zima, Christopher; Crowell, Richard E.; Belinsky, Steven A.

    2008-01-01

    Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of double-strand break repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as ≥ 3 of 8 genes methylated in sputum, compared to smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-Pkc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with ≥ 7 risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ATM was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify double-strand break DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, that is, in turn, associated with elevated risk for lung cancer. PMID:18413776

  17. 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

  18. Methylated MicroRNA Genes of the Developing Murine Palate

    PubMed Central

    Seelan, Ratnam S.; Mukhopadhyay, Partha; Warner, Dennis R.; Appana, Savitri N.; Brock, Guy N.; Pisano, M. Michele; Greene, Robert M.

    2016-01-01

    Environmental factors contribute to the etiology of cleft palate (CP). Environmental factors can also affect gene expression via alterations in DNA methylation suggesting a possible mechanism for the induction of CP. Identification of genes methylated during development of the secondary palate provides the basis for examination of the means by which environmental factors may adversely influence palatal ontogeny. We previously characterized the methylome of the developing murine secondary palate focusing primarily on protein-encoding genes. We now extend this study to include methylated microRNA (miRNA) genes. A total of 42 miRNA genes were found to be stably methylated in developing murine palatal tissue. Twenty eight of these were localized within host genes. Gene methylation was confirmed by pyrosequencing of selected miRNA genes. Integration of methylated miRNA gene and expression datasets identified 62 miRNAs, 69% of which were non-expressed. For a majority of genes (83%), upstream CpG islands (CGIs) were highly methylated suggesting down-regulation of CGI-associated promoters. DAVID and IPA analyses indicated that both expressed and non-expressed miRNAs target identical signaling pathways and biological processes associated with palatogenesis. Furthermore, these analyses also identified novel signaling pathways whose roles in palatogenesis remain to be elucidated. In summary, we identify methylated miRNA genes in the developing murine secondary palate, correlate miRNA gene methylation with expression of their cognate miRNA transcripts, and identify pathways and biological processes potentially mediated by these miRNAs. PMID:25642850

  19. The Human ARF Cell Cycle Regulatory Gene Promoter Is a CpG Island Which Can Be Silenced by DNA Methylation and Down-Regulated by Wild-Type p53

    PubMed Central

    Robertson, Keith D.; Jones, Peter A.

    1998-01-01

    The INK4a/ARF locus encodes two proteins involved in tumor suppression in a manner virtually unique in mammalian cells. Distinct first exons, driven from separate promoters, splice onto a common exon 2 and 3 but utilize different reading frames to produce two completely distinct proteins, both of which play roles in cell cycle control. INK4a, a critical element of the retinoblastoma gene pathway, binds to and inhibits the activities of CDK4 and CDK6, while ARF, a critical element of the p53 pathway, increases the level of functional p53 via interaction with MDM2. Here we clone and characterize the promoter of the human ARF gene and show that it is a CpG island characteristic of a housekeeping gene which contains numerous Sp1 sites. Both ARF and INK4a are coordinately expressed in cells except when their promoter regions become de novo methylated. In one of these situations, ARF transcription could be reactivated by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine, and the reactivation kinetics of ARF and INK4a were found to differ slightly in a cell line in which both genes were silenced by methylation. The ARF promoter was also found to be highly responsive to E2F1 expression, in keeping with previous results at the RNA level. Lastly, transcription from the ARF promoter was down-regulated by wild-type p53 expression, and the magnitude of the effect correlated with the status of the endogenous p53 gene. This finding points to the existence of an autoregulatory feedback loop between p53, MDM2, and ARF, aimed at keeping p53 levels in check. PMID:9774662

  20. 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

    Xu, Houguo; Dong, Xiaojing; 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.

  1. 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

  2. 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

  3. 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

  4. Genome-wide mapping reveals conservation of promoter DNA methylation following chicken domestication.

    PubMed

    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.

  5. 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

  6. 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.

  7. 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

  8. Methylated BNIP3 gene in colorectal cancer prognosis

    PubMed Central

    SHIMIZU, SAYAKA; IIDA, SATORU; ISHIGURO, MEGUMI; UETAKE, HIROYUKI; ISHIKAWA, TOSHIAKI; TAKAGI, YOKO; KOBAYASHI, HIROTOSHI; HIGUCHI, TETSURO; ENOMOTO, MASAYUKI; MOGUSHI, KAORU; MIZUSHIMA, HIROSHI; TANAKA, HIROSHI; SUGIHARA, KENICHI

    2010-01-01

    The DNA methylation of apoptosis-related genes in various cancers contributes to the disruption of the apoptotic pathway and results in resistance to chemotherapeutic agents. Irinotecan (CPT-11) is one of the key chemotherapy drugs used to treat metastatic colorectal cancer (CRC). However, a number of metastatic CRC patients do not benefit from this drug. Thus, the identification of molecular genetic parameters associated with the response to CPT-11 is of interest. To identify apoptosis-related genes that may contribute to CPT-11 resistance, microarray analysis was conducted using colon cancer cells in which 5-aza-2′deoxycytidine (DAC) enhanced sensitivity to CPT-11. Microarray analysis identified 10 apoptosis-related genes that were up-regulated following treatment with DAC. Among the genes, Bcl-2/adenovirus E1B 19 kDa protein interacting protein 3 (BNIP3), a Bcl-2 family pro-apoptotic protein, was identified as being involved in CPT-11 resistance following methylation of its promoter. An analysis of 112 primary CRC cases revealed that approximately 58% of cases showed BNIP3 methylation, and that patients with methylation exhibited a poorer outcome compared to those without methylation. In addition, in 30 patients who received first-line CPT-11 chemotherapy, patients with methylation exhibited resistance to chemotherapy compared to patients with no methylation. The results suggest that methylation of BNIP3 is a predictive factor in the prognosis and response to CPT-11 treatment in CRC patients. PMID:22966396

  9. 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

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

    PubMed

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

    2015-10-01

    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

  11. Genome-wide profiling of DNA methylation and gene expression in Crassostrea gigas male gametes

    PubMed Central

    Olson, Claire E.; Roberts, Steven B.

    2014-01-01

    DNA methylation patterns and functions are variable across invertebrate taxa. In order to provide a better understanding of DNA methylation in the Pacific oyster (Crassostrea gigas), we characterized the genome-wide DNA methylation profile in male gamete cells using whole-genome bisulfite sequencing. RNA-Seq analysis was performed to examine the relationship between DNA methylation and transcript expression. Methylation status of over 7.6 million CpG dinucleotides was described with a majority of methylated regions occurring among intragenic regions. Overall, 15% of the CpG dinucleotides were determined to be methylated and the mitochondrial genome lacked DNA methylation. Integrative analysis of DNA methylation and RNA-Seq data revealed a positive association between methylation status, both in gene bodies and putative promoter regions, and expression. This study provides a comprehensive characterization of the distribution of DNA methylation in the oyster male gamete tissue and suggests that DNA methylation is involved in gene regulatory activity. PMID:24987376

  12. Acquired Alterations of Hypothalamic Gene Expression of Insulin and Leptin Receptors and Glucose Transporters in Prenatally High-Glucose Exposed Three-Week Old Chickens Do Not Coincide with Aberrant Promoter DNA Methylation

    PubMed Central

    Ott, Raffael; Bogatyrev, Semen; Tzschentke, Barbara; Plagemann, Andreas

    2015-01-01

    Background Prenatal exposures may have a distinct impact for long-term health, one example being exposure to maternal ‘diabesity’ during pregnancy increasing offspring ‘diabesity’ risk. Malprogramming of the central nervous regulation of body weight, food intake and metabolism has been identified as a critical mechanism. While concrete disrupting factors still remain unclear, growing focus on acquired epigenomic alterations have been proposed. Due to the independent development from the mother, the chicken embryo provides a valuable model to distinctively establish causal factors and mechanisms. Aim The aim of this study was to determine the effects of prenatal hyperglycemia on postnatal hypothalamic gene expression and promoter DNA methylation in the chicken. Methods and Findings To temporarily induce high-glucose exposure in chicken embryos, 0.5 ml glucose solution (30 mmol/l) were administered daily via catheter into a vessel of the chorioallantoic egg membrane from days 14 to 17 of incubation. At three weeks of postnatal age, body weight, total body fat, blood glucose, mRNA expression (INSR, LEPR, GLUT1, GLUT3) as well as corresponding promoter DNA methylation were determined in mediobasal hypothalamic brain slices (Nucleus infundibuli hypothalami). Although no significant changes in morphometric and metabolic parameters were detected, strongly decreased mRNA expression occurred in all candidate genes. Surprisingly, however, no relevant alterations were observed in respective promoter methylation. Conclusion Prenatal hyperglycemia induces strong changes in later hypothalamic expression of INSR, LEPR, GLUT1, and GLUT3 mRNA. While the chicken provides an interesting approach for developmental malprogramming, the classical expression regulation via promoter methylation was not observed here. This may be due to alternative/interacting brain mechanisms or the thus far under-explored bird epigenome. PMID:25811618

  13. Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells

    PubMed Central

    Sung, Hye Youn; Park, Ae Kyung

    2014-01-01

    Purpose Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties. However, the precise regulatory mechanisms controlling metastasis genes and their role in metastatic transformation are largely unknown. To address epigenetically-regulated gene products involved in ovarian cancer metastasis, we examined the mechanisms regulating mucin 13 (MUC13) expression and its influence on aggressive behaviors of ovarian malignancies. Materials and Methods We injected SK-OV-3 ovarian cancer cells peritoneally into nude mice to mimic human ovarian tumor metastasis. Overexpression of MUC13 mRNA was detected in metastatic implants from the xenografts by expression microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The DNA methylation status within the MUC13 promoter region was determined using bisulfite sequencing PCR and quantitative methylation-specific PCR. We evaluated the effects of exogenous MUC13 on cell invasion and migration using in vitro transwell assays. Results MUC13 mRNA expression was up-regulated, and methylation of specific CpG sites within the promoter was reduced in the metastatic implants relative to those in wild-type SK-OV-3 cells. Addition of a DNA methyltransferase inhibitor to SK-OV-3 cells induced MUC13 expression, thereby implying epigenetic regulation of MUC13 by promoter methylation. MUC13 overexpression increased migration and invasiveness, compared to control cells, suggesting aberrant up-regulation of MUC13 is strongly associated with progression of aggressive behaviors in ovarian cancer. Conclusion We provide novel evidence for epigenetic regulation of MUC13 in ovarian cancer. We suggest that the DNA methylation status within the MUC13 promoter region may be a potential biomarker of aggressive behavior in ovarian cancer. PMID:25048476

  14. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome.

    PubMed

    Weber, Michael; Hellmann, Ines; Stadler, Michael B; Ramos, Liliana; Pääbo, Svante; Rebhan, Michael; Schübeler, Dirk

    2007-04-01

    To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation. PMID:17334365

  15. Methylation of miRNA genes and oncogenesis.

    PubMed

    Loginov, V I; Rykov, S V; Fridman, M V; Braga, E A

    2015-02-01

    Interaction between microRNA (miRNA) and messenger RNA of target genes at the posttranscriptional level provides fine-tuned dynamic regulation of cell signaling pathways. Each miRNA can be involved in regulating hundreds of protein-coding genes, and, conversely, a number of different miRNAs usually target a structural gene. Epigenetic gene inactivation associated with methylation of promoter CpG-islands is common to both protein-coding genes and miRNA genes. Here, data on functions of miRNAs in development of tumor-cell phenotype are reviewed. Genomic organization of promoter CpG-islands of the miRNA genes located in inter- and intragenic areas is discussed. The literature and our own results on frequency of CpG-island methylation in miRNA genes from tumors are summarized, and data regarding a link between such modification and changed activity of miRNA genes and, consequently, protein-coding target genes are presented. Moreover, the impact of miRNA gene methylation on key oncogenetic processes as well as affected signaling pathways is discussed.

  16. Role of PTCH1 gene methylation in gastric carcinogenesis.

    PubMed

    Zuo, Yun; Song, Yu; Zhang, Min; Xu, Zhen; Qian, Xiaolan

    2014-08-01

    The present study aimed to investigate the role of PTCH1 methylation in gastric carcinogenesis and the therapeutic effect of the methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), in the treatment of gastric cancer. Total RNA was extracted from 20 gastric cancer tissues, their corresponding adjacent normal tissues and a gastric cancer AGS cell line. PTCH1 mRNA expression was detected by quantitative PCR, and the PTCH1 methylation of the promoter was examined by methylation-specific PCR. The AGS cells were treated with 5-Aza-dC; apoptosis and the cell cycle were examined by flow cytometry, and the PTCH1 methylation level was observed. PTCH1 expression was negatively correlated with promoter methylation in the gastric cancer tissues, their corresponding adjacent normal tissues and the gastric cancer AGS cell line (r=-0.591, P=0.006). 5-Aza-dC treatment caused apoptosis and the G0/G1 phase arrest of the AGS cells, and also induced the demethylation and increased expression of PTCH1. In conclusion, the study found that the hypermethylation of the PTCH1 gene promoter region is one of the main causes of low PTCH1 expression in AGS cells. Demethylation agent 5-Aza-dC can reverse the methylation status of PTCH1 and regulate the expression of PTCH1, indicating its potential role in gastric cancer treatment. PMID:25013484

  17. Quantitative detection of TUSC3 promoter methylation -a potential biomarker for prognosis in lung cancer

    PubMed Central

    Duppel, Uta; Woenckhaus, Matthias; Schulz, Christian; Merk, Johannes; Dietmaier, Wolfgang

    2016-01-01

    Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding. PMID:27698890

  18. Quantitative detection of TUSC3 promoter methylation -a potential biomarker for prognosis in lung cancer

    PubMed Central

    Duppel, Uta; Woenckhaus, Matthias; Schulz, Christian; Merk, Johannes; Dietmaier, Wolfgang

    2016-01-01

    Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding.

  19. Aberrant Methylation of MGMT Promoter in HNSCC: A Meta-Analysis

    PubMed Central

    Cai, Fucheng; Xiao, Xiyue; Niu, Xun; Shi, Hao; Zhong, Yi

    2016-01-01

    Background O6-methylguanine-DNA methyl-transferase (MGMT) gene, a DNA repair gene, plays a critical role in the repair of alkylated DNA adducts that form following exposure to genotoxic agents. MGMT is generally expressed in various tumors, and its function is frequently lost because of hypermethylation in the promoter. The promoter methylation of MGMT has been extensively investigated in head and neck squamous cell carcinoma (HNSCC). However, the association between the promoter methylation of MGMT and HNSCC risk remains inconclusive and inconsistent. Therefore, we performed a meta-analysis to better clarify the association between the promoter methylation of MGMT and HNSCC risk. Methods A systematical search was conducted in PubMed, Web of Science, EMBASE, and Ovid for studies on the association between MGMT promoter methylation and HNSCC. Odds ratio (ORs) and 95% confidence intervals (CI) were calculated to estimate association between MGMT promoter methylation and risk of HNSCC. The meta-regression and subgroup analysis were undertaken to explore the potential sources of heterogeneity. Results Twenty studies with 1,030 cases and 775 controls were finally included in this study. The frequency of MGMT promoter methylation was 46.70% in HNSCC group and 23.23% in the control group. The frequency of MGMT promoter methylation in HNSCC group was significantly higher than the control group (OR = 2.83, 95%CI = 2.25–3.56). Conclusion This meta-analysis indicates that aberrant methylation of MGMT promoter was significantly associated with the risk of HNSCC, and it may be a potential molecular marker for monitoring the disease and may provide new insights to the treatment of HNSCC. PMID:27657735

  20. Identical sets of methylated and nonmethylated genes in Ciona intestinalis sperm and muscle cells

    PubMed Central

    2013-01-01

    Background The discovery of gene body methylation, which refers to DNA methylation within gene coding region, suggests an as yet unknown role of DNA methylation at actively transcribed genes. In invertebrates, gene bodies are the primary targets of DNA methylation, and only a subset of expressed genes is modified. Results Here we investigate the tissue variability of both the global levels and distribution of 5-methylcytosine (5mC) in the sea squirt Ciona intestinalis. We find that global 5mC content of early developmental embryos is high, but is strikingly reduced in body wall tissues. We chose sperm and adult muscle cells, with high and reduced levels of global 5mC respectively, for genome-wide analysis of 5mC targets. By means of CXXC-affinity purification followed by deep sequencing (CAP-seq), and genome-wide bisulfite sequencing (BS-seq), we designated body-methylated and unmethylated genes in each tissue. Surprisingly, body-methylated and unmethylated gene groups are identical in the sperm and muscle cells. Our analysis of microarray expression data shows that gene body methylation is associated with broad expression throughout development. Moreover, transgenic analysis reveals contrasting gene body methylation at an identical gene-promoter combination when integrated at different genomic sites. Conclusions We conclude that gene body methylation is not a direct regulator of tissue specific gene expression in C. intestinalis. Our findings reveal constant targeting of gene body methylation irrespective of cell type, and they emphasize a correlation between gene body methylation and ubiquitously expressed genes. Our transgenic experiments suggest that the promoter does not determine the methylation status of the associated gene body. PMID:24279449

  1. 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

  2. The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters.

    PubMed

    Bao, Xichen; Wu, Haitao; Zhu, Xihua; Guo, Xiangpeng; Hutchins, Andrew P; Luo, Zhiwei; Song, Hong; Chen, Yongqiang; Lai, Keyu; Yin, Menghui; Xu, Lingxiao; Zhou, Liang; Chen, Jiekai; Wang, Dongye; Qin, Baoming; Frampton, Jon; Tse, Hung-Fat; Pei, Duanqing; Wang, Huating; Zhang, Biliang; Esteban, Miguel A

    2015-01-01

    Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.

  3. Analysis of Promoter Non-CG Methylation in Prostate Cancer

    PubMed Central

    Truong, Matthew; Yang, Bing; Wagner, Jennifer; Desotelle, Joshua; Jarrard, David F.

    2013-01-01

    In vertebrates DNA methylation occurs primarily at CG dinucleotides, but recently non-CG methylation has been found at appreciable levels in embryonic stem cells. To assess non-CG methylation in cancer, we compared the extent of non-CG methylation at several biologically important CpG islands in prostate cancer and normal cell lines. An assessment of the promoter CpG islands Even-skipped homeobox 1 (EVX1) and filamin A-interacting protein 1-like (FILIP1L) demonstrates a 4-fold higher rate of non-CG methylation at EVX1 compared to FILIP1L across all cell lines. These loci are densely methylated at CG sites in cancer. No significant difference in non-CG methylation was demonstrated between cancer and normal. Treatment of cancer cell lines with 5-azacytidine significantly reduced methylation within EVX1 at CG and CC sites preferentially. We conclude that non-CG methylation does not correlate with CG methylation at hypermethylated promoter regions in cancer. Furthermore, global inhibition of DNA methyltransferases does not affect all methylated cytosines uniformly. PMID:23414321

  4. Genistein mediates the selective radiosensitizing effect in NSCLC A549 cells via inhibiting methylation of the keap1 gene promoter region

    PubMed Central

    Liu, Xiongxiong; Sun, Chao; Liu, Bingtao; Jin, Xiaodong; Li, Ping; Zheng, Xiaogang; Zhao, Ting; Li, Feifei; Li, Qiang

    2016-01-01

    Non-small cell lung cancer (NSCLC) cells often possess a hypermethylated Keap1 promoter, which decreases Keap1 mRNA and protein expression levels, thus impairing the Nrf2-Keap1 pathway and thereby leading to chemo- or radio-resistance. In this study, we showed that genistein selectively exhibited a radiosensitizing effect on NSCLC A549 cells but not on normal lung fibroblast MRC-5 cells. Genistein caused oxidative stress in A549 cells rather than MRC-5 cells, as determined by the oxidation of the ROS-sensitive probe DCFH-DA and oxidative damage marked by MDA, PCO or 8-OHdG content. In A549 instead of MRC-5 cells, genistein reduced the level of methylation in the Keap1 promoter region, leading to an increased mRNA expression, thus effectively inhibited the transcription of Nrf2 to the nucleus, which suppressed the Nrf2-dependent antioxidant and resulted in the upregulation of ROS. Importantly, when combined with radiation, genistein further increased the ROS levels in A549 cells whereas decreasing the radiation-induced oxidative stress in MRC-5 cells, possibly via increasing the expression levels of Nrf2, GSH and HO-1. Moreover, radiation combined with genistein significantly increased cell apoptosis in A549 but not MRC-5 cells. Together, the results herein show that the intrinsic difference in the redox status of A549 and MRC-5 cells could be the target for genistein to selectively sensitize A549 cells to radiation, thereby leading to an increase in radiosensitivity for A549 cells. PMID:27029077

  5. 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

  6. Epigenetic Methylation of Parathyroid CaR and VDR Promoters in Experimental Secondary Hyperparathyroidism.

    PubMed

    Hofman-Bang, Jacob; Gravesen, Eva; Olgaard, Klaus; Lewin, Ewa

    2012-01-01

    Secondary hyperparathyroidism (s-HPT) in uremia is characterized by decreased expression in the parathyroids of calcium sensing (CaR) and vitamin D receptors (VDR). Parathyroid hormone (PTH) is normalized despite low levels of CaR and VDR after experimental reversal of uremia. The expression of CaR in parathyroid cultures decreases rapidly. Methylation of promoter regions is often detected during epigenetic downregulation of gene expression. Therefore, using an experimental rat model, we examined changes in methylation levels of parathyroid CaR and VDR promoters in vivo and in vitro. Methods. Uremia was induced by 5/6 nephrectomy. Melting temperature profiling of CaR and VDR PCR products after bisulfite treatment of genomic DNA from rat parathyroids was performed. Real-time PCR measured expression of PTH, CaR, VDR, and klotho genes in vitro. Results. Parathyroids from uremic rats had similar low levels of methylation in vivo and in vitro. In culture, a significant downregulation of CaR, VDR, and klotho within two hours of incubation was observed, while housekeeping genes remained stable for 24 hours. Conclusion. In uremic s-HPT and in vitro, no overall changes in methylation levels in the promoter regions of parathyroid CaR and VDR genes were found. Thus, epigenetic methylation of these promoters does not explain decreased parathyroid expression of CaR and VDR genes in uremic s-HPT.

  7. Aberrantly methylated genes in human papillary thyroid cancer and their association with BRAF/RAS mutation

    PubMed Central

    Kikuchi, Yasuko; Tsuji, Eiichi; Yagi, Koichi; Matsusaka, Keisuke; Tsuji, Shingo; Kurebayashi, Junichi; Ogawa, Toshihisa; Aburatani, Hiroyuki; Kaneda, Atsushi

    2013-01-01

    Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7) was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2′-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P = 0.04, Fisher's exact test). Thus, we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer. PMID:24367375

  8. 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...

  9. DNA methylation of the LEP gene is associated with craving during alcohol withdrawal.

    PubMed

    Hillemacher, Thomas; Weinland, Christian; Lenz, Bernd; Kraus, Thomas; Heberlein, Annemarie; Glahn, Alexander; Muschler, Marc A N; Bleich, Stefan; Kornhuber, Johannes; Frieling, Helge

    2015-01-01

    Different studies have described evidence for an association between leptin serum levels and craving in alcohol dependent patients. As leptin expression is regulated by DNA methylation we investigated changes of DNA methylation of the LEP gene promoter region in alcohol dependent patients undergoing withdrawal. Results show that low methylation status is associated with increasing serum leptin levels and elevation of craving for alcohol in the referring patients group. These findings point towards a pathophysiological relevance of changes in DNA methylation of the LEP gene promoter region in alcohol dependence.

  10. 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

  11. Identification of genes specifically methylated in Epstein-Barr virus-associated gastric carcinomas.

    PubMed

    Okada, Toshiyuki; Nakamura, Munetaka; Nishikawa, Jun; Sakai, Kouhei; Zhang, Yibo; Saito, Mari; Morishige, Akihiro; Oga, Atsunori; Sasaki, Kosuke; Suehiro, Yutaka; Hinoda, Yuji; Sakaida, Isao

    2013-10-01

    We studied the comprehensive DNA methylation status in the naturally derived gastric adenocarcinoma cell line SNU-719, which was infected with the Epstein-Barr virus (EBV) by methylated CpG island recovery on chip assay. To identify genes specifically methylated in EBV-associated gastric carcinomas (EBVaGC), we focused on seven genes, TP73, BLU, FSD1, BCL7A, MARK1, SCRN1, and NKX3.1, based on the results of methylated CpG island recovery on chip assay. We confirmed DNA methylation of the genes by methylation-specific PCR and bisulfite sequencing in SNU-719. The expression of the genes, except for BCL7A, was upregulated by a combination of 5-Aza-2'-deoxycytidine and trichostatin A treatment in SNU-719. After the treatment, unmethylated DNA became detectable in all seven genes by methylation-specific PCR. We verified DNA methylation of the genes in 75 primary gastric cancer tissues from 25 patients with EBVaGC and 50 EBV-negative patients who were controls. The methylation frequencies of TP73, BLU, FSD1, BCL7A, MARK1, SCRN1, and NKX3.1 were significantly higher in EBVaGC than in EBV-negative gastric carcinoma. We identified seven genes with promoter regions that were specifically methylated in EBVaGC. Inactivation of these genes may suppress their function as tumor suppressor genes or tumor-associated antigens and help to develop and maintain EBVaGC. PMID:23829175

  12. Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP.

    PubMed

    Bian, Y S; Yan, P; Osterheld, M C; Fontolliet, C; Benhattar, J

    2001-01-01

    Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes. The combination of bisulfite modification and PCR results in the conversion of unmethylated cytosines to thymines, whereas methylated cytosines remain unchanged. This sequence conversion can lead to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of methylated and unmethylated DNA at known ratios revealed that the relative intensities of the corresponding bands following MS-SSCA were maintained. MS-SSCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed tissue sections. MS-SSCA is a rapid, specific, and semiquantitative approach that allows the detection of methylation of the p16 gene promoter. In reconstruction experiments, the method permits the detection of 10% or less of cells harboring a methylated p16 promoter. We have been successful in analyzing by MS-SSCA almost all (96%) tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results. In addition, when microdissection was performed, the clonality of this genetic alteration could be identified.

  13. LINE-1 and inflammatory gene methylation levels are early biomarkers of metabolic changes: association with adiposity.

    PubMed

    Carraro, Júlia Cristina Cardoso; Mansego, Maria Luisa; Milagro, Fermin Ignacio; Chaves, Larissa Oliveira; Vidigal, Fernanda Carvalho; Bressan, Josefina; Martínez, J Alfredo

    2016-11-01

    We analyzed whether global and inflammatory genes methylation can be early predictors of metabolic changes and their associations with the diet, in a cross-sectional study (n = 40). Higher global methylation was associated to adiposity, insulin resistance, and lower quality of the diet. Methylation of IL-6, SERPINE1 and CRP genes was related to adiposity traits and macronutrients intake. SERPINE1 hypermethylation was also related to some metabolic alterations. CRP methylation was a better predictor of insulin resistance than CRP plasma concentrations. Global and inflammatory gene promoter hypermethylation can be good early biomarkers of adiposity and metabolic changes and are associated to the quality of the diet.

  14. Promoter Methylation of SFRP3 Is Frequent in Hepatocellular Carcinoma

    PubMed Central

    Shih, Yu-Lueng; Lien, Gi-Shih; Suk, Fat-Moon; Hsieh, Chung-Bao; Yan, Ming-De

    2014-01-01

    Oncogenic activation of the Wnt/β-catenin signaling pathway is common in human cancers. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications in carcinogenesis. Because there have been no reports about the role of SFRP3 in hepatocellular carcinoma (HCC), we investigated the level of methylation and transcription of SFRP3. Four HCC cell lines, 60 HCCs, 23 cirrhosis livers, 37 chronic hepatitis livers, and 30 control livers were prescreened for SFRP3 promoter methylation by methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing. SFRP3 promoter methylation was observed in 100%, 60%, 39.1%, 16.2%, and 0% in HCC cell lines, primary HCCs, cirrhosis livers, chronic hepatitis livers, and control livers, respectively. Demethylation treatment with 5-aza-2′-deoxycytidine in HCC cells restored or increased the SFRP3 mRNA expression. We next used quantitative MS-PCR (QMSP) to analyze the methylation level of SFRP3 in 60 HCCs and their corresponding nontumor tissues. Methylation of SFRP3 promoter region in HCCs increased significantly compared with control tissues. There is a positive correlation between promoter hypermethylation and SFRP3 mRNA downregulation. Our data suggest that promoter hypermethylation of SFRP3 is a common event in HCCs and plays an important role in regulation of SFRP3 mRNA expression. PMID:24591760

  15. Reversible Regulation of Promoter and Enhancer Histone Landscape by DNA Methylation in Mouse Embryonic Stem Cells.

    PubMed

    King, Andrew D; Huang, Kevin; Rubbi, Liudmilla; Liu, Shuo; Wang, Cun-Yu; Wang, Yinsheng; Pellegrini, Matteo; Fan, Guoping

    2016-09-27

    DNA methylation is one of a number of modes of epigenetic gene regulation. Here, we profile the DNA methylome, transcriptome, and global occupancy of histone modifications (H3K4me1, H3K4me3, H3K27me3, and H3K27ac) in a series of mouse embryonic stem cells (mESCs) with varying DNA methylation levels to study the effects of DNA methylation on deposition of histone modifications. We find that genome-wide DNA demethylation alters occupancy of histone modifications at both promoters and enhancers. This is reversed upon remethylation by Dnmt expression. DNA methylation promotes H3K27me3 deposition at bivalent promoters, while opposing H3K27me3 at silent promoters. DNA methylation also reversibly regulates H3K27ac and H3K27me3 at previously identified tissue-specific enhancers. These effects require DNMT catalytic activity. Collectively, our data show that DNA methylation is essential and instructive for deposition of specific histone modifications across regulatory regions, which together influences gene expression patterns in mESCs. PMID:27681438

  16. Methylation patterns of testis-specific genes.

    PubMed Central

    Ariel, M; McCarrey, J; Cedar, H

    1991-01-01

    The methylation patterns of genes expressed in the mouse male germ line have been examined. Int-1, Hox-2.1, and Prm-1, all of which contain 5' CpG islands, were found to be completely unmethylated at many sites in these domains, both in somatic tissues and in sperm DNA. Many other testis-specific genes have a similar structure and are probably also constitutively unmethylated. Pgk-2, a non-CpG-island gene, is similar to somatic tissue-specific genes in that it is highly methylated in nonexpressing cell types but undermethylated in pachytene spermatocytes and round spermatids, where it is actively transcribed. At later stages of spermatogenesis, however, the gene becomes remethylated and thus acquires the full modification pattern in sperm DNA. In all these cases, the sperm DNA that emerges from the testis does not contain any germ-line-specific unmethylated sites and thus carries the methylation pattern typical of that in somatic tissues. Images PMID:2006171

  17. Interleukin-6 promotes tumorigenesis by altering DNA methylation in oral cancer cells.

    PubMed

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

    2011-09-01

    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 long interspersed nuclear element-1 (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 polymerase chain reaction (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.

  18. 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

  19. 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

  20. 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

  1. Establishment of Salvia castanea Diels f. tomentosa Stib. hairy root cultures and the promotion of tanshinone accumulation and gene expression with Ag⁺, methyl jasmonate, and yeast extract elicitation.

    PubMed

    Li, Bo; Wang, Bangqing; Li, Hongyan; Peng, Liang; Ru, Mei; Liang, Zongsuo; Yan, Xijun; Zhu, Yonghong

    2016-01-01

    Salvia castanea Diels f. tomentosa Stib. is an endemic medicinal plant distributed in China, and the notably high content of tanshinone IIA in the root is proven effective for the therapy of heart diseases. Hairy root induction of this Salvia species was inoculated with Agrobacterium rhizogenes strain ATCC 15834. Transformed hairy root was cultured in 6,7-V liquid medium for growth kinetics assessment and elicitation. An S curve was present in the hairy root cultures based on the fresh and dry weights with an interval of 3 days. An optimum concentration of the applied elicitors (15 μM Ag(+), 200 μM methyl jasmonate, and 200 mg l(-1) yeast extract elicitor) benefitted both the growth status and tanshinone accumulation in the hairy root cultures. Tanshinone IIA contents were mostly stimulated 1.8-fold and 1.99-fold compared with the control by Ag(+) and methyl jasmonate elicitation, respectively. Yeast extract dramatically enhanced dry mass accumulation, while it promoted cryptotanshinone content of 2.84 ± 0.33 mg g(-1) dry weight at most in the hairy root cultures. Selected elicitors diversely influenced tanshinone accumulation in the time courses of hairy root cultures within 7 days. Furthermore, transcripts of selected genes in the tanshinone biosynthetic pathway were remarkably upregulated with elicitation. Yeast extract elicitor heightened 13.9-fold of isopentenyl diphosphate isomerase expression level at 12 h, while it increased 16.7-fold of geranylgeranyl diphosphate synthase transcript at 24 h compared with that of the control, which was more effective than Ag(+) and methyl jasmonate. This study provided a convenient hairy root culture system of S. castanea Diels f. tomentosa Stib. for tanshinone production for the first time.

  2. 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

  3. Methylation dependent expression of the mom gene of bacteriophage Mu: deletions downstream from the methylation sites affect expression.

    PubMed Central

    Adley, C C; Bukhari, A I

    1984-01-01

    The expression of the DNA modification gene (mom) of bacteriophage Mu requires the cellular deoxyadenosine methylase (dam) and a transactivation factor from the phage. By hypothesis, the transcription of mom is activated by methylation of three GATC sequences upstream from the mom gene. We have introduced small deletions at a fourth GATC site located about 140 base pairs downstream from the primary methylation region. Some of the deletions severely affect the mom gene expression. We propose from this analysis that (1) some important elements, possibly the promoter, concerned with the expression of mom are located between nucleotides 840 and 880 from the right end of Mu and (2) the mom protein starts with the codon GTG located at position 810. We favor the hypothesis that methylation turns off transcription upstream, thereby allowing the main mom promoter to function. Images PMID:6328425

  4. Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas

    PubMed Central

    Muñoz, Jorge; Inda, María del Mar; Lázcoz, Paula; Zazpe, Idoya; Fan, Xing; Alfaro, Jorge; Tuñón, Teresa; Rey, Juan A.; Castresana, Javier S.

    2012-01-01

    While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations (mutations, homozygous deletions) are prevalent. PMID:22389839

  5. 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

  6. Promoter methylation and age-related downregulation of Klotho in rhesus monkey.

    PubMed

    King, Gwendalyn D; Rosene, Douglas L; Abraham, Carmela R

    2012-12-01

    While overall DNA methylation decreases with age, CpG-rich areas of the genome can become hypermethylated. Hypermethylation near transcription start sites typically decreases gene expression. Klotho (KL) is important in numerous age-associated pathways including insulin/IGF1 and Wnt signaling and naturally decreases with age in brain, heart, and liver across species. Brain tissues from young and old rhesus monkeys were used to determine whether epigenetic modification of the KL promoter underlies age-related decreases in mRNA and protein levels of KL. The KL promoter in genomic DNA from brain white matter did not show evidence of oxidation in vivo but did exhibit an increase in methylation with age. Further analysis identified individual CpG motifs across the region of interest with increased methylation in old animals. In vitro methyl modification of these individual cytosine residues confirmed that methylation of the promoter can decrease gene transcription. These results provide evidence that changes in KL gene expression with age may, at least in part, be the result of epigenetic changes to the 5' regulatory region. PMID:21922250

  7. 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

  8. 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.

  9. MGMT promoter methylation and correlation with protein expression in primary central nervous system lymphoma.

    PubMed

    Toffolatti, L; Scquizzato, E; Cavallin, S; Canal, F; Scarpa, M; Stefani, P M; Gherlinzoni, F; Dei Tos, A P

    2014-11-01

    The O (6)-methylguanine-DNA-methyltransferase (MGMT) gene encodes for a DNA repairing enzyme of which silencing by promoter methylation is involved in brain tumorigenesis. MGMT promoter methylation represents a favorable prognostic factor and has been associated with a better response to alkylating agents in glioma and systemic lymphoma. Primary central nervous system lymphoma (PCNSL) is a rare and aggressive extranodal malignant lymphoma. The current standard of care, based on high-dose methotrexate chemotherapy, has improved prognosis but outcome remains poor for a majority of patients. Therapeutic progress in this field is conditioned by limited biological and molecular knowledge about the disease. Temozolomide has recently emerged as an alternative option for PCNSL treatment. We aimed to analyze the MGMT gene methylation status in a series of 24 PCNSLs, to investigate the relationship between methylation status of the gene and immunohistochemical expression of MGMT protein and to evaluate the possible prognostic significance of these biomarkers. Our results confirm that methylation of the MGMT gene and loss of MGMT protein are frequent events in these lymphomas (54 % of our cases) and suggest that they are gender and age related. MGMT methylation showed high correlation with loss of protein expression (concordance correlation coefficient = -0.49; Fisher exact test: p < 0.01), different from what has been observed in other brain tumors. In the subgroup of ten patients who received high dose chemotherapy, the presence of methylated MGMT promoter (n = 4), seems to be associated with a prolonged overall survival (>60 months in three of four patients). The prognostic significance of these molecular markers in PCNSL needs to be further studied in groups of patients treated in a homogeneous way.

  10. Age-associated methylation change of TAP1 promoter in piglet.

    PubMed

    Dong, Wenhua; Yin, Xuemei; Sun, Li; Wang, Jing; Sun, Shouyong; Zhu, Guoqiang; Wu, Shenglong; Bao, Wenbin

    2015-11-15

    Diarrhea and edematous disease are two major causes of mortality in postweaning piglets. These conditions lead to huge economic losses in the swine industry. Escherichia coli F18 is the primary causative agent of these two diseases. Transported associated with antigen processing (TAP) plays an important role in the immune response and the TAP1 gene could be an effective anti-E. coli F18 molecular marker in pigs. The aim of this study was to determine the correlation between TAP1 gene promoter CpG island methylation status and mRNA expression in piglets. In this study, bisulfite sequencing PCR (BSP) was used to detect the methylation status of the TAP1 gene promoter CpG islands and fluorescence quantitative PCR was used to detect TAP1 expression in the jejunum of Sutai piglets from birth to weaning age. The fragment of the TAP1 gene promoter region under investigation has no mutation, has 13 putative transcription factor binding sites containing 19 CpG sites, and may be important for regulation of gene expression. With increasing age, the overall methylation levels decreased, while the TAP1 expression levels increased, indicating a negative correlation between TAP1 expression and promoter methylation levels. Variance analysis showed significant differences in the methylation status of CpG_4, CpG_13 and CpG_15 among the different age groups (P<0.05). Our data indicate that TAP1 expression is increased by demethylation of promoter CpG islands, with CpG_4, CpG_13 and CpG_15 implicated as the critical regulatory sites. PMID:26169022

  11. 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

  12. TET2 Promoter DNA Methylation and Expression in Childhood Acute Lymphoblastic Leukemia.

    PubMed

    Bahari, Gholamreza; Hashemi, Mohammad; Naderi, Majid; Taheri, Mohsen

    2016-01-01

    The ten-eleven-translocation-2 (TET2) gene is a novel tumor suppressor gene involved in several hematological malignancies of myeloid and lymphoid origin. Besides loss-of-function mutations and deletions, hypermethylation of the CpG island at the TET2 promoter has been found in human cancers. The TET2 encoded protein regulates DNA methylation. The present study aimed to examine DNA promoter methylation of TET2 in 100 childhood acute lymphoblastic leukemia (ALL) cases and 120 healthy children in southeast Iran. In addition, mRNA expression levels were assessed in 30 new cases of ALL and 32 controls. Our findings indicated that promoter methylation of TET2 significantly increases the risk of ALL (OR=2.60, 95% CI=1.31-5.12, p=0.0060) in comparison with absent methylation. Furthermore, the TET2 gene was significantly downregulated in childhood ALL compared to healthy children (p=0.0235). The results revealed that hypermethylation and downregulation of TET2 gene may play a role in predisposition to childhood ALL. Further studies with larger sample sizes and different ethnicities are needed to confirm our findings. PMID:27644645

  13. Albumin extinction without methylation of its gene.

    PubMed

    Ott, M O; Sperling, L; Weiss, M C

    1984-03-01

    In earlier work we identified at the 5' end of the rat albumin gene an Msp I site whose undermethylation appears to be necessary but not sufficient for stable expression of the gene in rat hepatoma cells. Here, we ask whether the block to expression of albumin production, which occurs when rat hepatoma cells are hybridized with cells that do not produce the protein, could be the result of de novo methylation of this site. In two types of somatic hybrids, rat hepatoma-mouse L cell fibroblasts, and rat hepatoma-dedifferentiated variant rat hepatoma cells, extinction occurs and is maintained during the first 5-15 generations after fusion. During this time the Msp I site of the now inactive rat albumin gene remained unmethylated.

  14. [Aberrant promoter methylation as biomarker for molecular cytological diagnosis of lung cancer].

    PubMed

    Grote, H J

    2006-01-01

    Aberrant promoter methylation represents a main mechanism of tumor suppressor gene inactivation and may serve as a new source for biomarker discovery. This study investigated its applicability as a molecular tool for lung cancer diagnostics on bronchial aspirates. A methylation assay was developed applying a quantitative methylation specific real-time PCR (QMSP). A total of 552 patients with the differential diagnosis of lung cancer were investigated. The QMSP findings on bronchial aspirates were compared with the methylation status of respective genes investigated in microdissected tumor tissues (QMSP, cloning and sequencing of promoter regions after bisulfite conversion). Among the genes tested a marker panel consisting of APC, p16(INK4a) and RASSF1A proved to be the best suited for lung cancer diagnostics. This panel allowed for a correct diagnosis of lung cancer in cases with an ambiguous or false negative conventional cytology. In a cohort study on 247 patients, the combination of histology (sensitivity 59 %), cytology (sensitivity 44 %) and QMSP-assay (sensitivity 53 %) raised the sensitivity of a single bronchoscopy for the diagnosis of lung cancer up to 81%. The methylation assay yielded its major diagnostic surplus with respect to peripheral tumors representing 59 % of all primaries detected. In patients without antecedent lung cancer its specificity considering malignancy was >99 %. Therefore, the QMSP-assay is a promising technique which could enhance the sensitivity and diagnostic impact of conventional cytology. The assay is applicable to residual material of regular diagnostic cytology even in retrospect.

  15. High-temperature effect on genes engaged in DNA methylation and affected by DNA methylation in Arabidopsis.

    PubMed

    Naydenov, Mladen; Baev, Vesselin; Apostolova, Elena; Gospodinova, Nadezhda; Sablok, Gaurav; Gozmanova, Mariyana; Yahubyan, Galina

    2015-02-01

    Along with its essential role in the maintenance of genome integrity, DNA methylation takes part in regulation of genes which are important for plant development and stress response. In plants, DNA methylation process can be directed by small RNAs in process known as RNA-directed DNA methylation (RdDM) involving two plant-specific RNA polymerases - PolIV and PolV. The aim of the present study was to investigate the effect of heat stress on the expression of genes encoding key players in DNA methylation - DNA methyltransferase (MET1, CMT3, and DRM2), the largest subunits of PoIIV and PolV (NRPD1 and NRPE1 respectively) and the DNA demethylase ROS1. We also examined the high-temperature effect on two protein-coding genes - At3g50770 and At5g43260 whose promoters contain transposon insertions and are affected by DNA-methylation, as well as on the AtSN1, a SINE-like retrotransposon. To assess the involvement of PolIV and PolV in heat stress response, the promoter methylation status and transcript levels of these genes were compared between wild type and double mutant lacking NRPD1 and NRPE1. The results demonstrate coordinated up-regulation of the DRM2, NRPD1 and NRPE1 in response to high temperature and suggest that PolIV and/or PolV might be required for the induction of DRM2 expression under heat stress. The ROS1 expression was confirmed to be suppressed in the mutant lacking active PolIV and PolV that might be a consequence of abolished DNA methylation. The increased expression of At3g50770 in response to elevated temperature correlated with reduced promoter DNA methylation, while the stress response of At5g43260 did not show inverse correlation between promoter methylation and gene expression. Our results also imply that PolIV and/or PolV could regulate gene expression under stress conditions not only through RdDM but also by acting in other regulatory processes.

  16. 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

  17. 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.

  18. 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.

  19. Promoter-restricted H3 Lys 4 di-methylation is an epigenetic mark for monoallelic expression.

    PubMed

    Rougeulle, Claire; Navarro, Pablo; Avner, Philip

    2003-12-15

    Methylation of histone tails has been implicated in long-term epigenetic memory. Methylated H3 Lys 4 (K4) is a generally conserved mark for euchromatic, transcriptionally active regions, although the effect of this modification is likely also to depend on its distribution both within the euchromatic region and more specifically within a given gene. Here we describe a profile of H3K4 di-methylation that is specific for monoallelically expressed genes. Both X-linked genes subject to X-inactivation and autosomal imprinted genes have di-methylated H3K4 restricted to their promoter regions. In contrast, high levels of H3K4 di-methylation are found in both promoters and exonic parts of autosomal genes and of X-linked genes that escape X-inactivation. We suggest that this pattern of promoter restricted H3 Lys 4 di-methylation, already present in totipotent cells, is causally related to the long-term programming of allelic expression and provides an epigenetic mark for monoallelically expressed genes.

  20. Ambient PM exposure and DNA methylation in tumor suppressor genes: a cross-sectional study

    PubMed Central

    2011-01-01

    Exposure to ambient air particles matter (PM) has been associated with increased risk of lung cancer. Aberrant tumor suppressor gene promoter methylation has emerged as a promising biomarker for cancers, including lung cancer. Whether exposure to PM is associated with peripheral blood leukocyte (PBL) DNA methylation in tumor suppressor genes has not been evaluated. In 63 male healthy steel workers with well-characterized exposure to metal-rich particles nearby Brescia, Italy, we evaluated whether exposure to PM and metal components was associated with PBL DNA methylation in 4 tumor suppressor genes (i.e., APC, p16, p53 and RASSF1A). Blood samples were obtained on the 1st (baseline) and 4th day (post-exposure) of the same work week and DNA methylation was measured using pyrosequencing. A linear mixed model was used to examine the correlations of the exposure with promoter methylation levels. Mean promoter DNA methylation levels of APC or p16 were significantly higher in post-exposure samples compared to that in baseline samples (p-values = 0.005 for APC, and p-value = 0.006 for p16). By contrast, the mean levels of p53 or RASSF1A promoter methylation was decreased in post-exposure samples compared to that in baseline samples (p-value = 0.015 for p53; and p-value < 0.001 for RASSF1A). In post-exposure samples, APC methylation was positively associated with PM10 (β = 0.27, 95% CI: 0.13-0.40), and PM1 (β = 0.23, 95% CI: 0.09-0.38). In summary, ambient PM exposure was associated with PBL DNA methylation levels of tumor suppressor genes of APC, p16, p53 and RASSF1A, suggesting that such methylation alterations may reflect processes related to PM-induced lung carcinogenesis. PMID:21878113

  1. DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues.

    PubMed

    Kont, Vivian; Murumägi, Astrid; Tykocinski, Lars-Oliver; Kinkel, Sarah A; Webster, Kylie E; Kisand, Kai; Tserel, Liina; Pihlap, Maire; Ströbel, Philipp; Scott, Hamish S; Marx, Alexander; Kyewski, Bruno; Peterson, Pärt

    2011-12-01

    Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs.

  2. 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

  3. Genome-wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with genes and centromeres in rice.

    PubMed

    Yan, Huihuang; Kikuchi, Shinji; Neumann, Pavel; Zhang, Wenli; Wu, Yufeng; Chen, Feng; Jiang, Jiming

    2010-08-01

    We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3-associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.

  4. De novo methylation, long-term promoter silencing, methylation patterns in the human genome, and consequences of foreign DNA insertion.

    PubMed

    Doerfler, W

    2006-01-01

    This chapter presents a personal account of the work on DNA methylation in viral and mammalian systems performed in the author's laboratory in the course of the past 30 years. The text does not attempt to give a complete and meticulous account of the work accomplished in many other laboratories; in that sense it is not a review of the field in a conventional sense. Since the author is also one of the editors of this series of Current Topics in Immunology and Microbiology on DNA methylation, to which contributions by many of our colleagues in this field have been invited, the author's conscience is alleviated that he has not cited many of the relevant and excellent reports by others. The choice of viral model systems in molecular biology is well founded. Over many decades, viruses have proved their invaluable and pioneering role as tools in molecular genetics. When our interest turned to the demonstration of genome-wide patterns of DNA methylation, we focused mainly on the human genome. The following topics in DNA methylation will be treated in detail: (1) The de novo methylation of integrated foreign genomes; (2) the long-term gene silencing effect of sequence-specific promoter methylation and its reversal; (3) the properties and specificity of patterns of DNA methylation in the human genome and their possible relations to pathogenesis; (4) the long-range global effects on cellular DNA methylation and transcriptional profiles as a consequence of foreign DNA insertion into an established genome; (5) the patterns of DNA methylation can be considered part of a cellular defense mechanism against foreign or repetitive DNA; which role has food-ingested DNA played in the elaboration of this mechanism? The interest in problems related to DNA methylation has spread-like the mechanism itself-into many neighboring fields. The nature of the transcriptional programs orchestrating embryonal and fetal development, chromatin structure, genetic imprinting, genetic disease, X

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

    PubMed Central

    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

  6. Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9.

    PubMed

    Mutskov, Vesco; Felsenfeld, Gary

    2004-01-14

    Transgenes stably integrated into cells or animals in many cases are silenced rapidly, probably under the influence of surrounding endogenous condensed chromatin. This gene silencing correlates with repressed chromatin structure marked by histone hypoacetylation, loss of methylation at H3 lysine 4, increase of histone H3 lysine 9 methylation as well as CpG DNA methylation at the promoter. However, the order and the timing of these modifications and their impact on transcription inactivation are less well understood. To determine the temporal order of these events, we examined a model system consisting of a transgenic cassette stably integrated in chicken erythroid cells. We found that histone H3 and H4 hypoacetylation and loss of methylation at H3 lysine 4 all occurred during the same window of time as transgene inactivation in both multicopy and low-copy-number lines. These results indicate that these histone modifications were the primary events in gene silencing. We show that the kinetics of silencing exclude histone H3 K9 and promoter DNA methylation as the primary causative events in our transgene system. PMID:14685282

  7. 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...

  8. 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

  9. Loss of expression and promoter methylation of SLIT2 are associated with sessile serrated adenoma formation.

    PubMed

    Beggs, Andrew D; Jones, Angela; Shepherd, Neil; Arnaout, Abed; Finlayson, Caroline; Abulafi, A Muti; Morton, Dion G; Matthews, Glenn M; Hodgson, Shirley V; Tomlinson, Ian P M

    2013-05-01

    Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing) and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1-4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson "two hit" hypothesis.

  10. EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

    PubMed Central

    Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A.; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forné, Thierry; Feil, Robert; Weber, Michael

    2016-01-01

    The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

  11. 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.

  12. 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

  13. Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression

    PubMed Central

    Tian, Kegui; Wang, Yuezeng; Huang, Yu; Sun, Boqiao; Li, Yuxin; Xu, Haopeng

    2008-01-01

    Background Previous results showed that over-expression of the WTH3 gene in MDR cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the WTH3 gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. WTH3 was also found to be directly targeted and up regulated by the p53 gene. Furthermore, over expression of the WTH3 gene promoted the apoptotic phenotype in various host cells. Methods To further confirm WTH3's drug resistant related characteristics, we recently employed the small hairpin RNA (shRNA) strategy to knockdown its expression in HEK293 cells. In addition, since the WTH3 promoter's p53-binding site was located in a CpG island that was targeted by methylation, we were interested in testing the possible effect this epigenetic modification had on the p53 transcription factor relative to WTH3 expression. To do so, the in vitro methylation method was utilized to examine the p53 transgene's influence on either the methylated or non-methylated WTH3 promoter. Results The results generated from the gene knockdown strategy showed that reduction of WTH3 expression increased MDR1 expression and elevated resistance to Doxorubicin as compared to the original control cells. Data produced from the methylation studies demonstrated that DNA methylation adversely affected the positive impact of p53 on WTH3 promoter activity. Conclusion Taken together, our studies provided further evidence that WTH3 played an important role in MDR development and revealed one of its transcription regulatory mechanisms, DNA methylation, which antagonized p53's positive impact on WTH3 expression. PMID:18992151

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines1*

    PubMed Central

    Wang, Yipeng; Yu, Qiuju; Cho, Ann H; Rondeau, Gaelle; Welsh, John; Adamson, Eileen; Mercola, Dan; McClelland, Michael

    2005-01-01

    Abstract DNA methylation and copy number in the genomes of three immortalized prostate epithelial and five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, and PC3M-LN4) were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme HpaII, followed by linker ligation, polymerase chain reaction (PCR) amplification, labeling, and hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5%) showed differential hybridization between immortalized prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, and TSPY) previously observed in prostate cancer and 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, and WIT-1). The majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, and GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors. PMID:16207477

  20. Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in oligodendroglial tumors.

    PubMed

    Kuo, Lu-Ting; Lu, Hsueh-Yi; Lee, Chien-Chang; Tsai, Jui-Chang; Lai, Hong-Shiee; Tseng, Ham-Min; Kuo, Meng-Fai; Tu, Yong-Kwang

    2016-08-01

    Aberrant methylation has been associated with transcriptional inactivation of tumor-related genes in a wide spectrum of human neoplasms. The influence of DNA methylation in oligodendroglial tumors is not fully understood. Genomic DNA was isolated from 61 oligodendroglial tumors for analysis of methylation using methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). We correlated methylation status with clinicopathological findings and outcome. The genes found to be most frequently methylated in oligodendroglial tumors were RASSF1A (80.3%), CASP8 (70.5%), and CDKN2A (52.5%). Kaplan-Meier survival curve analysis demonstrated longer duration of progression-free survival in patients with 19q loss, aged less than 38 years, and with a proliferative index of less than 5%. Methylation of the ESR1 promoter is significantly associated with shorter duration of overall survival and progression-free survival, and that methylation of IGSF4 and RASSF1A is significantly associated with shorter duration of progression-free survival. However, none of the methylation status of ESR1, IGSF4, and RASSF1A was of prognostic value for survival in a multivariate Cox model. A number of novel and interesting epigenetic alterations were identified in this study. The findings highlight the importance of methylation profiles in oligodendroglial tumors and their possible involvement in tumorigenesis. PMID:27367901

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. Association of genomic instability, and the methylation status of imprinted genes and mismatch-repair genes, with neural tube defects.

    PubMed

    Liu, Zhuo; Wang, Zhigang; Li, Yuanyuan; Ouyang, Shengrong; Chang, Huibo; Zhang, Ting; Zheng, Xiaoying; Wu, Jianxin

    2012-05-01

    We studied the genomic instability and methylation status of the mismatch-repair (MMR) genes hMLH1 and hMSH2, and the imprinted genes H19/IGF2, in fetuses with neural tube defects (NTDs) to explore the pathogenesis of the disease. Microsatellite instability (MSI) was observed in 23 of 50 NTD patients. Five NTD patients showed high-degree MSI (MSI-H) and 18 showed low-degree MSI (MSI-L). The frequencies of mutated microsatellite loci were 3/50 (6%) for BatT-25, 10/50 (20%) for Bat-26, 3/50 (6%) for Bat34C4, 6/50 (12%) for D2S123, 4/50 (8%) for D2S119, and 3/50 (6%) for D3S1611. The promoter regions of the hMLH1 and hMSH2 genes were unmethylated in NTD patients, as determined by methylation-specific PCR. The hMLH1 and hMSH2 promoter methylation patterns, the methylation levels of H19 DMR1, and IGF2 DMR0 were detected by bisulfite sequencing PCR, sub-cloning, and sequencing. The hMSH2 promoter sequence was unmethylated, and the hMLH1 promoter showed a specific methylation pattern at two CpG sites. The methylation levels of H19 DMR1 in the NTD and control groups are 73.3% ± 15.9 and 58.3% ± 11.2, respectively. The methylation level of the NTD group was higher than that of the control group (Student's t-test, P<0.05). There is no significant difference in IGF2 DMR0 methylation level between the two groups. All of the results presented here suggest that genomic instability, the MMR system, and hyper-methylation of the H19 DMR1 may be correlated with the occurrence of NTDs.

  6. DNA methylation is associated with transcription of Snail and Slug genes

    PubMed Central

    Chen, Ying; Wang, Kai; Qian, Chao-Nan; Leach, Richard

    2012-01-01

    Snail and Slug play critical roles in the epithelial to mesenchymal transition (EMT), the mesenchymal to epithelial transition (MET) and in the maintenance of mesenchymal morphology. In this research, we investigated the correlation of DNA methylation with the transcriptional level of these two genes during the EMT/MET process. First, we used several cell lines associated with EMT/MET processes of induced pluripotent stem cell generation and differentiation, trophoblast invasion, as well as cancer progression to examine the association between DNA methylation and transcription levels of these two genes. We found an inverse correlation between DNA methylation of first intron regions and transcription levels of Snail and Slug genes in these EMT/METs. To further verify the results, we treated two trophoblast cell line BeWo and HTR8/SVneo and one induced pluripotent stem cell line with 5-aza-2′-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferase, which caused increased expression of these two genes. Lastly, we cloned the promoters of both Snail and Slug into pGL3-Basic vector, after in vitro DNA methylation and transfection into IMR90 and HTR8/SVneo cells; we observed the significant reduction of their promoter activity due to DNA methylation. In summary, based on these results, DNA methylation is one of the molecular mechanisms regulating Snail and Slug genes during EMT/MET process. PMID:23261445

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

    PubMed

    Singhal, Sandeep K; Usmani, Nawaid; Michiels, Stefan; Metzger-Filho, Otto; Saini, Kamal S; Kovalchuk, Olga; Parliament, Matthew

    2016-01-19

    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.

  8. 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

  9. Exercise training alters DNA methylation patterns in genes related to muscle growth and differentiation in mice.

    PubMed

    Kanzleiter, Timo; Jähnert, Markus; Schulze, Gunnar; Selbig, Joachim; Hallahan, Nicole; Schwenk, Robert Wolfgang; Schürmann, Annette

    2015-05-15

    The adaptive response of skeletal muscle to exercise training is tightly controlled and therefore requires transcriptional regulation. DNA methylation is an epigenetic mechanism known to modulate gene expression, but its contribution to exercise-induced adaptations in skeletal muscle is not well studied. Here, we describe a genome-wide analysis of DNA methylation in muscle of trained mice (n = 3). Compared with sedentary controls, 2,762 genes exhibited differentially methylated CpGs (P < 0.05, meth diff >5%, coverage >10) in their putative promoter regions. Alignment with gene expression data (n = 6) revealed 200 genes with a negative correlation between methylation and expression changes in response to exercise training. The majority of these genes were related to muscle growth and differentiation, and a minor fraction involved in metabolic regulation. Among the candidates were genes that regulate the expression of myogenic regulatory factors (Plexin A2) as well as genes that participate in muscle hypertrophy (Igfbp4) and motor neuron innervation (Dok7). Interestingly, a transcription factor binding site enrichment study discovered significantly enriched occurrence of CpG methylation in the binding sites of the myogenic regulatory factors MyoD and myogenin. These findings suggest that DNA methylation is involved in the regulation of muscle adaptation to regular exercise training.

  10. 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

  11. DNA methylation mediated control of gene expression is critical for development of crown gall tumors.

    PubMed

    Gohlke, Jochen; Scholz, Claus-Juergen; Kneitz, Susanne; Weber, Dana; Fuchs, Joerg; Hedrich, Rainer; Deeken, Rosalia

    2013-01-01

    Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA-encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene

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

    PubMed

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

    2015-04-01

    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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  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. Applicability of the methylated CpG sites of paired box 5 (PAX5) promoter for prediction the prognosis of gastric cancer

    PubMed Central

    Deng, Jingyu; Liang, Han; Zhang, Rupeng; Dong, Qiuping; Hou, Yachao; Yu, Jun; Fan, Daiming; Hao, Xishan

    2014-01-01

    Paired box gene 5 (PAX5), a member of the paired box gene family, is involved in control of organ development and tissue differentiation. In previous study, PAX5 promoter methylation was found in gastric cancer (GC) cells and tissues. At present study, we found that the inconsistently methylated levels of PAX5 promoter were identified in the different GC tissues. The methylated CpG site count and the methylated statuses of four CpG sites (-236, -183, -162, and -152) were significantly associated with the survival of 460 GC patients, respectively. Ultimately, the methylated CpG -236 was the optimal prognostic predictor of patients identified by using the Cox regression with AIC value calculation. These findings indicated that the methylated CpG -236 of PAX5 promoter has the potential applicability for clinical evaluation the prognosis of GC. PMID:25277182

  1. 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

  2. Promoter methylation and expression changes of BRCA1 in cancerous tissues of patients with sporadic breast cancer

    PubMed Central

    LI, QIUYUN; WEI, WEI; JIANG, YI; YANG, HUAWEI; LIU, JIANLUN

    2015-01-01

    BRCA1 is a susceptibility gene that has a genetic predisposition for breast cancer. BRCA1 gene mutation is closely associated with familial hereditary breast cancer, but the BRCA1 gene mutation is rarely found in sporadic breast cancer. According to previous studies, decreased expression of BRCA1 was detected in certain types of sporadic breast cancer. Aberrant methylation of DNA promoter CpG islands is one of the mechanisms by which tumor suppressor gene expression and function is lost. The aim of the present study was to investigate BRCA1 gene expression, methylation status and clinical significance in sporadic types of breast cancer. Quantitative polymerase chain reaction (PCR) and bisulfite sequencing PCR were respectively used to detect expression differences of BRCA1 mRNA and BRCA1 methylation in the 49 cancerous and paired non-cancerous samples from patients with breast cancer. The associations of BRCA1 expression and methylation status with the clinicopathologic characteristics were analysed. BRCA1 mRNA expression levels in the 49 breast cancer tissues were lower than those in the paired non-cancerous tissues. There was a significant statistical difference (P=0.001). BRCA1 mRNA expression was not associated with the main clinicopathologic characteristics. Frequency of the BRCA1 promoter methylation in the breast cancerous tissues was significantly higher than that in the non-cancerous tissues (P=0.007); BRCA1 gene methylation status was negatively correlated with mRNA expression (P=0.029); and BRCA1 methylation exhibited no association with all clinicopathological features. DNA promoter hypermethylation may be the potential mechanism accounting for BRCA1 expression silence in part of sporadic types of breast cancer. Some patients with hypermethylated BRCA1 may display favorable clinicopathological status. PMID:25789047

  3. Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation

    PubMed Central

    Nguyen, Carvell T.; Gonzales, Felicidad A.; Jones, Peter A.

    2001-01-01

    Silencing of tumor-suppressor genes by hypermethylation of promoter CpG islands is well documented in human cancer and may be mediated by methyl-CpG-binding proteins, like MeCP2, that are associated in vivo with chromatin modifiers and transcriptional repressors. However, the exact dynamic between methylation and chromatin structure in the regulation of gene expression is not well understood. In this study, we have analyzed the methylation status and chromatin structure of three CpG islands in the p14(ARF)/p16(INK4A) locus in a series of normal and cancer cell lines using methylation-sensitive digestion, MspI accessibility in intact nuclei and chromatin immunoprecipitation (ChIP) assays. We demonstrate the existence of an altered chromatin structure associated with the silencing of tumor-suppressor genes in human cancer cell lines involving CpG island methylation, chromatin condensation, histone deacetylation and MeCP2 binding. The data showed that MeCP2 could bind to methylated CpG islands in both promoters and exons; MeCP2 does not interfere with transcription when bound at an exon, suggesting a more generalized role for the protein beyond transcriptional repression. In the absence of methylation, it is demonstrated that CpG islands located in promoters versus exons display marked differences in the levels of acetylation of associated histone H3, suggesting that chromatin remodeling can be achieved by methylation-independent processes and perhaps explaining why non-promoter CpG islands are more susceptible to de novo methylation than promoter islands. PMID:11713309

  4. G9a orchestrates PCL3 and KDM7A to promote histone H3K27 methylation

    PubMed Central

    Pan, Mei-Ren; Hsu, Ming-Chuan; Chen, Li-Tzong; Hung, Wen-Chun

    2015-01-01

    Methylation of histone H3-lysine 9 (H3K9) and H3K27 by the methyltransferase G9a and polycomb repressive complex 2 (PRC2) inhibits transcription of target genes. A crosstalk between G9a and PRC2 via direct physical interaction has been shown recently. Here, we demonstrate an alternative mechanism by which G9a promotes H3K27 methylation. Overexpression of G9a increases both H3K9 and H3K27 methylation, reduces E-cadherin expression, and induces epithelial-mesenchymal transition in PANC-1 pancreatic cancer cells. Conversely, the depletion of G9a or ectopic expression of methyltransferase-dead G9a in G9a-overexpressing gemcitabine-resistant PANC-1-R cells exhibits opposite effects. G9a promotes H3K27 methylation of the E-cadherin promoter by upregulating PCL3 to increase PRC2 promoter recruitment and by downregulating the H3K27 demethylase KDM7A to silence E-cadherin gene. The depletion of PCL3 or overexpression of KDM7A elevated expression of E-cadherin in PANC-1-R cells while ectopic expression of PCL3 or knockdown of KDM7A downregulated E-cadherin in PANC-1 cells. Collectively, we provide evidence that G9a orchestrates the dynamic balance within histone-modifying enzymes to regulate H3K27 methylation and gene expression. PMID:26688070

  5. Association between P16INK4a Promoter Methylation and Ovarian Cancer: A Meta-Analysis of 12 Published Studies

    PubMed Central

    Niu, Xun; Shi, Hao; Zhong, Yi

    2016-01-01

    Background Ovarian cancer is the primary cause of death in women diagnosed with gynecological malignancies worldwide. Absence of early symptoms prevents prompt diagnosis or successful therapeutic intervention. P16INK4a is a well-known tumor suppressor gene (TSG). Aberrant methylation of TSG promoter is an important epigenetic silencing mechanism leading to ovarian cancer progression. Studies have reported differences in methylation frequencies of the p16INK4a promoter between ovarian cancer and the corresponding control group. However, the association between p16INK4a promoter methylation and ovarian cancer remains unclear and controversial. Therefore, a meta-analysis was conducted to clarify the relationship between p16INK4a promoter methylation and ovarian cancer. Methods PubMed, Web of Science, EMBASE and CNKI were searched to identify eligible studies for the evaluation of the association between p16INK4a promoter methylation and ovarian cancer. Odds ratio (ORs) and 95% confidence intervals (95%CI) were calculated to determine the strength of association between p16INK4a promoter methylation and ovarian cancer. Results A total of 612 ovarian cancer patients and 289 controls from 12 eligible studies were included in the meta-analysis. Overall, a significant association was observed between p16INK4a methylation status and ovarian cancer risk using a fixed-effects model (OR = 2.02, 95% CI = 1.39–2.94). Conclusion The results of our meta-analysis show that aberrant methylation of p16INK4a promoter was significantly associated with ovarian cancer. It may represent a promising molecular marker to monitor the disease and provides new insights into the treatment of human ovarian cancer. PMID:27648827

  6. 35S Promoter Methylation in Kanamycin-Resistant Kalanchoe (Kalanchoe pinnata L.) Plants Expressing the Antimicrobial Peptide Cecropin P1 Transgene.

    PubMed

    Shevchuk, T V; Zakharchenko, N S; Tarlachkov, S V; Furs, O V; Dyachenko, O V; Buryanov, Y I

    2016-09-01

    Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases. PMID:27682168

  7. DNA methylation and nucleosome occupancy regulate the cancer germline antigen gene MAGEA11

    PubMed Central

    James, Smitha R; Cedeno, Carlos D; Sharma, Ashok; Zhang, Wa; Mohler, James L; Odunsi, Kunle; Wilson, Elizabeth M; Karpf, Adam R

    2013-01-01

    MAGEA11 is a cancer germline (CG) antigen and androgen receptor co-activator. Its expression in cancers other than prostate, and its mechanism of activation, has not been reported. In silico analyses reveal that MAGEA11 is frequently expressed in human cancers, is increased during tumor progression, and correlates with poor prognosis and survival. In prostate and epithelial ovarian cancers (EOC), MAGEA11 expression was associated with promoter and global DNA hypomethylation, and with activation of other CG genes. Pharmacological or genetic inhibition of DNA methyltransferases (DNMTs) and/or histone deacetylases (HDACs) activated MAGEA11 in a cell line specific manner. MAGEA11 promoter activity was directly repressed by DNA methylation, and partially depended on Sp1, as pharmacological or genetic targeting of Sp1 reduced MAGEA11 promoter activity and endogenous gene expression. Importantly, DNA methylation regulated nucleosome occupancy specifically at the -1 positioned nucleosome of MAGEA11. Methylation of a single Ets site near the transcriptional start site (TSS) correlated with -1 nucleosome occupancy and, by itself, strongly repressed MAGEA11 promoter activity. Thus, DNA methylation regulates nucleosome occupancy at MAGEA11, and this appears to function cooperatively with sequence-specific transcription factors to regulate gene expression. MAGEA11 regulation is highly instructive for understanding mechanisms regulating CG antigen genes in human cancer. PMID:23839233

  8. Arabidopsis RPT2a, 19S proteasome subunit, regulates gene silencing via DNA methylation.

    PubMed

    Sako, Kaori; Maki, Yuko; Kanai, Tomoyuki; Kato, Eriko; Maekawa, Shugo; Yasuda, Shigetaka; Sato, Takeo; Watahiki, Masaaki K; Yamaguchi, Junji

    2012-01-01

    The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation. PMID:22615900

  9. Monozygotic twins discordant for constitutive BRCA1 promoter methylation, childhood cancer and secondary cancer.

    PubMed

    Galetzka, Danuta; Hansmann, Tamara; El Hajj, Nady; Weis, Eva; Irmscher, Benjamin; Ludwig, Marco; Schneider-Rätzke, Brigitte; Kohlschmidt, Nicolai; Beyer, Vera; Bartsch, Oliver; Zechner, Ulrich; Spix, Claudia; Haaf, Thomas

    2012-01-01

    We describe monozygotic twins discordant for childhood leukemia and secondary thyroid carcinoma. We used bisulfite pyrosequencing to compare the constitutive promoter methylation of BRCA1 and several other tumor suppressor genes in primary fibroblasts. The affected twin displayed an increased BRCA1 methylation (12%), compared with her sister (3%). Subsequent bisulfite plasmid sequencing demonstrated that 13% (6 of 47) BRCA1 alleles were fully methylated in the affected twin, whereas her sister displayed only single CpG errors without functional implications. This between-twin methylation difference was also found in irradiated fibroblasts and untreated saliva cells. The BRCA1 epimutation may have originated by an early somatic event in the affected twin: approximately 25% of her body cells derived from different embryonic cell lineages carry one epigenetically inactivated BRCA1 allele. This epimutation was associated with reduced basal protein levels and a higher induction of BRCA1 after DNA damage. In addition, we performed a genome-wide microarray analysis of both sisters and found several copy number variations, i.e., heterozygous deletion and reduced expression of the RSPO3 gene in the affected twin. This monozygotic twin pair represents an impressive example of epigenetic somatic mosaicism, suggesting a role for constitutive epimutations, maybe along with de novo genetic alterations in recurrent tumor development.

  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. 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

  12. 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.

  13. Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

    PubMed

    Balgkouranidou, I; Matthaios, D; Karayiannakis, A; Bolanaki, H; Michailidis, P; Xenidis, N; Amarantidis, K; Chelis, L; Trypsianis, G; Chatzaki, E; Lianidou, E S; Kakolyris, S

    2015-08-01

    Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p < 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p = 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. PMID:26073472

  14. Differential methylation of the gene encoding myo-inositol 3-phosphate synthase (Isyna1) in rat tissues

    PubMed Central

    Seelan, Ratnam S; Pisano, M Michele; Greene, Robert M; Casanova, Manuel F; Parthasarathy, Ranga N

    2011-01-01

    Aims Myo-inositol levels are frequently altered in several brain disorders. Myo-inositol 3-phosphate synthase, encoded by the Isyna1 gene, catalyzes the synthesis of myo-inositol in cells. Very little is known about the mechanisms regulating Isyna1 expression in brain and other tissues. In this study, we have examined the role of DNA methylation in regulating Isyna1 expression in rat tissues. Materials & methods Transfection analysis using in vitro methylated promoter constructs, Southern blot analysis of genomic DNA from various tissues digested with a methylation-sensitive enzyme and CpG methylation profiling of genomic DNA from different tissues were used to determine differential methylation of Isyna1 in tissues. Transfection analysis using plasmids harboring mutated CpG residues in the 5’-upstream region of Isyna1 was used to identify critical residues mediating promoter activity. Results The −700 bp to −500 bp region (region 1) of Isyna1 exhibited increased methylation in brain cortex compared with other tissues; it also exhibited sex-specific methylation differences between matched male and female brain cortices. Mutation analysis identified one CpG residue in region 1 necessary for promoter activity in neuronal cells. A tissue-specific differentially methylated region (T-DMR) was found to be localized between +450 bp and +650 bp (region 3). This DMR was comparatively highly methylated in spleen, moderately methylated in brain cortex and poorly methylated in testis, consistent with mRNA levels observed in these tissues. Conclusion Rat Isyna1 exhibits tissue-specific DNA methylation. Brain DNA was uniquely methylated in the 5’-upstream region and displayed gender specificity. A T-DMR was identified within the gene body of Isyna1. These findings suggest that Isyna1 is regulated, in part, by DNA methylation and that significant alterations in methylation patterns during development could have a major impact on inositol phosphate synthase expression in

  15. DNA Methylation and Histone Modifications Are Associated with Repression of the Inhibin α Promoter in the Rat Corpus Luteum

    PubMed Central

    Meldi, Kristen M.; Gaconnet, Georgia A.

    2012-01-01

    The transition from follicle to corpus luteum after ovulation is associated with profound morphological and functional changes and is accompanied by corresponding changes in gene expression. The gene encoding the α subunit of the dimeric reproductive hormone inhibin is maximally expressed in the granulosa cells of the preovulatory follicle, is rapidly repressed by the ovulatory LH surge, and is expressed at only very low levels in the corpus luteum. Although previous studies have identified transient repressors of inhibin α gene transcription, little is known about how this repression is maintained in the corpus luteum. This study examines the role of epigenetic changes, including DNA methylation and histone modification, in silencing of inhibin α gene expression. Bisulfite sequencing reveals that methylation of the inhibin α proximal promoter is low in preovulatory and ovulatory follicles but is elevated in the corpus luteum. Increased methylation during luteinization is observed within the cAMP response element in the promoter, and EMSA demonstrate that methylation of this site inhibits cAMP response element binding protein binding in vitro. Chromatin immunoprecipitation reveals that repressive histone marks H3K9 and H3K27 trimethylation are increased on the inhibin α promoter in primary luteal cells, whereas the activation mark H3K4 trimethylation is decreased. The changes in histone modification precede the alterations in DNA methylation, suggesting that they facilitate the recruitment of DNA methyltransferases. We show that the DNA methyltransferase DNMT3a is present in the ovary and in luteal cells when the inhibin α promoter becomes methylated and observe recruitment of DNMT3a to the inhibin promoter during luteinization. PMID:22865368

  16. DNA Methylation Levels of Melanoma Risk Genes Are Associated with Clinical Characteristics of Melanoma Patients.

    PubMed

    de Araújo, Érica S S; Pramio, Dimitrius T; Kashiwabara, André Y; Pennacchi, Paula C; Maria-Engler, Silvya S; Achatz, Maria I; Campos, Antonio H J F M; Duprat, João P; Rosenberg, Carla; Carraro, Dirce M; Krepischi, Ana C V

    2015-01-01

    In melanoma development, oncogenic process is mediated by genetic and epigenetic mutations, and few studies have so far explored the role of DNA methylation either as predisposition factor or biomarker. We tested patient samples for germline CDKN2A methylation status and found no evidence of inactivation by promoter hypermethylation. We have also investigated the association of clinical characteristics of samples with the DNA methylation pattern of twelve genes relevant for melanomagenesis. Five genes (BAP1, MGMT, MITF, PALB2, and POT1) presented statistical association between blood DNA methylation levels and either CDKN2A-mutation status, number of lesions, or Breslow thickness. In tumors, five genes (KIT, MGMT, MITF, TERT, and TNF) exhibited methylation levels significantly different between tumor groups including acral compared to nonacral melanomas and matched primary lesions and metastases. Our data pinpoint that the methylation level of eight melanoma-associated genes could potentially represent markers for this disease both in peripheral blood and in tumor samples. PMID:26106605

  17. 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

  18. Influence of lifestyle on the FAIM2 promoter methylation between obese and lean children: a cohort study

    PubMed Central

    Wu, Lijun; Zhao, Xiaoyuan; Shen, Yue; Huang, Guimin; Zhang, Meixian; Yan, Yinkun; Hou, Dongqing; Meng, Linghui; Liu, Junting; Cheng, Hong; Mi, Jie

    2015-01-01

    Objective An obesity-related gene, Fas apoptotic inhibitory molecule 2 (FAIM2), is regulated by nutritional state and the methylation levels of the FAIM2 promoter are significantly associated with obesity. Lifestyle factors, such as sedentary behaviour and physical activity, might modify epigenetic patterns that have been related to obesity. Whether the molecular mechanisms by which FAIM2 affects obesity are involved in lifestyle is unclear. This study investigates the potential differences of the FAIM2 promoter methylation with sedentary behaviour and physical activity in obese and lean children. Design Cohort study. Setting Institute of Pediatrics in China. Participants 59 obese cases and 39 lean controls aged 8–18 years recruited from a cross-sectional survey of children from Beijing in 2013. Primary and secondary outcome measures The FAIM2 promoter methylation was quantified using the Sequenom MassARRAY platform. Sedentary behaviour and physical activity were investigated using a questionnaire. The influences of different lifestyles on methylation variations in obese and lean children were examined by multiple linear regression. Results The methylation levels at seven CpG sites of the FAIM2 promoter were significantly associated with sedentary behaviour, especially the methylation levels at site −975, site −413, sites −362 and −360, and sites −353 and −349 (p=0.00004, 0.00009, 0.0006 and 0.00005, respectively). There were significant differences between the methylation levels at four CpG sites in obese and lean participants with high or moderate physical activity level <150 min/week. Conclusions This study provides the first evidence that there are significant differences in the associations of the FAIM2 promoter methylation with sedentary behaviour and physical activity between obese and lean children. Our results suggest that lifestyle may possibly be mediating the process of the FAIM2 involved in obesity. PMID:25922107

  19. Genes with stable DNA methylation levels show higher evolutionary conservation than genes with fluctuant DNA methylation levels.

    PubMed

    Zhang, Ruijie; Lv, Wenhua; Luan, Meiwei; Zheng, Jiajia; Shi, Miao; Zhu, Hongjie; Li, Jin; Lv, Hongchao; Zhang, Mingming; Shang, Zhenwei; Duan, Lian; Jiang, Yongshuai

    2015-11-24

    Different human genes often exhibit different degrees of stability in their DNA methylation levels between tissues, samples or cell types. This may be related to the evolution of human genome. Thus, we compared the evolutionary conservation between two types of genes: genes with stable DNA methylation levels (SM genes) and genes with fluctuant DNA methylation levels (FM genes). For long-term evolutionary characteristics between species, we compared the percentage of the orthologous genes, evolutionary rate dn/ds and protein sequence identity. We found that the SM genes had greater percentages of the orthologous genes, lower dn/ds, and higher protein sequence identities in all the 21 species. These results indicated that the SM genes were more evolutionarily conserved than the FM genes. For short-term evolutionary characteristics among human populations, we compared the single nucleotide polymorphism (SNP) density, and the linkage disequilibrium (LD) degree in HapMap populations and 1000 genomes project populations. We observed that the SM genes had lower SNP densities, and higher degrees of LD in all the 11 HapMap populations and 13 1000 genomes project populations. These results mean that the SM genes had more stable chromosome genetic structures, and were more conserved than the FM genes.

  20. Correlating gene-specific DNA methylation changes with expression and transcriptional activity of astrocytic KCNJ10 (Kir4.1)

    PubMed Central

    Nwaobi, Sinifunanya E.; Olsen, Michelle L.

    2016-01-01

    Short abstract DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression. Long abstract DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able

  1. 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

  2. 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.

  3. 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

  4. Methylation of serum SST gene is an independent prognostic marker in colorectal cancer

    PubMed Central

    Liu, Yanqun; Chew, Min Hoe; Tham, Chee Kian; Tang, Choong Leong; Ong, Simon YK; Zhao, Yi

    2016-01-01

    There is an increasing demand for accurate prognostication for colorectal cancer (CRC). This study sought to assess prognostic potentials of methylation targets in the serum of CRC patients. A total of 165 CRC patients were enrolled in this prospective study. Promoter methylation levels of seven genes in pre-operative sera and matched tumor tissues were evaluated by quantitative methylation-specific PCR. Kaplan-Meier test, and univariate and multivariate Cox proportional hazards regression models were used for survival analyses. After a median follow-up of 56 months, 43 patients (28.7%) experienced tumor recurrence. In univariate survival analyses, serum methylation levels of SST and MAL were significantly predictive of cancer-specific death (P<0.005 for both). The former was also a significant predictor for tumor recurrence (P=0.007). Independent prognostic effects of serum methylation levels of SST were revealed by multivariate Cox regression model (P=0.031 and P=0.003 for cancer death and recurrence, respectively). When focusing on stage II and III patients, prognostication with serum methylated SST remained significant. Methylated SST detected in all serum samples can be traced back to the matched primary tumor tissues. We believe that methylated SST detected in the pre-operative sera of CRC patients appear to be a novel promising prognostic marker and probably can be auxiliary to tumor staging system and serum carcinoembryonic antigen towards better risk stratification. PMID:27725914

  5. 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

  6. 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

  7. Methylation of the oxytocin receptor gene and oxytocin blood levels in the development of psychopathy.

    PubMed

    Dadds, Mark R; Moul, Caroline; Cauchi, Avril; Dobson-Stone, Carol; Hawes, David J; Brennan, John; Ebstein, Richard E

    2014-02-01

    Child conduct problems (CPs) are a robust predictor of adult mental health; the concurrence of callous-unemotional (CU) traits confers specific risk for psychopathy. Psychopathy may be related to disturbances in the oxytocin (OXT) system. Evidence suggests that epigenetic changes in the OXT receptor gene (OXTR) are associated with lower circulating OXT and social-cognitive difficulties. We tested methylation levels of OXTR in 4- to 16-year-old males who met DSM criteria for a diagnosis of oppositional-defiant or conduct disorder and were stratified by CU traits and age. Measures were DNA methylation levels of six CpG sites in the promoter region of the OXTR gene (where a CpG site is a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its lenth, linked together by phosphate binding), and OXT blood levels. High CU traits were associated with greater methylation of the OXTR gene for two cytosine nucleotide and guanine nucleotide phosphate linked sites and lower circulating OXT in older males. Higher methylation correlated with lower OXT levels. We conclude that greater methylation of OXTR characterizes adolescent males with high levels of CU and CPs, and this methylation is associated with lower circulating OXT and functional impairment in interpersonal empathy. The results add genetic evidence that high CU traits specify a distinct subgroup within CP children, and they suggest models of psychopathy may be informed by further identification of these epigenetic processes and their functional significance. PMID:24059811

  8. FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.

    PubMed

    Gong, C; Fujino, K; Monteiro, L J; Gomes, A R; Drost, R; Davidson-Smith, H; Takeda, S; Khoo, U S; Jonkers, J; Sproul, D; Lam, E W-F

    2015-09-24

    FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly

  9. DNA methylation in the Neuropeptide S Receptor 1 (NPSR1) promoter in relation to asthma and environmental factors.

    PubMed

    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

  10. 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

  11. 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

  12. The SNF2 family ATPase LSH promotes cell-autonomous de novo DNA methylation in somatic cells

    PubMed Central

    Termanis, Ausma; Torrea, Natalia; Culley, Jayne; Kerr, Alastair; Ramsahoye, Bernard; Stancheva, Irina

    2016-01-01

    Methylation of DNA at carbon 5 of cytosine is essential for mammalian development and implicated in transcriptional repression of genes and transposons. New patterns of DNA methylation characteristic of lineage-committed cells are established at the exit from pluripotency by de novo DNA methyltransferases enzymes, DNMT3A and DNMT3B, which are regulated by developmental signaling and require access to chromatin-organized DNA. Whether or not the capacity for de novo DNA methylation of developmentally regulated loci is preserved in differentiated somatic cells and can occur in the absence of exogenous signals is currently unknown. Here, we demonstrate that fibroblasts derived from chromatin remodeling ATPase LSH (HELLS)-null mouse embryos, which lack DNA methylation from centromeric repeats, transposons and a number of gene promoters, are capable of reestablishing DNA methylation and silencing of misregulated genes upon re-expression of LSH. We also show that the ability of LSH to bind ATP and the cellular concentration of DNMT3B are critical for cell-autonomous de novo DNA methylation in somatic cells. These data suggest the existence of cellular memory that persists in differentiated cells through many cell generations and changes in transcriptional state. PMID:27179028

  13. 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

  14. 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

  15. Epigenetic genes regulated by the BRAFV600E signaling are associated with alterations in the methylation and expression of tumor suppressor genes and patient survival in melanoma.

    PubMed

    Liu, Dingxie; Liu, Xuan; Xing, Mingzhao

    2012-08-17

    We have previously reported that the BRAFV600E signaling causes genome-wide aberrations in gene methylation in melanoma cells. To explore the potential molecular mechanisms for this epigenetic effect of BRAFV600E, in this in silico study we analyzed 11 microarray datasets retrieved from NCBI GEO database and examined the relationship of the expression of the epigenetic genes (genes involved in epigenetic regulation) with BRAFV600E signaling, methylation and expression of tumor-suppressor genes (TSGs) in melanoma, and patient survival with this cancer. Among 273 epigenetic genes examined, 12 genes were down-regulated (named DD genes) and 16 were up-regulated (UU genes) by suppression of the BRAFV600E signaling using inhibitors. While the expression of 245 non-DD/UU genes overall had no correlation with the expression and methylation of a set of potential TSGs, the expression of DD genes was significantly correlated negatively with the TSG expression and positively with TSG methylation. Expression of UU genes was positively, albeit weakly, associated with the TSG expression. Overall, no correlation was found between UU gene expression and TSG methylation. Importantly, the expression of DD genes, but not UU genes, was significantly associated with decreased survival of patients with melanoma. Interestingly, the promoters of DD genes contain more binding motifs of c-fos and myc, two BRAFV600E signaling-related transcription factors, than those of UU and non-DD/UU genes. Thus, these results link epigenetic genes to methylation and suppression of tumor suppressor genes as a mechanism involved in BRAFV600E-promoted melanoma tumorigenesis and uncover a novel molecular signature that predicts a poor prognosis of melanoma.

  16. DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus.

    PubMed

    Gonzalez, Diego; Collier, Justine

    2013-04-01

    DNA methylation regulates many processes, including gene expression, by superimposing secondary information on DNA sequences. The conserved CcrM enzyme, which methylates adenines in GANTC sequences, is essential to the viability of several Alphaproteobacteria. In this study, we find that Caulobacter crescentus cells lacking the CcrM enzyme accumulate low levels of the two conserved FtsZ and MipZ proteins, leading to a severe defect in cell division. This defect can be compensated by the expression of the ftsZ gene from an inducible promoter or by spontaneous suppressor mutations that promote FtsZ accumulation. We show that CcrM promotes the transcription of the ftsZ and mipZ genes and that the ftsZ and mipZ promoter regions contain a conserved CGACTC motif that is critical to their activities and to their regulation by CcrM. In addition, our results suggest that the ftsZ promoter has the lowest activity when the CGACTC motif is non-methylated, an intermediate activity when it is hemi-methylated and the highest activity when it is fully methylated. The regulation of ftsZ expression by DNA methylation may explain why CcrM is essential in a subset of Alphaproteobacteria.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Roles of Cell Division and Gene Transcription in the Methylation of CpG Islands

    PubMed Central

    Bender, Christina M.; Gonzalgo, Mark L.; Gonzales, Felicidad A.; Nguyen, Carvell T.; Robertson, Keith D.; Jones, Peter A.

    1999-01-01

    De novo methylation of CpG islands within the promoters of eukaryotic genes is often associated with their transcriptional repression, yet the methylation of CpG islands located downstream of promoters does not block transcription. We investigated the kinetics of mRNA induction, demethylation, and remethylation of the p16 promoter and second-exon CpG islands in T24 cells after 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment to explore the relationship between CpG island methylation and gene transcription. The rates of remethylation of both CpG islands were associated with time but not with the rate of cell division, and remethylation of the p16 exon 2 CpG island occurred at a higher rate than that of the p16 promoter. We also examined the relationship between the remethylation of coding sequence CpG islands and gene transcription. The kinetics of remethylation of the p16 exon 2, PAX-6 exon 5, c-ABL exon 11, and MYF-3 exon 3 loci were examined following 5-Aza-CdR treatment because these genes contain exonic CpG islands which are hypermethylated in T24 cells. Remethylation occurred most rapidly in the p16, PAX-6, and c-ABL genes, shown to be transcribed prior to drug treatment. These regions also exhibited higher levels of remethylation in single-cell clones and subclones derived from 5-Aza-CdR-treated T24 cells. Our data suggest that de novo methylation is not restricted to the S phase of the cell cycle and that transcription through CpG islands does not inhibit their remethylation. PMID:10490608

  2. Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.

    PubMed

    Cho, Hyun-Soo; Shimazu, Tadahiro; Toyokawa, Gouji; Daigo, Yataro; Maehara, Yoshihiko; Hayami, Shinya; Ito, Akihiro; Masuda, Ken; Ikawa, Noriko; Field, Helen I; Tsuchiya, Eiju; Ohnuma, Shin-ichi; Ponder, Bruce A J; Yoshida, Minoru; Nakamura, Yusuke; Hamamoto, Ryuji

    2012-01-01

    Although heat-shock protein 70 (HSP70), an evolutionarily highly conserved molecular chaperone, is known to be post-translationally modified in various ways such as phosphorylation, ubiquitination and glycosylation, physiological significance of lysine methylation has never been elucidated. Here we identify dimethylation of HSP70 at Lys-561 by SETD1A. Enhanced HSP70 methylation was detected in various types of human cancer by immunohistochemical analysis, although the methylation was barely detectable in corresponding non-neoplastic tissues. Interestingly, methylated HSP70 predominantly localizes to the nucleus of cancer cells, whereas most of the HSP70 protein locates to the cytoplasm. Nuclear HSP70 directly interacts with Aurora kinase B (AURKB) in a methylation-dependent manner and promotes AURKB activity in vitro and in vivo. We also find that methylated HSP70 has a growth-promoting effect in cancer cells. Our findings demonstrate a crucial role of HSP70 methylation in human carcinogenesis. PMID:22990868

  3. 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

  4. Significant association between DRD3 gene body methylation and schizophrenia.

    PubMed

    Dai, Dongjun; Cheng, Jia; Zhou, Kena; Lv, Yuelong; Zhuang, Qidong; Zheng, Rongjiong; Zhang, Kai; Jiang, Danjie; Gao, Shugui; Duan, Shiwei

    2014-12-30

    The current study was the first one to reveal the contribution of DRD3 methylation to the risk of different (SCZ) subtypes. This study comprised a total of 30 paranoid (15 males and 15 females) and 29 undifferentiated (15 males and 14 females) SCZ patients and 26 age- and gender-matched controls. Our results showed a significant association of CpG2 with SCZ. A breakdown analysis by gender showed that CpG2 and CpG3 methylation were significantly higher in male patients than male controls, and that CpG5 methylation was significantly higher in female patients than female controls. A further breakdown analysis by both gender and SCZ subtype showed that CpG2 and CpG3 methylation were significantly higher in male paranoid SCZ and male undifferentiated SCZ than male controls. In contrast, CpG2 and CpG3 methylation were significantly lower in female undifferentiated SCZ than female controls. Additionally, CpG5 methylation was significantly higher in female paranoid SCZ than female controls. In conclusion, our findings supported that DRD3 gene body hypermethylation was significantly associated with the risk of SCZ. Future study is needed to clarify the mechanisms by which DRD3 gene body hypermethylation contributes to the risk of SCZ.

  5. 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

  6. Cytosine methylation in CTF and Sp1 recognition sites of an HSV tk promoter: effects on transcription in vivo and on factor binding in vitro.

    PubMed Central

    Ben-Hattar, J; Beard, P; Jiricny, J

    1989-01-01

    We methylated specific cytosine residues within or immediately around the CTF and Sp1 binding sites of the Herpes simplex virus thymidine kinase promoter. The efficiency of transcription in vivo was reduced at least 50-fold compared with transcription from the unmethylated promoter. However, methylation within the CTF recognition site had no effect on the affinity of CTF for this site in vitro. Methylation of the Sp1 site resulted in only a small decrease in the affinity of this factor for its recognition site. In vivo studies showed that the same gene inserted in different vector DNAs was regulated differently by methylation in the promoter. These results show that cytosine methylation can inhibit transcription by a mechanism other than directly blocking the binding of transcription factors. Images PMID:2557588

  7. Polyisoprenylated methylated protein methyl esterase overexpression and hyperactivity promotes lung cancer progression

    PubMed Central

    Amissah, Felix; Duverna, Randolph; Aguilar, Byron J; Poku, Rosemary A; Kiros, Gebre-Egziabher; Lamango, Nazarius S

    2014-01-01

    The involvement of hyperactive polyisoprenylated proteins in cancers has stimulated the search for drugs to target and suppress their excessive activities. Polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition has been shown to modulate polyisoprenylated protein function. For PMPMEase inhibition to be effective against cancers, polyisoprenylated proteins, the signaling pathways they mediate and/or PMPMEase must be overexpressed, hyperactive and be involved in at least some cases of cancer. PMPMEase activity in lung cancer cells and its expression in lung cancer cells and cancer tissues were investigated. PMPMEase was found to be overexpressed and significantly more active in lung cancer A549 and H460 cells than in normal lung fibroblasts. In a tissue microarray study, PMPMEase immunoreactivity was found to be significantly higher in lung cancer tissues compared to the normal controls (p < 0.0001). The mean scores ± SEM were 118.8 ± 7.7 (normal), 232.1 ± 25.1 (small-cell lung carcinomas), 352.1 ± 9.4 (squamous cell carcinomas), 311.7 ± 9.8 (adenocarcinomas), 350.0 ± 24.2 (papillary adenocarcinomas), 334.7 ± 30.1 (adenosquamous carcinomas), 321.9 ± 39.7 (bronchioloalveolar carcinomas), and 331.3 ± 85.0 (large-cell carcinomas). Treatment of lung cancer cells with L-28, a specific PMPMEase inhibitor, resulted in concentration-dependent cell death (EC50 of 8.5 μM for A549 and 2.8 μM for H460 cells). PMPMEase inhibition disrupted actin filament assembly, significantly inhibited cell migration and altered the transcription of cancer-related genes. These results indicate that elevated PMPMEase activity spur cell growth and migration, implying the possible use of PMPMEase as a protein biomarker and drug target for lung cancer. PMID:24660102

  8. 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

  9. 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

  10. 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

  11. Genome-wide DNA methylation analysis identifies hypomethylated genes regulated by FOXP3 in human regulatory T cells.

    PubMed

    Zhang, Yuxia; Maksimovic, Jovana; Naselli, Gaetano; Qian, Junyan; Chopin, Michael; Blewitt, Marnie E; Oshlack, Alicia; Harrison, Leonard C

    2013-10-17

    Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg, we analyzed genome-wide methylation in human naive nTreg (rTreg) and conventional naive CD4(+) T cells (Naive). We detected 2315 differentially methylated cytosine-guanosine dinucleotides (CpGs) between these 2 cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naive but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3-binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets, we showed that increased expression of the immune suppressive receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGIT locus. Differential methylation analysis provides insight into previously undefined human Treg signature genes and their mode of regulation.

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

    PubMed

    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.

  13. 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

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

    PubMed

    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

  15. DNA Methylation Profiling Revealed Promoter Hypermethylation-induced Silencing of p16, DDAH2 and DUSP1 in Primary Oral Squamous Cell Carcinoma

    PubMed Central

    Khor, Goot Heah; Froemming, Gabriele Ruth Anisah; Zain, Rosnah Binti; Abraham, Mannil Thomas; Omar, Effat; Tan, Su Keng; Tan, Aik Choon; Vincent-Chong, Vui King; Thong, Kwai Lin

    2013-01-01

    Background: Hypermethylation in promoter regions of genes might lead to altered gene functions and result in malignant cellular transformation. Thus, biomarker identification for hypermethylated genes would be very useful for early diagnosis, prognosis, and therapeutic treatment of oral squamous cell carcinoma (OSCC). The objectives of this study were to screen and validate differentially hypermethylated genes in OSCC and correlate the hypermethylation-induced genes with demographic, clinocopathological characteristics and survival rate of OSCC. Methods: DNA methylation profiling was utilized to screen the differentially hypermethylated genes in OSCC. Three selected differentially-hypermethylated genes of p16, DDAH2 and DUSP1 were further validated for methylation status and protein expression. The correlation between demographic, clinicopathological characteristics, and survival rate of OSCC patients with hypermethylation of p16, DDAH2 and DUSP1 genes were analysed in the study. Results: Methylation profiling demonstrated 33 promoter hypermethylated genes in OSCC. The differentially-hypermethylated genes of p16, DDAH2 and DUSP1 revealed positivity of 78%, 80% and 88% in methylation-specific polymerase chain reaction and 24% and 22% of immunoreactivity in DDAH2 and DUSP1 genes, respectively. Promoter hypermethylation of p16 gene was found significantly associated with tumour site of buccal, gum, tongue and lip (P=0.001). In addition, DDAH2 methylation level was correlated significantly with patients' age (P=0.050). In this study, overall five-year survival rate was 38.1% for OSCC patients and was influenced by sex difference. Conclusions: The study has identified 33 promoter hypermethylated genes that were significantly silenced in OSCC, which might be involved in an important mechanism in oral carcinogenesis. Our approaches revealed signature candidates of differentially hypermethylated genes of DDAH2 and DUSP1 which can be further developed as potential

  16. DNA methylation patterns of protein coding genes and long noncoding RNAs in female schizophrenic patients.

    PubMed

    Liao, Qi; Wang, Yunliang; Cheng, Jia; Dai, Dongjun; Zhou, Xingyu; Zhang, Yuzheng; Gao, Shugui; Duan, Shiwei

    2015-02-01

    Schizophrenia (SCZ) is a complex mental disorder contributed by both genetic and epigenetic factors. Long noncoding RNAs (lncRNAs) was recently found playing an important regulatory role in mental disorders. However, little was known about the DNA methylation of lncRNAs, although numerous SCZ studies have been performed on genetic polymorphisms or epigenetic marks in protein coding genes. We presented a comprehensive genome wide DNA methylation study of both protein coding genes and lncRNAs in female patients with paranoid and undifferentiated SCZ. Using the methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), 8,163 and 764 peaks were identified in paranoid and undifferentiated SCZ, respectively (p < 1 × 10-5). Gene ontology analysis showed that the hypermethylated regions were enriched in the genes related to neuron system and brain for both paranoid and undifferentiated SCZ (p < 0.05). Among these peaks, 121 peaks were located in gene promoter regions that might affect gene expression and influence the SCZ related pathways. Interestingly, DNA methylation of 136 and 23 known lncRNAs in Refseq database were identified in paranoid and undifferentiated SCZ, respectively. In addition, ∼20% of intergenic peaks annotated based on Refseq genes were overlapped with lncRNAs in UCSC and gencode databases. In order to show the results well for most biological researchers, we created an online database to display and visualize the information of DNA methyation peaks in both types of SCZ (http://www.bioinfo.org/scz/scz.htm). Our results showed that the aberrant DNA methylation of lncRNAs might be another important epigenetic factor for SCZ.

  17. Identification of direction in gene networks from expression and methylation

    PubMed Central

    2013-01-01

    Background Reverse-engineering gene regulatory networks from expression data is difficult, especially without temporal measurements or interventional experiments. In particular, the causal direction of an edge is generally not statistically identifiable, i.e., cannot be inferred as a statistical parameter, even from an unlimited amount of non-time series observational mRNA expression data. Some additional evidence is required and high-throughput methylation data can viewed as a natural multifactorial gene perturbation experiment. Results We introduce IDEM (Identifying Direction from Expression and Methylation), a method for identifying the causal direction of edges by combining DNA methylation and mRNA transcription data. We describe the circumstances under which edge directions become identifiable and experiments with both real and synthetic data demonstrate that the accuracy of IDEM for inferring both edge placement and edge direction in gene regulatory networks is significantly improved relative to other methods. Conclusion Reverse-engineering directed gene regulatory networks from static observational data becomes feasible by exploiting the context provided by high-throughput DNA methylation data. An implementation of the algorithm described is available at http://code.google.com/p/idem/. PMID:24182195

  18. Gene methylation of SFRP2, P16, DAPK1, HIC1, and MGMT and KRAS mutations in sporadic colorectal cancer.

    PubMed

    Pehlivan, Sacide; Artac, Mehmet; Sever, Tugce; Bozcuk, Hakan; Kilincarslan, Can; Pehlivan, Mustafa

    2010-09-01

    The aim of this study was to investigate the methylation of the SFRP2, P16, DAPK1, HIC1, and MGMT genes, as well as the mutation of amino acid codons 12 and 13 of the KRAS gene in normal and tumor tissue DNA of patients diagnosed with sporadic colorectal cancer (SCRC). The methylation of gene regions and the KRAS mutations of normal (N) and tumor tissue (T) DNA obtained from 17 patients diagnosed with SCRC and 20 healthy controls were investigated using the polymerase chain reaction and reverse-hybridization methods. There was an Asp mutation in four patients, an Asp and Ser mutations in one patient in codon 12 of the KRAS gene, and an Asp mutation in codon 13 in eight patients. Overall promoter methylation (OPM) in the SFRP2 gene was observed in one N and four T, whereas partial promoter methylation (PPM) was observed in two N and five T. OPM in the P16 gene was present in one T. In the DAPK1 gene, OPM existed in seven T and five N, while PPM was present in two N. In the HIC1 gene, OPM was demonstrated in three T, while PPM was noted in two N; however, no methylation existed in N. In the MGMT gene, OPM occurred in five T and two N, and PPM was present in one T. KRAS mutations in Turkish patients with SCRC are similar to those of other population groups. Methylations in the genes, which underwent methylation analysis, were higher in T in comparison with N, and it has been suggested that significant results would be obtained by making a study with a larger population.

  19. Relationships between MGMT promoter methylation and gastric cancer: a meta-analysis

    PubMed Central

    Yu, Dan; Cao, Tao; Han, Ya-Di; Huang, Fu-Sheng

    2016-01-01

    A DNA repair enzyme, O6-methylguanine-DNA methyltransferase (MGMT), plays an important role in the development of gastric cancers. However, the role of MGMT promoter methylation in the occurrence of gastric cancer and its relationships with clinicopathologic characteristics has not been fully clarified. Thus, we performed a meta-analysis to evaluate the associations between MGMT promoter methylation and gastric cancer. Electronic databases, including PubMed and Web of Science, were used to systematically search related clinical studies published in English until April 1, 2016. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to evaluate the associations between MGMT promoter methylation and gastric cancer risk or clinicopathologic characteristics. A total of 16 studies including 1,935 patients and 1,948 control persons were included in the analysis. Our study suggested that MGMT promoter methylation frequency was associated with gastric cancer (OR=3.46, 95% CI: 2.13–5.61, P<0.001). Moreover, the frequency of MGMT promoter methylation in the no lymph node metastasis group was lower than that in lymph node metastasis group, with marginal significance (OR=0.65, 95% CI: 0.42–1.01, P=0.05). Additionally, the methylation rate of the MGMT promoter was much lower in patients without distant metastases than in those with metastases (OR=0.27, 95% CI: 0.18–0.40, P<0.001). No significant association of MGMT promoter methylation with Lauren classification, tumor location, tumor invasion, or Helicobacter pylori infection was found. In conclusion, the methylation status of the MGMT promoter was related to gastric cancer risk, distant metastasis, and lymph node metastasis, which indicates that MGMT promoter methylation may play an important role in gastric cancer development. PMID:27785051

  20. Investigation of DNA damage response and apoptotic gene methylation pattern in sporadic breast tumors using high throughput quantitative DNA methylation analysis technology

    PubMed Central

    2010-01-01

    Background- Sporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR), apoptosis and immunesurvelliance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR) and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology. Results- The study identified five genes with statistically significant difference between normal and tumor tissues. Hypermethylation of DR5 (P = 0.001), DCR1 (P = 0.00001), DCR2 (P = 0.0000000005) and BRCA2 (P = 0.007) and hypomethylation of DR4 (P = 0.011) in sporadic breast tumor tissues suggested a weak/aberrant activation of the DDR/apoptotic pathway in breast tumorigenesis. Negative correlation was observed between methylation status and transcript expression levels for TRAIL, DR4, CASP8, ATM, CHEK2, BRCA1 and BRCA2 CpG sites. Categorization of the gene methylation with respect to the clinicopathological parameters showed an increase in aberrant methylation pattern in advanced tumors. These uncharacteristic methylation patterns corresponded with decreased death receptor apoptosis (P = 0.047) and DNA damage repair potential (P = 0.004) in advanced tumors. The observation of BRCA2 -26 G/A 5'UTR polymorphism concomitant with the presence of methylation in the promoter region was novel and emerged as a strong candidate for susceptibility to sporadic breast tumors. Conclusion- Our study indicates that methylation of DDR-apoptotic gene promoters in sporadic breast cancer is not a random phenomenon. Progressive epigenetic alterations in advancing tumors result in

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

    PubMed

    Chater-Diehl, Eric J; Laufer, Benjamin I; 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

  2. 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

  3. Colon cancer cell apoptosis is induced by combined exposure to the n-3 fatty acid docosahexaenoic acid and butyrate through promoter methylation.

    PubMed

    Cho, Youngmi; Turner, Nancy D; Davidson, Laurie A; Chapkin, Robert S; Carroll, Raymond J; Lupton, Joanne R

    2014-03-01

    DNA methylation and histone acetylation contribute to the transcriptional regulation of genes involved in apoptosis. We have demonstrated that docosahexaenoic acid (DHA, 22:6 n-3) and butyrate enhance colonocyte apoptosis. To determine if DHA and/or butyrate elevate apoptosis through epigenetic mechanisms thereby restoring the transcription of apoptosis-related genes, we examined global methylation; gene-specific promoter methylation of 24 apoptosis-related genes; transcription levels of Cideb, Dapk1, and Tnfrsf25; and global histone acetylation in the HCT-116 colon cancer cell line. Cells were treated with combinations of (50 µM) DHA or linoleic acid (18:2 n-6), (5 mM) butyrate or an inhibitor of DNA methyltransferases, and 5-aza-2'-deoxycytidine (5-Aza-dC, 2 µM). Among highly methylated genes, the combination of DHA and butyrate significantly reduced methylation of the proapoptotic Bcl2l11, Cideb, Dapk1, Ltbr, and Tnfrsf25 genes compared to untreated control cells. DHA treatment reduced the methylation of Cideb, Dapk1, and Tnfrsf25. These data suggest that the induction of apoptosis by DHA and butyrate is mediated, in part, through changes in the methylation state of apoptosis-related genes.

  4. 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

  5. Methylation pattern of methylene tetrahydrofolate reductase and small nuclear ribonucleoprotein polypeptide N promoters in oligoasthenospermia: a case-control study.

    PubMed

    Botezatu, Anca; Socolov, Razvan; Socolov, Demetra; Iancu, Iulia Virginia; Anton, Gabriela

    2014-02-01

    Alterations in DNA methylation patterns in several genes may lead to abnormal male sexual development and infertility. This study investigated the promoter methylation status of MTHFR and SNRPN in infertile men from Romania by quantitative methylation-specific PCR in order to investigate possible correlations with sperm abnormalities. The study groups included patients (n=27) with a median age of 31 years (range 26-41 years) as well as controls (n=11) with a median age of 30 years (range 24-37 years) recruited from couples seeking advice for infertility. DNA was isolated from sperm samples and promoter methylation was assessed using direct. Significant trends were detected for both genes that indicate a tendency towards promoter hypermethylation in spermatozoa with low motility (MTHFR P=0.0032, r=0.23; SNRPN P=0.0003, r=0.32) and poor morphology (MTHFR P=0.0012, r=0.27; SNRPN P=0.0003, r=0.33) but no trend was found in cases of low sperm count (MTHFR r=0.007; SNRPN r=0.06). The data indicate that the methylation patterns of the promoters of MTHFR and SNRPN are associated with changes in sperm motility and morphology, which could lead to male infertility. A large number of studies are now focused on the causes of male infertility. Among these are epigenetic modifications, which are important contributors to reproductive pathology in the male by providing dynamic changes of the phenotype according to the environmental and metabolic factors. The most known epigenetic modification is DNA methylation and alterations in this pattern in several genes could induce male infertility. The present study aims to investigate the promoter methylation status of the genes for methylene tetrahydrofolate reductase (MTHFR) and small nuclear ribonucleoprotein polypeptide N (SNRPN) in infertile males from Romania, in order to establish a correlation with sperm parameters. MTHFR is an enzyme involved in the folate pathway and in de novo nucleotide biosynthesis but also a good example for

  6. 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

  7. 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

  8. 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

  9. Coordinated DNA methylation and gene expression changes in smoker alveolar macrophages: specific effects on VEGF receptor 1 expression

    PubMed Central

    Philibert, Robert A.; Sears, Rory A.; Powers, Linda S.; Nash, Emma; Bair, Thomas; Gerke, Alicia K.; Hassan, Ihab; Thomas, Christie P.; Gross, Thomas J.; Monick, Martha M.

    2012-01-01

    Cigarette smoking is implicated in numerous diseases, including emphysema and lung cancer. The clinical expression of lung disease in smokers is not well explained by currently defined variations in gene expression or simple differences in smoking exposure. Alveolar macrophages play a critical role in the inflammation and remodeling of the lung parenchyma in smoking-related lung disease. Significant gene expression changes in alveolar macrophages from smokers have been identified. However, the mechanism for these changes remains unknown. One potential mechanism for smoking-altered gene expression is via changes in cytosine methylation in DNA regions proximal to gene-coding sequences. In this study, alveolar macrophage DNA from heavy smokers and never smokers was isolated and methylation status at 25,000 loci determined. We found differential methylation in genes from immune-system and inflammatory pathways. Analysis of matching gene expression data demonstrated a parallel enrichment for changes in immune-system and inflammatory pathways. A significant number of genes with smoking-altered mRNA expression had inverse changes in methylation status. One gene highlighted by this data was the FLT1, and further studies found particular up-regulation of a splice variant encoding a soluble inhibitory form of the receptor. In conclusion, chronic cigarette smoke exposure altered DNA methylation in specific gene promoter regions in human alveolar macrophages. PMID:22427682

  10. Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription.

    PubMed

    Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J; van Hooff, Sander; van Leenen, Dik; Dargemont, Catherine; Géli, Vincent; Holstege, Frank C P

    2012-09-01

    Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by

  11. 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

  12. Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression.

    PubMed

    Koczor, Christopher A; Fields, Earl; Jedrzejczak, Mark J; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A; Lewis, William

    2015-11-01

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10d, 3mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change>1.5, p<0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. PMID:26307267

  13. 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

    2015-01-01

    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

  14. 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

    2015-01-01

    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.

  15. 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

  16. Elevated Klotho promoter methylation is associated with severity of chronic kidney disease.

    PubMed

    Chen, Jing; Zhang, Xiaoyan; Zhang, Han; Lin, Jing; Zhang, Chen; Wu, Qing; Ding, Xiaoqiang

    2013-01-01

    Klotho (KL) expression is down-regulated in the renal tissues of chronic kidney disease (CKD) animal models and patients with end-stage renal disease. The putative role of KL promoter hypermethylation in the progression of CKD remains unclear. The present study aimed to determine renal and peripheral blood mononuclear cells (PBMC) levels of KL promoter methylation and analyze their relationship with clinical and histological severity in patients with CKD. Using bisulfite pyrosequencing, renal and PBMC levels of KL promoter methylation were quantified in 47 patients with CKD. 47 nephrectomy specimens of patients with renal cell carcinoma and 48 PBMC specimens of healthy volunteers were used as renal tissue and PBMC controls, respectively. Renal expression of KL protein was assayed by immunohistochemistry staining. Receiver operating characteristic (ROC) curve was used to identify the optimal cut-off value of PBMC KL promoter methylation level for renal KL promoter hypermethylation. Higher levels of KL promoter methylation were observed in renal tissue and PBMC in patients with CKD compared with controls (8.79±3.24 vs. 5.17±1.11%, P<0.001; 7.20±2.79 vs. 3.27±0.79%, P<0.001). In these patients, renal KL methylation level correlated inversely with renal KL immunostaining intensity (ρ=-0.794, P<0.001). Estimated glomerular filtration rate correlated inversely with renal and PBMC levels of KL promoter methylation (r=-0.829, P<0.001; r=-0.645, P<0.001), while tubulointerstistial fibrosis score correlated positively (ρ=0.826, P<0.001; ρ=0.755, P<0.001). PBMC KL promoter methylation level correlated positively with renal KL promoter methylation level in patients with CKD (r=0.787, P<0.001). In ROC curve, the area under curve was 0.964 (P<0.001) and the optimal cut-off value was 5.83% with a sensitivity of 93.8% and specificity of 86.7% to predict renal KL promoter hypermethylation. The degree of KL promoter methylation is associated with clinical and histological

  17. Effects of the Social Environment and Stress on Glucocorticoid Receptor Gene Methylation: A Systematic Review.

    PubMed

    Turecki, Gustavo; Meaney, Michael J

    2016-01-15

    The early-life social environment can induce stable changes that influence neurodevelopment and mental health. Research focused on early-life adversity revealed that early-life experiences have a persistent impact on gene expression and behavior through epigenetic mechanisms. The hypothalamus-pituitary-adrenal axis is sensitive to changes in the early-life environment that associate with DNA methylation of a neuron-specific exon 17 promoter of the glucocorticoid receptor (GR) (Nr3c1). Since initial findings were published in 2004, numerous reports have investigated GR gene methylation in relationship to early-life experience, parental stress, and psychopathology. We conducted a systematic review of this growing literature, which identified 40 articles (13 animal and 27 human studies) published since 2004. The majority of these examined the GR exon variant 1F in humans or the GR17 in rats, and 89% of human studies and 70% of animal studies of early-life adversity reported increased methylation at this exon variant. All the studies investigating exon 1F/17 methylation in conditions of parental stress (one animal study and seven human studies) also reported increased methylation. Studies examining psychosocial stress and psychopathology had less consistent results, with 67% of animal studies reporting increased exon 17 methylation and 17% of human studies reporting increased exon 1F methylation. We found great consistency among studies investigating early-life adversity and the effect of parental stress, even if the precise phenotype and measures of social environment adversity varied among studies. These results are encouraging and warrant further investigation to better understand correlates and characteristics of these associations.

  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. Candidate luminal B breast cancer genes identified by genome, gene expression and DNA methylation profiling.

    PubMed

    Cornen, Stéphanie; Guille, Arnaud; Adélaïde, José; Addou-Klouche, Lynda; Finetti, Pascal; Saade, Marie-Rose; Manai, Marwa; Carbuccia, Nadine; Bekhouche, Ismahane; Letessier, Anne; Raynaud, Stéphane; 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.

  20. Analysis of methylation and mRNA expression status ofFADD andFAS genes in patients with oral squamous cell carcinoma

    PubMed Central

    Saberi, Eshaghali; Jamali, Sara; Rigi-Ladez, Mohammad A.; Augend, Arsalan

    2014-01-01

    Background: Apoptosis is an important mechanism that is responsible for the physiological deletion of harmful, damaged, or unwanted cells. Changed expression of apoptosis-related genes may lead to abnormal cell proliferation and finally to tumorigenesis. Our aims were to analyze the promoter methylation and gene expression profiles of FADD and FAS genes in risk of OSCC. Material and Methods: we analyze the promoter methylation status of FADD and FAS genes using Methylation - Specific PCR (MSP) in 86 OSCC tissues were kept in paraffin and 68 normal oral tissues applied as control. Also, FADD and FAS genes expression were analyzed in 19 cases and 20 normal specimens by Real-Time Reverse-Transcripts PCR. Results: Aberrant promoter methylation of FADD and FAS genes were detected in 12.79 % (11 of 86) and 60.46 % (52 of 86) of the OSCC cases, respectively, with a significant difference between cases and healthy controls for both FADD and FAS genes (P<0.001). The gene expression analysis showed statistically significant difference between cases and healthy controls for both FADD (p<0.02) and FAS (p<0.007) genes. Conclusions: To the best our knowledge, the data of this study are the first report regarding, the effect of promoter hypermethylation of the FADD and FAS genes in development of OSCC. To confirm the data, it is recommended doing further study in large sample sizes in various genetic populations. Key words:OSCC, FADD, FAS, DNA methylation, gene expression. PMID:25129245

  1. 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.

  2. DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass

    PubMed Central

    Navarro-Martín, Laia; Viñas, Jordi; Ribas, Laia; Díaz, Noelia; Gutiérrez, Arantxa; Di Croce, Luciano; Piferrer, Francesc

    2011-01-01

    Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination. PMID:22242011

  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. 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

  5. Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells

    PubMed Central

    Berdasco, María; Alcázar, Rubén; García-Ortiz, María Victoria; Ballestar, Esteban; Fernández, Agustín F.; Roldán-Arjona, Teresa; Tiburcio, Antonio F.; Altabella, Teresa; Buisine, Nicolas; Quesneville, Hadi; Baudry, Antoine; Lepiniec, Loïc; Alaminos, Miguel; Rodríguez, Roberto; Lloyd, Alan; Colot, Vincent; Bender, Judith; Canal, María Jesús; Esteller, Manel; Fraga, Mario F.

    2008-01-01

    Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells. PMID:18827894

  6. Promoter Methylation in Prostate Cancer and its Application for the Early Detection of Prostate Cancer Using Serum and Urine Samples

    PubMed Central

    Ahmed, Hafiz

    2010-01-01

    Prostate cancer is the second most common cancer and the second leading cause of cancer death in men. However, prostate cancer can be effectively treated and cured, if it is diagnosed in its early stages when the tumor is still confined to the prostate. Combined with the digital rectal examination, the PSA test has been widely used to detect prostate cancer. But, the PSA screening method for early detection of prostate cancer is not reliable due to the high prevalence of false positive and false negative results. Epigenetic alterations including hypermethylation of gene promoters are believed to be the early events in neoplastic progression and thus these methylated genes can serve as biomarkers for the detection of cancer from clinical specimens. This review discusses DNA methylation of several gene promoters during prostate carcinogenesis and evaluates the usefulness of monitoring methylated DNA sequences, such as GSTP1, RASSF1A, RARβ2 and galectin-3, for early detection of prostate cancer in tissue biopsies, serum and urine. PMID:20657713

  7. 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

  8. Identification of Highly Methylated Genes across Various Types of B-Cell Non-Hodgkin Lymphoma

    PubMed Central

    Bethge, Nicole; Honne, Hilde; Hilden, Vera; Trøen, Gunhild; Eknæs, Mette; Liestøl, Knut; Holte, Harald; Delabie, Jan; Smeland, Erlend B.; Lind, Guro E.

    2013-01-01

    Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients. PMID:24260260

  9. A combination of transcriptome and methylation analyses reveals embryologically-relevant candidate genes in MRKH patients

    PubMed Central

    2011-01-01

    Background The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is present in at least 1 out of 4,500 female live births and is the second most common cause for primary amenorrhea. It is characterized by vaginal and uterine aplasia in an XX individual with normal secondary characteristics. It has long been considered a sporadic anomaly, but familial clustering occurs. Several candidate genes have been studied although no single factor has yet been identified. Cases of discordant monozygotic twins suggest that the involvement of epigenetic factors is more likely. Methods Differences in gene expression and methylation patterns of uterine tissue between eight MRKH patients and eight controls were identified using whole-genome microarray analyses. Results obtained by expression and methylation arrays were confirmed by qRT-PCR and pyrosequencing. Results We delineated 293 differentially expressed and 194 differentially methylated genes of which nine overlap in both groups. These nine genes are mainly embryologically relevant for the development of the female genital tract. Conclusion Our study used, for the first time, a combined whole-genome expression and methylation approach to reveal the etiology of the MRKH syndrome. The findings suggest that either deficient estrogen receptors or the ectopic expression of certain HOXA genes might lead to abnormal development of the female reproductive tract. In utero exposure to endocrine disruptors or abnormally high maternal hormone levels might cause ectopic expression or anterior transformation of HOXA genes. It is, however, also possible that different factors influence the anti-Mullerian hormone promoter activity during embryological development causing regression of the Müllerian ducts. Thus, our data stimulate new research directions to decipher the pathogenic basis of MRKH syndrome. PMID:21619687

  10. 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

  11. Demethylation of the human eotaxin-3 gene promoter leads to the elevated expression of eotaxin-3

    PubMed Central

    Lim, Eunjin; Rothenberg, Marc E.

    2014-01-01

    DNA demethylation has been primarily studied in the context of development biology, cell fate, and cancer, with less attention on inflammation. Herein, we investigate the association between DNA methylation and production of the chemoattractant cytokine eotaxin-3 in the tissue of patients with allergic disease. Regions of the human eotaxin-3 promoter were found to be hypomethylated in primary epithelial cells obtained from allergic tissue compared with normal control tissue (CTL). The demethylation of a specific CpG site (designated CpG 2), which is juxtaposed to a key cyclic AMP-responsive element (CRE) site, was significantly demethylated in patient-derived compared to CTL-derived epithelial cells. Levels of methylation at CpG 2 inversely correlated with basal and IL-13–induced eotaxin-3 gene expression. Conversely, global inhibition of methylation with 5-azacytidine (5-AzaC) promoted eotaxin-3 production in association with decreasing CpG 2 methylation. In addition, the basal and IL-13-induced eotaxin-3 transcriptional activity was suppressed by promoter methylation using a methylation-free in vitro system. Further, electrophoretic mobility shift assays (EMSA) demonstrated that the attachment of CREB binding protein (CBP) and activating transcription factor 2 (ATF-2) to the CRE site was methylation dependent. Taken together, these data identify a contributory role for DNA methylation in regulating eotaxin-3 production in human allergic inflammation. PMID:24323578

  12. DNA methylation impacts gene expression and ensures hypoxic survival of Mycobacterium tuberculosis.

    PubMed

    Shell, Scarlet S; Prestwich, Erin G; Baek, Seung-Hun; Shah, Rupal R; Sassetti, Christopher M; Dedon, Peter C; Fortune, Sarah M

    2013-01-01

    DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N⁶-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces the expression of a number of genes. Each has a MamA site located at a conserved position relative to the sigma factor -10 binding site and transcriptional start site, suggesting that MamA modulates their expression through a shared, not locus-specific, mechanism. While strains lacking MamA grow normally in vitro, they are attenuated in hypoxic conditions, suggesting that methylation promotes survival in discrete host microenvironments. Interestingly, we demonstrate strikingly different patterns of DNA methyltransferase activity in different lineages of M. tuberculosis, which have been associated with preferences for distinct host environments and different disease courses in humans. Thus, MamA is the major functional adenine methyltransferase in M. tuberculosis strains of the Euro-American lineage while strains of the Beijing lineage harbor a point mutation that largely inactivates MamA but possess a second functional DNA methyltransferase. Our results indicate that MamA influences gene expression in M. tuberculosis and plays an important but strain-specific role in fitness during hypoxia.

  13. DNA-methylation gene network dysregulation in peripheral blood lymphocytes of schizophrenia patients.

    PubMed

    Auta, J; Smith, R C; Dong, E; Tueting, P; Sershen, H; Boules, S; Lajtha, A; Davis, J; Guidotti, A

    2013-10-01

    The epigenetic dysregulation of the brain genome associated with the clinical manifestations of schizophrenia (SZ) includes altered DNA promoter methylation of several candidate genes. We and others have reported that two enzymes that belong to the DNA-methylation/demethylation network pathways-DNMT1 (DNA-methyltransferase) and ten-eleven translocator-1(TET1) methylcytosine deoxygenase are abnormally increased in corticolimbic structures of SZ postmortem brain. The objective of this study was to investigate whether the expression of these components of the DNA-methylation-demethylation pathways known to be altered in the brain of SZ patients are also altered in peripheral blood lymphocytes (PBL). The data show that increases in DNMT1 and TET1 and in glucocorticoid receptor (GCortR) and brain derived neurotrophic factor (BDNF) mRNAs in PBL of SZ patients are comparable to those reported in the brain of SZ patients. The finding that the expressions of DNMT1 and TET1 are increased and SZ candidate genes such as BDNF and GCortR are altered in the same direction in both the brain and PBL together with recent studies showing highly correlated patterns of DNA methylation across the brain and blood, support the hypothesis that a common epigenetic dysregulation may be operative in the brain and peripheral tissues of SZ patients.

  14. DNA-Methylation Gene Network Dysregulation in Peripheral Blood Lymphocytes of Schizophrenia Patients

    PubMed Central

    Auta, J.; Smith, R.C.; Dong, E.; Tueting, P.; Sershen, H.; Boules, S.; Lajtha, A.; Davis, J.; Guidotti, A.

    2014-01-01

    The epigenetic dysregulation of the brain genome associated with the clinical manifestations of schizophrenia (SZ) includes altered DNA promoter methylation of several candidate genes. We and others have reported that two enzymes that belong to the DNA-methylation/demethylation network pathways -- DNMT1 (DNA-methyltransferase) and ten-eleven translocator-1(TET1) methylcytosine deoxygenase are abnormally increased in corticolimbic structures of SZ postmortem brain. The objective of this study was to investigate whether the expression of these components of the DNA-methylation demethylation pathways known to be altered in the brain of SZ patients are also altered in peripheral blood lymphocytes (PBL). The data show that increases in DNMT1 and TET1 and in glucocorticoid receptor (GCortR) and brain derived neurotrophic factor (BDNF) mRNAs in PBL of SZ patients are comparable to those reported in the brain of SZ patients. The finding that the expression of DNMT1and TET1are increased and SZ candidate genes such as BDNF and GCortR are altered in the same direction in both the brain and PBL together with recent studies showing highly correlated patterns of DNA methylation across brain and blood, support the hypothesis that a common epigenetic dysregulation may be operative in the brain and peripheral tissues of SZ patients PMID:23938174

  15. Hypermethylation of p16 and DAPK promoter gene regions in patients with non-invasive urinary bladder cancer

    PubMed Central

    Jabłonowski, Zbigniew; Reszka, Edyta; Gromadzińska, Jolanta; Wąsowicz, Wojciech; Sosnowski, Marek

    2011-01-01

    Introduction The aim of the study was to examine the frequency of methylation status in promoter regions of p16 and DAPK genes in patients with non-invasive bladder cancer. Material and methods Forty-two patients (92.9% men, 73.8% smokers, 71.4% T1G1, 19.1% T1G2, 9.5% T1G3) and 36 healthy controls were studied. Isolation of genomic DNA from blood serum and methylation-specific PCR (MSP) were applied. Methylation status – methylated and unmethylated promoter regions of p16 and DAPK genes were analysed. Results Seventeen out of 42 patients (40.5%) had the methylated p16 gene, while methylation of the DAPK gene was seen in 27 of 42 cases (64.3%). In 12 patients (28.6%) both analysed genes were methylated. A statistically significant (p = 0.046) higher frequency of DAPK gene methylation (71.4%) was observed in patients with lower grade (G1) bladder cancer. Conclusions Detection of the aberrant hypermethylation of DAPK and p16 genes in blood DNA from non-invasive bladder cancer patients might offer an effective means for earlier auxiliary diagnosis of the malignancy. PMID:22295037

  16. 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.

  17. 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

  18. Methylation of an intragenic alternative promoter regulates transcription of GARP.

    PubMed

    Haupt, Sonja; Söntgerath, Viktoria Sophie Apollonia; Leipe, Jan; Schulze-Koops, Hendrik; Skapenko, Alla

    2016-02-01

    Alternative promoter usage has been proposed as a mechanism regulating transcriptional and translational diversity in highly elaborated systems like the immune system in humans. Here, we report that transcription of human glycoprotein A repetitions predominant (GARP) in regulatory CD4 T cells (Tregs) is tightly regulated by two alternative promoters. An intragenic promoter contains several CpGs and acts as a weak promoter that is demethylated and initiates transcription Treg-specifically. The strong up-stream promoter containing a CpG-island is, in contrast, fully demethylated throughout tissues. Transcriptional activity of the strong promoter was surprisingly down-regulated upon demethylation of the weak promoter. This demethylation-induced transcriptional attenuation regulated the magnitude of GARP expression and correlated with disease activity in rheumatoid arthritis. Treg-specific GARP transcription was initiated by synergistic interaction of forkhead box protein 3 (Foxp3) with nuclear factor of activated T cells (NFAT) and was underpinned by permissive chromatin remodeling caused by release of the H3K4 demethylase, PLU-1. Our findings describe a novel function of alternative promoters in regulating the extent of transcription. Moreover, since GARP functions as a transporter of transforming growth factor β (TGFβ), a cytokine with broad pleiotropic traits, GARP transcriptional attenuation by alternative promoters might provide a mechanism regulating peripheral TGFβ to avoid unwanted harmful effects.

  19. Chemotaxis in Escherichia coli: Methylation of che gene products

    PubMed Central

    Silverman, Michael; Simon, Melvin

    1977-01-01

    The products of three chemotaxis-specific genes in Escherichia coli, cheM, cheD, and cheZ, are methylated. The cheZ gene codes for the synthesis of a 24,000 molecular weight polypeptide that appears in the cytoplasm. cheM codes for the synthesis of a membrane-bound polypeptide with a molecular weight of 61,000. cheD codes for another membrane-bound polypeptide with an apparent molecular weight of 64,000. CheM- mutants show chemotaxis toward some attractants (Tar- phenotype), while CheD- mutants respond to other attractants (Tsr- phenotype). The double mutant (CheD-, CheM-) does not respond to any attractant or repellent tested. Therefore, these polypeptides play a central role in chemotaxis. They collect information from two subsets of chemoreceptors and act as the last step in the chemoreceptor pathway and the first step in the general processing of signals for transmission to the flagellar rotor. It is suggested that they may be involved in both an initial process that reflects the instantaneous state of the chemoreceptors and in an integrative, adaptive process. Two other genes, cheX and cheW, are required for the methylation of the cheD and cheM gene products. Images PMID:333434

  20. LOXL2 Oxidizes Methylated TAF10 and Controls TFIID-Dependent Genes during Neural Progenitor Differentiation.

    PubMed

    Iturbide, Ane; Pascual-Reguant, Laura; Fargas, Laura; Cebrià, Joan Pau; Alsina, Berta; García de Herreros, Antonio; Peiró, Sandra

    2015-06-01

    Protein function is often regulated and controlled by posttranslational modifications, such as oxidation. Although oxidation has been mainly considered to be uncontrolled and nonenzymatic, many enzymatic oxidations occur on enzyme-selected lysine residues; for instance, LOXL2 oxidizes lysines by converting the ε-amino groups into aldehyde groups. Using an unbiased proteomic approach, we have identified methylated TAF10, a member of the TFIID complex, as a LOXL2 substrate. LOXL2 oxidation of TAF10 induces its release from its promoters, leading to a block in TFIID-dependent gene transcription. In embryonic stem cells, this results in the inactivation of the pluripotency genes and loss of the pluripotent capacity. During zebrafish development, the absence of LOXL2 resulted in the aberrant overexpression of the neural progenitor gene Sox2 and impaired neural differentiation. Thus, lysine oxidation of the transcription factor TAF10 is a controlled protein modification and demonstrates a role for protein oxidation in regulating pluripotency genes.

  1. Epigenomic Modifications Predict Active Promoters and Gene Structure in Toxoplasma gondii

    PubMed Central

    Gissot, Mathieu; Kelly, Krystyna A; Ajioka, James W; Greally, John M; Kim, Kami

    2007-01-01

    Mechanisms of gene regulation are poorly understood in Apicomplexa, a phylum that encompasses deadly human pathogens like Plasmodium and Toxoplasma. Initial studies suggest that epigenetic phenomena, including histone modifications and chromatin remodeling, have a profound effect upon gene expression and expression of virulence traits. Using the model organism Toxoplasma gondii, we characterized the epigenetic organization and transcription patterns of a contiguous 1% of the T. gondii genome using custom oligonucleotide microarrays. We show that methylation and acetylation of histones H3 and H4 are landmarks of active promoters in T. gondii that allow us to deduce the position and directionality of gene promoters with >95% accuracy. These histone methylation and acetylation “activation” marks are strongly associated with gene expression. We also demonstrate that the pattern of histone H3 arginine methylation distinguishes certain promoters, illustrating the complexity of the histone modification machinery in Toxoplasma. By integrating epigenetic data, gene prediction analysis, and gene expression data from the tachyzoite stage, we illustrate feasibility of creating an epigenomic map of T. gondii tachyzoite gene expression. Further, we illustrate the utility of the epigenomic map to empirically and biologically annotate the genome and show that this approach enables identification of previously unknown genes. Thus, our epigenomics approach provides novel insights into regulation of gene expression in the Apicomplexa. In addition, with its compact genome, genetic tractability, and discrete life cycle stages, T. gondii provides an important new model to study the evolutionarily conserved components of the histone code. PMID:17559302

  2. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver.

    PubMed

    Ohashi, Koji; Munetsuna, Eiji; Yamada, Hiroya; Ando, Yoshitaka; Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki; Suzuki, Koji; Teradaira, Ryoji; Hashimoto, Shuji

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status.

  3. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver.

    PubMed

    Ohashi, Koji; Munetsuna, Eiji; Yamada, Hiroya; Ando, Yoshitaka; Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki; Suzuki, Koji; Teradaira, Ryoji; Hashimoto, Shuji

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. PMID:26519879

  4. Genetic and epigenetic characterization of low-grade gliomas reveals frequent methylation of the MLH3 gene.

    PubMed

    Lhotska, Halka; Zemanova, Zuzana; Cechova, Hana; Ransdorfova, Sarka; Lizcova, Libuse; Kramar, Filip; Krejcik, Zdenek; Svobodova, Karla; Bystricka, Dagmar; Hrabal, Petr; Dohnalova, Alena; Michalova, Kyra

    2015-11-01

    Diffuse astrocytomas and oligodendrogliomas (WHO grade II) are the most common histological subtypes of low-grade gliomas (LGGs). Several molecular and epigenetic markers have been identified that predict tumor progression. Our aim was in detail to investigate the genetic and epigenetic background of LGGs and to identify new markers that might play a role in tumor behavior. Twenty-three patients with oligodendroglioma or oligoastrocytoma (LGO) and 22 patients with diffuse astrocytoma (LGA) were investigated using several molecular-cytogenetic and molecular methods to assess their copy number variations, mutational status and level of promoter methylation. The most frequent findings were a 1p/19q codeletion in 83% of LGO and copy-neutral loss of heterozygosity (CN-LOH) of 17p in 72% of LGA. Somatic mutations in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) genes were detected in 96% of LGO and 91% of LGA. The O-6-methylguanine-DNA-methyltransferase (MGMT) promoter was methylated in 83% of LGO and 59% of LGA. MutL homolog 3 (MLH3) promoter methylation was observed in 61% of LGO and 27% of LGA. Methylation of the MGMT promoter, 1p/19q codeletion, mutated IDH1, and CN-LOH of 17p were the most frequent genetic aberrations in LGGs. The findings were more diverse in LGA than in LGO. To the best of our knowledge, this is the first time description of methylation of the MLH3 gene promoter in LGGs. Further studies are required to determine the role of the methylated MLH3 promoter and the other aberrations detected.

  5. 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

  6. Characterization of CpG island DNA methylation of impairment-related genes in a rat model of cognitive aging

    PubMed Central

    Haberman, Rebecca P.; Quigley, Caitlin K.; Gallagher, Michela

    2012-01-01

    Cognitive abilities, particularly memory formation, vary substantially in the elderly, with some individuals exhibiting dramatic decline with age while others maintain function well into late life. Epigenetic modifications suggest an intriguing mechanism to account for the range of cognitive outcomes in aging as they are responsive to environmental influences and affect gene transcription in cognitively relevant brain regions. Leveraging a well-characterized rat model of neurocognitive aging that recapitulates the range of outcomes seen in humans, we previously identified gene expression profiles in the CA3 subregion of the hippocampus that distinguish between young and aged subjects as well as between impaired and preserved spatial memory function. To investigate the influence of epigenetics on these profiles, we examined genomic CpG DNA methylation in the promoter regions of three neurophysiologically relevant genes (Gabra5, Hspa5 and Syn1) whose expression levels decrease with age and correlate with spatial memory performance. Consistent with mRNA decreases, DNA methylation increased in aged rats relative to young in CpG dense regions of all target promoters examined. However, no correlation with cognition was found. Focused analysis of the Gabra5 gene found that methylation changes were limited to the CpG island and varied substantially across individual CpGs. Methylation at one CpG correlated with learning and demonstrated a significant difference between memory impaired aged rats and those with intact learning. These data provide evidence that broad age-dependent DNA methylation changes occur in CpG dense promoter regions of cognitively relevant genes but suggest that methylation at single CpGs may be more pertinent to individual cognitive differences. PMID:22869088

  7. 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

  8. 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

  9. 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

  10. Integrin α9 gene promoter is hypermethylated and downregulated in nasopharyngeal carcinoma

    PubMed Central

    Hu, Li-Fu; Moumad, Khalid; Pavlova, Tatiana V.; Kashuba, Vladimir; Almgren, Malin; Zabarovsky, Eugene R.; Ernberg, Ingemar

    2015-01-01

    Epigenetic silencing of tumor suppressor genes (TSGs) by promoter methylation can be an early event in the multi-step process of carcinogenesis. Human chromosome 3 contains clusters of TSGs involved in many cancer types including nasopharyngeal carcinoma (NPC), the most common cancer in Southern China. Among ten candidate TSGs identified in chromosome 3 using NotI microarray, ITGA9 and WNT7A could be validated. 5′-aza-2′ deoxycytidine treatment restored the expression of ITGA9 and WNT7A in two NPC cell lines. Immunostaining showed strong expression of these genes in the membrane and cytoplasm of adjacent control nasopharyngeal epithelium cells, while they were weakly expressed in NPC tumor cells. The ITGA9 promoter showed marked differentially methylation between tumor and control tissue, whereas no differentially methylation could be detected for the WNT7A promoter. The expression level of ITGA9 in NPC tumors was downregulated 4.9-fold, compared to the expression in control. ITGA9 methylation was detected by methylation specific PCR (MSP) in 56% of EBV positive NPC- cases with 100% specificity. Taken together, this suggests that ITGA9 might be a TSG in NPC that is involved in tumor cell biology. The possibility of using ITGA9 methylation as a marker for early detection of NPC should further be explored. PMID:26372814

  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. 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

  13. Methylome analysis reveals alterations in DNA methylation in the regulatory regions of left ventricle development genes in human dilated cardiomyopathy.

    PubMed

    Jo, Bong-Seok; Koh, In-Uk; Bae, Jae-Bum; Yu, Ho-Yeong; Jeon, Eun-Seok; Lee, Hae-Young; Kim, Jae-Joong; Choi, Murim; Choi, Sun Shim

    2016-08-01

    Dilated cardiomyopathy (DCM) is one of the main causes of heart failure (called cardiomyopathies) in adults. Alterations in epigenetic regulation (i.e., DNA methylation) have been implicated in the development of DCM. Here, we identified a total of 1828 differentially methylated probes (DMPs) using the Infinium 450K HumanMethylation Bead chip by comparing the methylomes between 18 left ventricles and 9 right ventricles. Alterations in DNA methylation levels were observed mainly in lowly methylated regions corresponding to promoter-proximal regions, which become hypermethylated in severely affected left ventricles. Subsequent mRNA microarray analysis showed that the effect of DNA methylation on gene expression regulation is not unidirectional but is controlled by the functional sub-network context. DMPs were significantly enriched in the transcription factor binding sites (TFBSs) we tested. Alterations in DNA methylation were specifically enriched in the cis-regulatory regions of cardiac development genes, the majority of which are involved in ventricular development (e.g., TBX5 and HAND1).

  14. 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.

  15. 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.

  16. Promoter methylation and large intragenic rearrangements of DPYD are not implicated in severe toxicity to 5-fluorouracil-based chemotherapy in gastrointestinal cancer patients

    PubMed Central

    2010-01-01

    Background Severe toxicity to 5-fluorouracil (5-FU) based chemotherapy in gastrointestinal cancer has been associated with constitutional genetic alterations of the dihydropyrimidine dehydrogenase gene (DPYD). Methods In this study, we evaluated DPYD promoter methylation through quantitative methylation-specific PCR and screened DPYD for large intragenic rearrangements in peripheral blood from 45 patients with gastrointestinal cancers who developed severe 5-FU toxicity. DPYD promoter methylation was also assessed in tumor tissue from 29 patients Results Two cases with the IVS14+1G > A exon 14 skipping mutation (c.1905+1G > A), and one case carrying the 1845 G > T missense mutation (c.1845G > T) in the DPYD gene were identified. However, DPYD promoter methylation and large DPYD intragenic rearrangements were absent in all cases analyzed. Conclusions Our results indicate that DPYD promoter methylation and large intragenic rearrangements do not contribute significantly to the development of 5-FU severe toxicity in gastrointestinal cancer patients, supporting the need for additional studies on the mechanisms underlying genetic susceptibility to severe 5-FU toxicity. PMID:20809970

  17. Methylation of the PTPRO Gene in Human Hepatocellular Carcinoma and Identification of VCP as Its Substrate

    PubMed Central

    Hsu, Shu-hao; Motiwala, Tasneem; Roy, Satavisha; Claus, Rainer; Mustafa, Mufaddal; Plass, Christoph; Freitas, Michael A.; Ghoshal, Kalpana; Jacob, Samson T.

    2014-01-01

    We have previously reported that the gene encoding protein tyrosine phosphatase receptor type-O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate-trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate-trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild-type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy. PMID:23533167

  18. EGFR Promoter Methylation, EGFR Mutation, and HPV Infection in Chinese Cervical Squamous Cell Carcinoma.

    PubMed

    Zhang, Wei; Jiang, Yinghao; Yu, Qingmiao; Qiang, Shaoying; Liang, Ping; Gao, Yane; Zhao, Xingye; Liu, Wenchao; Zhang, Ju

    2015-10-01

    Therapy strategy toward epidermal growth factor receptor (EGFR) inhibition in cervical cancer has been ongoing. EGFR promoter methylation status and EGFR tyrosine kinase inhibitor-sensitive mutations in cervical cancer may be significant for clinical outcome prediction using anti-EGFR treatment. In this study, EGFR tyrosine kinase inhibitor-sensitive mutations, EGFR exons 18, 19, and 21 mutations, were detected by sequencing in a total of 293 Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation status was detected by an EGFR asymmetric PCR and hybridization-fluorescence polarization assay and sequencing in 293 Chinese cervical squamous cell carcinoma tissue samples. High-risk human papillomavirus (HPV) genotypes in 293 Chinese cervical squamous cell carcinoma tissue samples were detected by an asymmetric GP5+/6+ PCR and hybridization-fluorescence polarization assay. No EGFR exons 18, 19, and 21 mutations were detected, EGFR promoter methylation status was identified in 98 samples, and HPV 16 infection was the first frequent HPV genotype. The methylated EGFR promoter was identified most frequently in cervical squamous cell carcinoma samples with HPV 16 infection (53.4%). Statistical significant difference of EGFR promoter methylation prevalence was found between HPV 16 and other HPV genotypes (P<0.01). This study suggested that there was no EGFR tyrosine kinase inhibitor-sensitive mutation in EGFR exons 18, 19, and 21 in Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation was common and it might be associated with HPV 16 infection in Chinese cervical squamous cell carcinoma. The results provided a novel understanding and an applicable pharmacogenomic tool for individualized management of cervical cancer patients.

  19. DNA methylation of Alzheimer disease and tauopathy-related genes in postmortem brain.

    PubMed

    Barrachina, Marta; Ferrer, Isidre

    2009-08-01

    DNA methylation occurs predominantly at cytosines that precede guanines in dinucleotide CpG sites; it is one of the most important mechanisms for epigenetic DNA regulation during normal development and for aberrant DNA in cancer. To determine the feasibility of DNA methylation studies in the postmortem human brain, we evaluated brain samples with variable postmortem artificially increased delays up to 48 hours. DNA methylation was analyzed in selected regions of MAPT, APP, and PSEN1 in the frontal cortex and hippocampus of controls (n=26) and those with Alzheimer disease at Stages I to II (n=17); Alzheimer disease at Stages III to IV (n=15); Alzheimer disease at Stages V to VI (n=12); argyrophilic grain disease (n=10); frontotemporal lobar degeneration linked to tau mutations (n=6); frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (n=4); frontotemporal lobar degeneration with motor neuron disease (n=3); Pick disease (n=3); Parkinson disease (n=8); dementia with Lewy bodies, pure form (n=5); and dementia with Lewy bodies, common form (n=15). UCHL1 (ubiquitin carboxyl-terminal hydrolase 1 gene) was analyzed in the frontal cortex of controls and those with Parkinson disease and related synucleinopathies. DNA methylation sites were very reproducible in every case. No differences in the percentage of CpG methylation were found between control and disease samples or among the different pathological entities in any region analyzed. Because small changes in methylation of DNA promoters in vulnerable cells might have not been detected in total homogenates, however, these results should be interpreted with caution, particularly as they relate to chronic degenerative diseases in which small modifications may be sufficient to modulate disease progression.

  20. DNMT3B isoforms without catalytic activity stimulate gene body methylation as accessory proteins in somatic cells.

    PubMed

    Duymich, Christopher E; Charlet, Jessica; Yang, Xiaojing; Jones, Peter A; Liang, Gangning

    2016-04-28

    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.

  1. 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.

  2. 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

  3. 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

  4. ERK and RAF1 genes: analysis of methylation and expression profiles in patients with oral squamous cell carcinoma.

    PubMed

    Kordi-Tamandani, D M; Saberi, E; Jamali, S; Ladiz, M A Rigi

    2014-01-01

    The Ras/RAF/MEK/ERK1/2 pathway is important in the control of growth signals, differentiation and cell survival. Over-expression and activation of this pathway have been reported in different types of cancer. This study analyses the promoter methylation and RNA expression profiles of ERK and RAF1 genes with risk of oral squamous cell carcinoma (OSCC) along with the promoter methylation status of ERK and RAF1 genes using a methylation-specific polymerase chain reaction (MS-PCR) in 86 paraffin-wax embedded samples of OSCC and 68 normal control tissues. Furthermore, ERK and RAF1 expression was analysed in 19 cases and 20 normal samples by real-time reverse transcription PCR. Frequency of promoter methylation was detected for ERK (93.02% and 6.98%) and RAF1 (95.35% and 4.65%) genes in cases and controls, respectively. Messenger RNA (mRNA) expression analysis indicated statistically significant difference between cases and controls for ERK (P < 0.002) and RAF1 (P < 0.006). The authors believe that this is the first report to show that expression of ERK and RAF1 is involved in risk of OSCC. PMID:25265753

  5. 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.

  6. 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

  7. Rapid Divergence of Prolamin Gene Promoters of Maize After Gene Amplification and Dispersal

    PubMed Central

    Wu, Yongrui; Messing, Joachim

    2012-01-01

    Seeds have evolved to accommodate complicated processes like senescence, dormancy, and germination. Central to these is the storage of carbohydrates and proteins derived from sugars and amino acids synthesized during photosynthesis. In the grasses, the bulk of amino acids is stored in the prolamin superfamily that specifically accumulates in seed endosperm during senescence. Their promoters contain a conserved cis-element, called prolamin-box (P-box), recognized by the trans-activator P-box binding factor (PBF). Because of the lack of null mutants in all grass species, its physiological role in storage–protein gene expression has been elusive. In contrast, a null mutant of another endosperm-specific trans-activator Opaque2 (O2) has been shown to be required for the transcriptional activation of subsets of this superfamily by binding to the O2 box. Here, we used RNAi to knockdown Pbf expression and found that only 27-kDa γ- and 22-kDa α-zein gene expression were affected, whereas the level of other zeins remained unchanged. Still, transgenic seeds had an opaque seed phenotype. Combination of PbfRNAi and o2 resulted in further reduction of α-zein expression. We also tested the interaction of promoters and constitutively expressed PBF and O2. Whereas transgenic promoters could be activated, endogenous promoters appeared to be not accessible to transcriptional activation, presumably due to differential chromatin states. Although analysis of the methylation of binding sites of PBF and O2 correlated with the expression of endogenous 22-kDa α-zein promoters, a different mechanism seems to apply to the 27-kDa γ-zein promoter, which does not undergo methylation changes. PMID:22798485

  8. 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.

  9. 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

  10. Relationships among folate, alcohol consumption, gene variants in one-carbon metabolism and p16 INK4a methylation and expression in healthy breast tissues

    PubMed Central

    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

  11. Pentraxin 3 (PTX3) promoter methylation associated with PTX3 plasma levels and neutrophil to lymphocyte ratio in coronary artery disease

    PubMed Central

    Guo, Tang-Meng; Huang, Li-Li; Liu, Kai; Ke, Li; Luo, Zhi-Jian; Li, Yun-Qiao; Chen, Xing-Lin; Cheng, Bei

    2016-01-01

    Background Pentraxin 3 (PTX3) is expressed in the heart under inflammatory conditions and plays an important role in atherogenesis. Patients with increased PTX3 levels may suffer from higher rates of cardiac events. Regulation of specific genes by promoter methylation is important in atherogenesis. The factors influencing PTX3 levels and the association between epigenetics and PTX3 levels have not been investigated. Methods Blood samples were collected from 64 patients admitted to the Department of Cardiology, 35 who had coronary artery disease (CAD), and 29 who were CAD-free. Plasma levels of PTX3 were measured by ELISA. PTX3 promoter methylation was evaluated via methyl-specific PCR. The severity of coronary artery lesion was evaluated by angiography. Results The level of PTX3 promoter methylation in the CAD group was 62.69% ± 20.57%, significantly lower than that of the CAD-free group, which was 72.45% ± 11.84% (P = 0.03). Lower PTX3 promoter methylation levels in the CAD group were associated with higher plasma PTX3 concentrations (r = −0.29, P = 0.02). Furthermore, lower PTX3 promoter methylation levels were associated with higher neutrophil to lymphocyte ratio (NLR) in men (r = −0.58, P = 0.002). Conclusions The present study provides new evidence that methylation of the PTX3 promoter is associated with PTX3 plasma levels and NLR in coronary artery disease. This study also shows that modification of epigenetics by chronic inflammation might be a significant molecular mechanism in the atherosclerotic processes that influence plasma PTX3 concentrations. PMID:27781062

  12. Sensitivity of human prostate cancer cells to chemotherapeutic drugs depends on EndoG expression regulated by promoter methylation

    PubMed Central

    Wang, Xiaoying; Tryndyak, Volodymyr; Apostolov, Eugene O.; Yin, Xiaoyan; Shah, Sudhir V.; Pogribny, Igor P.; Basnakian, Alexei G.

    2016-01-01

    Analysis of promoter sequences of all known human cytotoxic endonucleases showed that endonuclease G (EndoG) is the only endonuclease that contains a CpG island, a segment of DNA with high G+C content and a site for methylation, in the promoter region. A comparison of three human prostate cancer cell lines showed that EndoG is highly expressed in 22Rv1 and LNCaP cells. In PC3 cells, EndoG was not expressed and the EndoG CpG island was hypermethylated. The expression of EndoG correlated positively with sensitivity to cisplatin and etoposide, and the silencing of EndoG by siRNA decreased the sensitivity of the cells to the chemotherapeutic agents in the two EndoG-expressing cell lines. 5-aza-2′-deoxycytidine caused hypomethylation of the EndoG promoter in PC3 cells, induced EndoG mRNA and protein expression, and made the cells sensitive to both cisplatin and etoposide. The acetylation of histones by trichostatin A, the histone deacetylase inhibitor, induced EndoG expression in 22Rv1 cells, while it had no such effect in PC3 cells. These data are the first indication that EndoG may be regulated by methylation of its gene promoter, and partially by histone acetylation, and that EndoG is essential for prostate cancer cell death in the used models. PMID:18565644

  13. Changes in gene expression and methylation in the blood of patients with first-episode psychosis.

    PubMed

    Ota, Vanessa Kiyomi; Noto, Cristiano; Gadelha, Ary; Santoro, Marcos Leite; Spindola, Leticia Maria; Gouvea, Eduardo Sauerbronn; Stilhano, Roberta Sessa; Ortiz, Bruno Bertolucci; Silva, Patricia Natalia; Sato, João Ricardo; Han, Sang Won; Cordeiro, Quirino; Bressan, Rodrigo Affonseca; Belangero, Sintia Iole

    2014-11-01

    Schizophrenia is a severe mental health disorder with high heritability. The investigation of individuals during their first-episode psychosis (FEP), before the progression of psychotic disorders and especially before treatment with antipsychotic medications, is particularly helpful for understanding this complex disease and for the identification of potential biomarkers. In this study, we compared the expression of genes that are involved in neurotransmission and neurodevelopment of antipsychotic-naive FEP in the peripheral blood of patients (n=51) and healthy controls (n=51). In addition, we investigated the differentially expressed genes with respect to a) DNA methylation, b) the correlation between gene expression and clinical variables (PANSS), and c) gene expression changes after risperidone treatment. Expression levels of 11 genes were quantified with SYBR Green. For methylation analysis, bisulfite sequencing was performed. A significant decrease in GCH1 mRNA levels was observed in FEP patients relative to controls. Also, when we compare the FEP patients after risperidone treatment with controls, this difference remains significant, and no significant differences were observed in GCH1 mRNA levels when comparing patients before and after risperidone treatment. Additionally, although the differences were non-significant after Bonferroni correction, the expression of GCH1 seemed to be correlated with PANSS scores, and the GCH1 promoter region was more methylated in FEP than in controls, thus corroborating the results obtained at the mRNA level. Few studies have been conducted on GCH1, and future studies are needed to clarify its potential role in the progression of schizophrenia. PMID:25270546

  14. 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

  15. Methylation and Gene Expression Responses to Ethanol Feeding and Betaine Supplementation in the Cystathionine Beta Synthase-Deficient Mouse

    PubMed Central

    Medici, Valentina; Schroeder, Diane I.; Woods, Rima; LaSalle, Janine M.; Geng, Yongzhi; Shibata, Noreene M.; Peerson, Janet; Hodzic, Emir; Dayal, Sanjana; Tsukamoto, Hidekazu; Kharbanda, Kusum K.; Tillman, Brittany; French, Samuel W.; Halsted, Charles H.

    2014-01-01

    Background Alcoholic steatohepatitis (ASH) is caused in part by the effects of ethanol on hepatic methionine metabolism. Methods To investigate the phenotypic and epigenetic consequences of altered methionine metabolism in this disease, we studied the effects of 4-wk intragastric ethanol feeding with and without the methyl donor betaine in cystathionine beta synthase (CβS) heterozygous C57BL/6J mice. Results The histopathology of early ASH was induced by ethanol feeding and prevented by betaine supplementation, while ethanol feeding reduced and betaine supplementation maintained the hepatic methylation ratio of the universal methyl donor S-adenosylmethionine (SAM) to the methyltransferase inhibitor S-adenosylhomocysteine (SAH). MethylC-Seq genomic sequencing of heterozygous liver samples from each diet group found 2–4% reduced methylation in gene bodies but not promoter regions of all autosomes of ethanol fed mice, each of which were normalized in samples from mice fed the betaine supplemented diet. The transcript levels of inducible nitric oxide synthase (Nos2) and DNA methyltransferase 1 (Dnmt1) were increased, while those of peroxisome proliferator receptor-a (Pparα) were reduced in ethanol fed mice, and each was normalized in mice fed the betaine supplemented diet. DNA pyrosequencing of CβS heterozygous samples found reduced methylation in a gene body of Nos2 by ethanol feeding that was restored by betaine supplementation, and was correlated inversely with its expression and positively with SAM: SAH ratios. Conclusions The present studies have demonstrated relationships among ethanol induction of ASH with aberrant methionine metabolism that was associated with gene body DNA hypomethylation in all autosomes and was prevented by betaine supplementation. The data imply that ethanol-induced changes in selected gene transcript levels and hypomethylation in gene bodies during the induction of ASH is a result of altered methionine metabolism that can be reversed

  16. System-Wide Associations between DNA-Methylation, Gene Expression, and Humoral Immune Response to Influenza Vaccination.

    PubMed

    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

  17. System-Wide Associations between DNA-Methylation, Gene Expression, and Humoral Immune Response to Influenza Vaccination.

    PubMed

    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.

  18. 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

  19. The expression of IFN-gamma, IL-4, Foxp3 and perforin genes are not correlated with DNA methylation status in patients with immune thrombocytopenic purpura.

    PubMed

    Zhao, Haifeng; Du, Weiting; Wang, Donghai; Gu, Dongsheng; Xue, Feng; Ge, Jing; Sui, Tao; Yang, Renchi

    2010-01-01

    We investigated the relationship between the expression of IFN-gamma, IL-4, Foxp3 and perforin gene and the methylation status of their promoters in immune thrombocytopenic purpura (ITP) patients. Gene expression and DNA methylation were determined by quantitative PCR and bisulfite genomic sequencing, respectively. The expression of IFN-gamma was higher, while the expression of IL-4 was notably lower in ITP patients when compared with the controls. Most importantly, the Th1/Th2 (IFN-gamma/IL-4) was remarkably higher in ITP patients, showing that the ITP patients were mainly in the Th1 polarization response. Although the methylation rate of CpGs located at -409 of perforin was higher (p = 0.0376) and that of IFN-gamma were relatively higher (p = 0.0582) in ITP, we did not find the negative correlation between the expression of those genes and the methylation of their promoters. PMID:20100010

  20. 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

  1. hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

    PubMed Central

    2010-01-01

    Background Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells. Methods Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter. Results We found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity. By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls. Conclusions Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these

  2. Analysis of genes involved in methyl halide degradation in Aminobacter lissarensis CC495.

    PubMed

    Warner, Karen L; Larkin, Michael J; Harper, David B; Murrell, J Colin; McDonald, Ian R

    2005-10-01

    Aminobacter lissarensis CC495 is an aerobic facultative methylotroph capable of growth on glucose, glycerol, pyruvate and methylamine as well as the methyl halides methyl chloride and methyl bromide. Previously, cells grown on methyl chloride have been shown to express two polypeptides with apparent molecular masses of 67 and 29 kDa. The 67 kDa protein was purified and identified as a halomethane:bisulfide/halide ion methyltransferase. This study describes a single gene cluster in A. lissarensis CC495 containing the methyl halide utilisation genes cmuB, cmuA, cmuC, orf 188, paaE and hutI. The genes correspond to the same order and have a high similarity to a gene cluster found in Aminobacter ciceronei IMB-1 and Hyphomicrobium chloromethanicum strain CM2 indicating that genes encoding methyl halide degradation are highly conserved in these strains. PMID:16102909

  3. Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences

    PubMed Central

    Günther, Katharina; Rust, Mareike; Leers, Joerg; Boettger, Thomas; Scharfe, Maren; Jarek, Michael; Bartkuhn, Marek; Renkawitz, Rainer

    2013-01-01

    The heterogeneous collection of nucleosome remodelling and deacetylation (NuRD) complexes can be grouped into the MBD2- or MBD3-containing complexes MBD2–NuRD and MBD3–NuRD. MBD2 is known to bind to methylated CpG sequences in vitro in contrast to MBD3. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here, we show that MBD2–NuRD, in contrast to MBD3–NuRD, converts open chromatin with euchromatic histone modifications into tightly compacted chromatin with repressive histone marks. Genome-wide, a strong enrichment for MBD2 at methylated CpG sequences is found, whereas CpGs bound by MBD3 are devoid of methylation. MBD2-bound genes are generally lower expressed as compared with MBD3-bound genes. When depleting cells for MBD2, the MBD2-bound genes increase their activity, whereas MBD2 plus MBD3-bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes. PMID:23361464

  4. N-methyl-D-aspartate promotes the survival of cerebellar granule cells in culture.

    PubMed

    Balázs, R; Jørgensen, O S; Hack, N

    1988-11-01

    Our previous studies on the survival-promoting influence of elevated concentrations of extracellular K+ ([K+]e) on cultured cerebellar granule cells led to the proposal that depolarization in vitro mimics the effect of the earliest afferent inputs received by the granule cells in vivo. This, in turn, might be mediated through the stimulation of excitatory amino acid receptors, in particular the N-methyl-D-aspartate-preferring subtype gating ion channels which are also permeable to Ca2+. Here we report that N-methyl-D-aspartate indeed has a dramatic effect on the survival in culture of cells derived from dissociated cerebella of 7-8-day-old rats and cultured in media containing 'low' [K+]e (5-15 mM). In addition to the visual inspection of the cultures, the effect of N-methyl-D-aspartate was quantitatively evaluated, using estimates related to the number of viable cells (determination of DNA and of reduction rate of a tetrazolium salt). Furthermore, proteins which are relatively enriched in either nerve cells (neuronal cell adhesion molecule, D3-protein and synaptin) or in glia (glutamine synthetase) were also measured. The findings showed that the rescue of cells by N-methyl-D-aspartate involved primarily nerve cells and that the survival requirement for N-methyl-D-aspartate, as for high K+, developed between 2 and 4 days in vitro. The effect depended on both the concentration of N-methyl-D-aspartate and the degree of depolarization of the cells: both the potency and the efficacy of N-methyl-D-aspartate were increased as [K+]e was raised from 5 to 15 mM, at which range K+ on its own has little if any influence on granule cell survival. These characteristics are consistent with the voltage-dependence of ion conductance through the N-methyl-D-aspartate receptor-linked channel. The most pronounced effect of N-methyl-D-aspartate was obtained in the presence of 15 mM K+, when cell survival approached that obtained in 'control' cultures (grown in 25 mM K

  5. 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

  6. RUNX3 promoter methylation correlation with pathogenesis of hepatocellular carcinoma in Asians.

    PubMed

    Lu, W; Liu, Y; Liu, L-L; Zhuang, P-H

    2016-01-01

    The aim of this study was to elucidate the role of RUNX3 promoter methylation in the pathogenesis of hepatocellular carcinoma (HCC) among Asians. For this purpose, we performed a comprehensive search of Chinese and English language scientific literature databases using stringent selection criteria; ultimately, we identified relevant studies that specifically assessed the correlation between RUNX3 promoter methylation and HCC. All data was retrieved and analyzed by two independent investigators using the STATA software (version 12.0). Initially, 132 studies (103 in Chinese, 29 in English) were retrieved; 122 were eliminated through a stepwise filtering process. Finally, 10 studies conducted in Asian populations (5 Chinese, 4 Japanese, 1 Korean) fulfilled all the inclusion criteria of our meta-analysis. The studies included 588 HCC patients (641 cancer tissues; 593 adjacent normal tissues) and 184 healthy controls. We observed that RUNX3 promoter methylation was significantly higher in cancer tissues than in adjacent normal tissues (RR = 6.35, 95%CI = 3.62-11.14, P < 0.001) and normal control tissues (RR = 17.31, 95%CI = 7.08-42.34, P < 0.001). RUNX3 promoter methylation status did not differ significantly between patients with different TNM stages (RR = 0.88, 95%CI = 0.70-1.10, P = 0.269) and histological grades (RR = 0.86, 95%CI = 0.65-1.14, P = 0.304), suggesting that RUNX3 promoter methylation is linked to the origin of HCC but not to its progression from non-metastatic to metastatic stages. This in turn indicated that RUNX3 could be an early diagnostic marker distinguishing benign from malignant hepatocellular carcinoma. PMID:27420934

  7. 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. PMID:27635200

  8. DNA Methylation Impacts Gene Expression and Ensures Hypoxic Survival of Mycobacterium tuberculosis

    PubMed Central

    Shell, Scarlet S.; Prestwich, Erin G.; Baek, Seung-Hun; Shah, Rupal R.; Sassetti, Christopher M.; Dedon, Peter C.; Fortune, Sarah M.

    2013-01-01

    DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N6-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces the expression of a number of genes. Each has a MamA site located at a conserved position relative to the sigma factor −10 binding site and transcriptional start site, suggesting that MamA modulates their expression through a shared, not locus-specific, mechanism. While strains lacking MamA grow normally in vitro, they are attenuated in hypoxic conditions, suggesting that methylation promotes survival in discrete host microenvironments. Interestingly, we demonstrate strikingly different patterns of DNA methyltransferase activity in different lineages of M. tuberculosis, which have been associated with preferences for distinct host environments and different disease courses in humans. Thus, MamA is the major functional adenine methyltransferase in M. tuberculosis strains of the Euro-American lineage while strains of the Beijing lineage harbor a point mutation that largely inactivates MamA but possess a second functional DNA methyltransferase. Our results indicate that MamA influences gene expression in M. tuberculosis and plays an important but strain-specific role in fitness during hypoxia. PMID:23853579

  9. 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

  10. 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.

  11. 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

  12. Serotonin 1B Receptor Gene (HTR1B) Methylation as a Risk Factor for Callous-Unemotional Traits in Antisocial Boys

    PubMed Central

    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

  13. Sex differential in methylation patterns of selected genes in Singapore Chinese.

    PubMed

    Sarter, Barbara; Long, Tiffany I; Tsong, Wan H; Koh, Woon-Puay; Yu, Mimi C; Laird, Peter W

    2005-08-01

    To date there have been few reports of a gender difference in methylation levels of genes. When examining the methylation levels of four autosomal genes (ESR1, MTHFR, CALCA and MGMT) in the white blood cells of a random sample of Singapore Chinese Health Study cohort participants (n = 291), we encountered an unexpected gender differential. Using MethyLight technology, we calculated a gene-specific percentage of methylated reference (PMR) value, which quantified the relative level of gene methylation for each study subject (134 males and 157 females). Two summary methylation indices were constructed by assigning gene-specific rank scores. We then used ANCOVA to compare logarithmically transformed individual PMR values and summary methylation indices by age and gender simultaneously. Adjustment was made for plasma homocysteine. For ESR1, for which a large proportion of subjects were negative for methylation, we also used polytomous regression to compare methylation across age and gender. Increasing age and the male gender independently predicted increasing PMR values for CALCA and MGMT. For the MTHFR gene, male gender was associated with higher PMR values (P = 0.002), while age was not (P = 0.75). Neither age nor gender had any statistically significant influence on the PMR values for ESR1 (P = 0.13 and 0.96, respectively). Our data suggest that gender is at least as strong a predictor of methylation level in the four genes under study as age, with males showing higher PMRs.

  14. The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis

    PubMed Central

    Zhang, Cui-Jun; Zhou, Jin-Xing; Liu, Jun; Ma, Ze-Yang; Zhang, Su-Wei; Dou, Kun; Huang, Huan-Wei; Cai, Tao; Liu, Renyi; Zhu, Jian-Kang; He, Xin-Jian

    2013-01-01

    DNA methylation in transposons and other DNA repeats is conserved in plants as well as in animals. In Arabidopsis thaliana, an RNA-directed DNA methylation (RdDM) pathway directs de novo DNA methylation. We performed a forward genetic screen for suppressors of the DNA demethylase mutant ros1 and identified a novel Zinc-finger and OCRE domain-containing Protein 1 (ZOP1) that promotes Pol IV-dependent siRNA accumulation, DNA methylation, and transcriptional silencing. Whole-genome methods disclosed the genome-wide effects of zop1 on Pol IV-dependent siRNA accumulation and DNA methylation, suggesting that ZOP1 has both RdDM-dependent and -independent roles in transcriptional silencing. We demonstrated that ZOP1 is a pre-mRNA splicing factor that associates with several typical components of the splicing machinery as well as with Pol II. Immunofluorescence assay revealed that ZOP1 overlaps with Cajal body and is partially colocalized with NRPE1 and DRM2. Moreover, we found that the other development-defective splicing mutants tested including mac3a3b, mos4, mos12 and mos14 show defects in RdDM and transcriptional silencing. We propose that the splicing machinery rather than specific splicing factors is involved in promoting RdDM and transcriptional silencing. PMID:23524848

  15. Genetic Determinants for Promoter Hypermethylation in the Lungs of Smokers: A Candidate Gene-Based Study

    PubMed Central

    Leng, Shuguang; Stidley, Christine A.; Liu, Yushi; Edlund, Christopher K.; Willink, Randall P.; Han, Younghun; Landi, Maria Teresa; Thun, Michael; Picchi, Maria A.; Bruse, Shannon E.; Crowell, Richard E.; Van Den Berg, David; Caporaso, Neil E.; Amos, Christopher I.; Siegfried, Jill M.; Tesfaigzi, Yohannes; Gilliland, Frank D.; Belinsky, Steven A.

    2011-01-01

    The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here we identified genetic determinants for this epigenetic process and examined their biological effects on gene regulation. A two-stage approach involving discovery and replication was employed to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response in members of the Lovelace Smokers Cohort (n=1434). Molecular validation of three identified variants was conducted using primary bronchial epithelial cells. Association of study-wide significance (P<8.2×10−5) was identified for rs1641511, rs3730859, and rs1883264 in TP53, LIG1, and BIK, respectively. These SNPs were significantly associated with altered expression of the corresponding genes in primary bronchial epithelial cells. In addition, rs3730859 in LIG1 was also moderately associated with increased risk for lung cancer among Caucasian smokers. Together, our findings suggest that genetic variation in DNA replication and apoptosis pathways impacts the propensity for gene promoter hypermethylation in the aerodigestive tract of smokers. The incorporation of genetic biomarkers for gene promoter hypermethylation with clinical and somatic markers may improve risk assessment models for lung cancer. PMID:22139380

  16. 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...

  17. 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

  18. 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.

  19. 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

  20. Methylation patterns of immunoglobulin genes in lymphoid cells: correlation of expression and differentiation with undermethylation.

    PubMed

    Storb, U; Arp, B

    1983-11-01

    Different states of eukaryotic gene expression are often correlated with different levels of methylation of DNA sequences containing structural genes and their flanking regions. To assess the potential role of DNA methylation in the expression of immunoglobulin genes, which require complex rearrangements prior to expression, methylation patterns were examined in cell lines representing different stages of lymphocyte maturation. Methylation of the second cytosine in the sequence 5' C-C-G-G 3' was determined by using Hpa II/Msp I endonuclease digestion. Four CH genes (C mu, C delta, C gamma 2b, and C alpha), C kappa, V kappa, C lambda, and V lambda genes were analyzed. The results lead to the following conclusions: (i) transcribed immunoglobulin genes are undermethylated; (ii) the C gene allelic to an expressed C gene is always also undermethylated; and (iii) all immunoglobulin loci tend to become increasingly undermethylated as B cells mature.

  1. 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.

  2. 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

  3. Roux-En Y Gastric Bypass Surgery Induces Genome-Wide Promoter-Specific Changes in DNA Methylation in Whole Blood of Obese Patients

    PubMed Central

    Nilsson, Emil K.; Ernst, Barbara; Voisin, Sarah; Almén, Markus Sällman; Benedict, Christian; Mwinyi, Jessica; Fredriksson, Robert; Schultes, Bernd; Schiöth, Helgi B.

    2015-01-01

    Context DNA methylation has been proposed to play a critical role in many cellular and biological processes. Objective To examine the influence of Roux-en-Y gastric bypass (RYGB) surgery on genome-wide promoter-specific DNA methylation in obese patients. Promoters are involved in the initiation and regulation of gene transcription. Methods Promoter-specific DNA methylation in whole blood was measured in 11 obese patients (presurgery BMI >35 kg/m2, 4 females), both before and 6 months after RYGB surgery, as well as once only in a control group of 16 normal-weight men. In addition, body weight and fasting plasma glucose were measured after an overnight fast. Results The mean genome-wide distance between promoter-specific DNA methylation of obese patients at six months after RYGB surgery and controls was shorter, as compared to that at baseline (p<0.001). Moreover, postsurgically, the DNA methylation of 51 promoters was significantly different from corresponding values that had been measured at baseline (28 upregulated and 23 downregulated, P<0.05 for all promoters, Bonferroni corrected). Among these promoters, an enrichment for genes involved in metabolic processes was found (n = 36, P<0.05). In addition, the mean DNA methylation of these 51 promoters was more similar after surgery to that of controls, than it had been at baseline (P<0.0001). When controlling for the RYGB surgery-induced drop in weight (-24% of respective baseline value) and fasting plasma glucose concentration (-16% of respective baseline value), the DNA methylation of only one out of 51 promoters (~2%) remained significantly different between the pre-and postsurgery time points. Conclusions Epigenetic modifications are proposed to play an important role in the development of and predisposition to metabolic diseases, including type II diabetes and obesity. Thus, our findings may form the basis for further investigations to unravel the molecular effects of gastric bypass surgery. Clinical Trial

  4. 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

  5. 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.

  6. Inbreeding effects on gene-specific DNA methylation among tissues of Chinook salmon.

    PubMed

    Venney, Clare J; Johansson, Mattias L; Heath, Daniel D

    2016-09-01

    Inbreeding depression is the loss of fitness resulting from the mating of genetically related individuals. Traditionally, the study of inbreeding depression focused on genetic effects, although recent research has identified DNA methylation as also having a role in inbreeding effects. Since inbreeding depression and DNA methylation change with age and environmental stress, DNA methylation is a likely candidate for the regulation of genes associated with inbreeding depression. Here, we use a targeted, multigene approach to assess methylation at 22 growth-, metabolic-, immune- and stress-related genes. We developed PCR-based DNA methylation assays to test the effects of intense inbreeding on intragenic gene-specific methylation in inbred and outbred Chinook salmon. Inbred fish had altered methylation at three genes, CK-1, GTIIBS and hsp70, suggesting that methylation changes associated with inbreeding depression are targeted to specific genes and are not whole-genome effects. While we did not find a significant inbreeding by age interaction, we found that DNA methylation generally increases with age, although methylation decreased with age in five genes, CK-1, IFN-ɣ, HNRNPL, hsc71 and FSHb, potentially due to environmental context and sexual maturation. As expected, we found methylation patterns differed among tissue types, highlighting the need for careful selection of target tissue for methylation studies. This study provides insight into the role of epigenetic effects on ageing, environmental response and tissue function in Chinook salmon and shows that methylation is a targeted and regulated cellular process. We provide the first evidence of epigenetically based inbreeding depression in vertebrates. PMID:27480590

  7. Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations.

    PubMed

    Ryotokuji, Takeshi; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kurosawa, Saiko; Kobayashi, Yutaka; Kawata, Eri; Tajika, Kenji; Gomi, Seiji; Kanda, Junya; Kobayashi, Anna; Omori, Ikuko; Marumo, Atsushi; Fujiwara, Yusuke; Yui, Shunsuke; Terada, Kazuki; Fukunaga, Keiko; Hirakawa, Tsuneaki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Tamai, Hayato; Nakayama, Kazutaka; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti

    2016-09-01

    In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations - mutations of the genes that regulate gene expression through DNA methylation - is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (P<0.0001) and in patients with intermediate cytogenetic risk (P<0.0001) accompanied by a high white blood cell count (P=0.0032). DNA-methylation regulatory gene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia.

  8. Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations

    PubMed Central

    Ryotokuji, Takeshi; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kurosawa, Saiko; Kobayashi, Yutaka; Kawata, Eri; Tajika, Kenji; Gomi, Seiji; Kanda, Junya; Kobayashi, Anna; Omori, Ikuko; Marumo, Atsushi; Fujiwara, Yusuke; Yui, Shunsuke; Terada, Kazuki; Fukunaga, Keiko; Hirakawa, Tsuneaki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Tamai, Hayato; Nakayama, Kazutaka; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti

    2016-01-01

    In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations – mutations of the genes that regulate gene expression through DNA methylation – is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (P<0.0001) and in patients with intermediate cytogenetic risk (P<0.0001) accompanied by a high white blood cell count (P=0.0032). DNA-methylation regulatory gene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia. PMID:27247325

  9. 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.

  10. 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

  11. Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure.

    PubMed

    Ozden, Sibel; Turgut Kara, Neslihan; Sezerman, Osman Ugur; Durasi, İlknur Melis; Chen, Tao; Demirel, Goksun; Alpertunga, Buket; Chipman, J Kevin; Mally, Angela

    2015-12-01

    Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC-MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity.

  12. Promoter methylation is not associated with FLCN irregulation in lung cyst lesions of primary spontaneous pneumothorax.

    PubMed

    Ding, Yibing; Zou, Wei; Zhu, Chengchu; Min, Haiyan; Ma, Dehua; Chen, Baofu; Ye, Minhua; Pan, Yanqing; Cao, Lei; Wan, Yueming; Zhu, Qiuxiang; Xia, Haizhen; Zhang, Wenwen; Feng, Ying; Gao, Qian; Yi, Long

    2015-11-01

    Germline mutations in FLCN are responsible for ~10% of patients with primary spontaneous pneumothorax (PSP), characterized by multiple lung cysts in the middle/lower lobes and recurrent pneumothorax. These clinical features are also observed in a substantial portion of patients with sporadic PSP exhibiting no FLCN coding mutations. To assess the potential underlying mechanisms, 71 patients with PSP were selected, including 69 sporadic and 2 familial cases, who bared FLCN mutation‑like lung cysts, however, harbored no FLCN protein‑altering mutations. Notably, in a significant proportion of the patients, FLCN irregulation was observed at the transcript and protein levels. Genetic analyses of the cis‑regulatory region of FLCN were performed by sequencing and multiplex ligation‑dependent probe amplification assay. No inheritable DNA defect was detected, with the exception of a heterozygous deletion spanning the FLCN promoter, which was identified in a family with PSP. This mutation caused a reduction in the expression of FLCN in the lung cysts. Pedigree analysis demonstrated that haploinsufficiency of FLCN was pathogenic. To determine whether epigenetic mechanisms may be involved in the irregulation of FLCN, the promoter methylation status was measured in the remainder of the patients. No evidence of FLCN promoter methylation was demonstrated. The present study suggested that FLCN irregulation in lung cysts of PSP is not associated with promoter methylation.

  13. 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. PMID:27630389

  14. 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

  15. 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

  16. Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors