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Sample records for gene promoter methylation

  1. Sequential gene promoter methylation during HPV-induced cervical carcinogenesis.

    PubMed

    Henken, F E; Wilting, S M; Overmeer, R M; van Rietschoten, J G I; Nygren, A O H; Errami, A; Schouten, J P; Meijer, C J L M; Snijders, P J F; Steenbergen, R D M

    2007-11-19

    We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARbeta and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas.

  2. Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

    PubMed Central

    Henken, F E; Wilting, S M; Overmeer, R M; van Rietschoten, J G I; Nygren, A O H; Errami, A; Schouten, J P; Meijer, C J L M; Snijders, P J F; Steenbergen, R D M

    2007-01-01

    We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARβ and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas. PMID:17971771

  3. Promoter Methylation Analysis of IDH Genes in Human Gliomas.

    PubMed

    Flanagan, Simon; Lee, Maggie; Li, Cheryl C Y; Suter, Catherine M; Buckland, Michael E

    2012-01-01

    Mutations in isocitrate dehydrogenase (IDH)-1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132) or IDH2 (R172). But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a "toxic gain-of-function" to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumor suppressor gene. As most, if not all, tumor suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumors, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumor suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumors, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumors examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumors. These findings do not support a tumor suppressor role for IDH genes in human gliomas.

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

    PubMed

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

    2016-04-01

    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.

  5. 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...

  6. 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

  7. Role of CTGF gene promoter methylation in the development of hepatic fibrosis

    PubMed Central

    Shi, Cuicui; Li, Guangming; Tong, Yanyan; Deng, Yilin; Fan, Jiangao

    2016-01-01

    Connective tissue growth factor (CTGF) plays a critical role in the hepatic stellate cells (HSCs)-mediated development of hepatic fibrosis. Nevertheless, the effects of CTGF gene promoter methylation in the pathogenesis of hepatic fibrosis remain largely unknown. In the current study, we isolated and overexpressed CTGF in primary HSCs. We analyzed the CTGF gene promoter methylation inHSCs that undergo a phenotypic change into myofibroblast-like cellsthat express α-smooth muscle actin (α-SMA) in vitro and in vivo in a CCl4-induced rat hepatic fibrosis model. We found that CTGF promoted the phenotypic changes of HSCs into myofibroblasts in vitro, while inhibition of CTGF promoter methylation augmented the process, suggesting that CTGF gene promoter methylation may negatively regulate hepatic fibrosis. In vivo, CCl4 induced hepatic fibrosis in rats, and the severity of hepatic fibrosis inversely correlated with the levels of CTGF gene promoter methylation in HSCs. Together, our data demonstrate that CTGF gene promoter methylation may prevent the development of hepatic fibrosis, and low level of CTGF gene promoter methylation in HSCs may be a predisposing factor for developing liver fibrotic disease. PMID:27069546

  8. Quantitative assessment of the relationship between RASSF1A gene promoter methylation and bladder cancer (PRISMA)

    PubMed Central

    Zhan, Leyun; Zhang, Bingyi; Tan, Yaojun; Yang, Chengliang; Huang, Chenhong; Wu, Qiongya; Zhang, Yulin; Chen, Xiaobo; Zhou, Mi; Shu, Aihua

    2017-01-01

    Abstract Background: Methylation of the Ras-association domain family 1 isoform A (RASSF1A) gene promoter region is thought to participate in the initiation and development of many different cancers. However, in bladder cancer the role of RASSF1A methylation was unclear. To evaluate the relationship between RASSF1A methylation and bladder cancer, a quantitative assessment of an independent meta-analysis was performed. In addition, a DNA methylation microarray database from the cancer genome atlas (TCGA) project was used to validate the results of the meta-analysis. Methods: We searched published articles from computerized databases, and DNA methylation data were extracted from TCGA project. All data were analyzed by R software. Results: The results of the meta-analysis indicated that the frequency of RASSF1A gene methylation in bladder cancer patients is significantly higher than in healthy controls. The hazard ratio (HR) was 2.24 (95% CI = [1.45; 3.48], P = 0.0003) for overall survival (OS), and the RASSF1A gene promoter methylation status was strongly associated with the TNM stage and differentiation grade of the tumor. The similar results were also found by the data from TCGA project. Conclusion: There was a significant relationship between the methylation of the RASSF1A gene promoter and bladder cancer. Therefore, RASSF1A gene promoter methylation will be a potential biomarker for the clinical diagnosis of bladder cancer. PMID:28207521

  9. 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 %.

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. 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

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

    PubMed Central

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

    2014-01-01

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

  14. Analysis of APC and IGFBP7 promoter gene methylation in Swedish and Vietnamese colorectal cancer patients.

    PubMed

    Dimberg, Jan; Hong, Thai Trinh; Skarstedt, Marita; Löfgren, Sture; Zar, Niklas; Matussek, Andreas

    2013-01-01

    The tumour suppressor gene adenomatous polyposis coli (APC) is a key component that drives colorectal carcinogenesis. The reported DNA methylation in the promoter of APC varies greatly among studies of colorectal cancer (CRC) in different populations. Insulin-like growth factor binding protein 7 (IGFBP7), also known as IGFBP-related protein 1 (IGFBP-rP1), is expressed in various tissue types, including the lung, brain, prostate and gastrointestinal tract, and has been suggested to play a tumour suppressor role against colorectal carcinogenesis. Studies have indicated that IGFBP7 is inactivated by DNA methylation in human colon, lung and breast cancer. In the present study, we used the methylation-specific polymerase chain reaction to study the methylation status of the APC and IGFBP7 gene promoters in cancerous and paired normal tissue to evaluate its impact on clinical factors and association with ethnicity, represented by Swedish and Vietnamese CRC patients. We also investigated the distribution of CpG islands and the CpG dinucleotide density of each CpG island in the regions which were the subject of our investigation. Overall, normal tissue from Swedish patients exhibited a significantly higher frequency of IGFBP7 gene methylation in comparison with that of Vietnamese patients. Moreover, a significantly higher number of cancer tissues from Vietnamese individuals showed higher levels of methylation versus the paired normal tissue compared with that of Swedish patients. When we studied the methylation in cancer compared with the matched normal tissue in individuals, we found that a significantly higher number of Vietnamese patients had a higher degree of IGFBP7 gene methylation in cancer versus matched normal tissue in comparison with Swedish patients. Taken together, our results suggest that the methylation of the APC and IGFBP7 gene promoter region in cancerous tissue, in combination with the predominance of methylation in normal tissue, may serve as a

  15. 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

  16. Association between promoter methylation of DAPK gene and HNSCC: A meta-analysis

    PubMed Central

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

    2017-01-01

    Background The death-associated protein kinase (DAPK) is a tumor suppressor gene, which is a mediator of cell death of INF-γ–induced apoptosis. Aberrant methylation of DAPK promoter has been reported in patients with head and neck squamous cell carcinoma (HNSCC). However, the results of these studies are inconsistent. Hence, the present study aimed to evaluate the association between the promoter methylation of DAPK gene and HNSCC. Methods Relevant studies were systematically searched in PubMed, Web of Science, Ovid, and Embase. The association between DAPK promoter methylation and HNSCC was assessed by odds ratio (ORs) and 95% confidence intervals (CI). To evaluate the potential sources of heterogeneity, we conducted the meta-regression analysis and subgroup analysis. Results Eighteen studies were finally included in the meta-analysis. The frequency of DAPK promoter methylation in patients with HNSCC was 4.09-fold higher than the non-cancerous controls (OR = 3.96, 95%CI = 2.26–6.95). A significant association between DAPK promoter methylation and HNSCC was found among the Asian region and the Non-Asia region (Asian region, OR = 4.43, 95% CI = 2.29–8.58; Non-Asia region, OR = 3.39, 95% CI = 1.18–9.78). In the control source, the significant association between DAPK promoter methylation and HNSCC was seen among the autologous group and the heterogeneous group (autologous group, OR = 2.71, 95% CI = 1.49–4.93; heterogeneous group, OR = 9.50, 95% CI = 2.98–30.27). DAPK promoter methylation was significantly correlated with alcohol status (OR = 1.85, 95% CI = 1.07–3.21). Conclusion The results of this meta-analysis suggested that aberrant methylation of DAPK promoter was associated with HNSCC. PMID:28249042

  17. 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.

  18. Correcting Transcription Factor Gene Sets for Copy Number and Promoter Methylation Variations

    PubMed Central

    Rathi, Komal S.; Gaykalova, Daria A.; Hennesey, Patrick; Califano, Joseph A.; Ochs, Michael F.

    2014-01-01

    Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, since some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation and demonstrate the improvement in inference of transcription factor activity in a simulated data set based on the background expression observed in normal head and neck tissue. PMID:25195578

  19. Correcting transcription factor gene sets for copy number and promoter methylation variations.

    PubMed

    Rathi, Komal S; Gaykalova, Daria A; Hennessey, Patrick; Califano, Joseph A; Ochs, Michael F

    2014-09-01

    Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, as some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation, and demonstrate the improvement in inference of transcription factor activity in a simulated dataset based on the background expression observed in normal head and neck tissue.

  20. Dnmt3L antagonizes DNA methylation at bivalent promoters and favors DNA methylation at gene bodies in ESCs.

    PubMed

    Neri, Francesco; Krepelova, Anna; Incarnato, Danny; Maldotti, Mara; Parlato, Caterina; Galvagni, Federico; Matarese, Filomena; Stunnenberg, Hendrik G; Oliviero, Salvatore

    2013-09-26

    The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methyltransferase that cooperates with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESCs), but its function in these cells is unknown. Through genome-wide analysis of Dnmt3L knockdown in ESCs, we found that Dnmt3L is a positive regulator of methylation at the gene bodies of housekeeping genes and, more surprisingly, is also a negative regulator of methylation at promoters of bivalent genes. Dnmt3L is required for the differentiation of ESCs into primordial germ cells (PGCs) through the activation of the homeotic gene Rhox5. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low methylation levels at the H3K27me3 regions. Thus, in ESCs, Dnmt3L counteracts the activity of de novo DNA methylases to maintain hypomethylation at promoters of bivalent developmental genes.

  1. Identification of MGMT promoter methylation sites correlating with gene expression and IDH1 mutation in gliomas.

    PubMed

    Zhang, Jie; Yang, Jian-Hui; Quan, Jia; Kang, Xing; Wang, Hui-Juan; Dai, Peng-Gao

    2016-10-01

    O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation was reported to be an independent prognostic and predictive factor in glioma patients who received temozolomide treatment. However, the predictive value of MGMT methylation was recently questioned by several large clinical studies. The purpose of this study is to identify MGMT gene promoter CpG sites or region whose methylation were closely correlated with its gene expression to elucidate this contradictory clinical observations. The methylation status for all CpG dinucleotides in MGMT promoter and first exon region were determined in 42 Chinese glioma patients, which were then correlated with MGMT gene expression, IDH1 mutation, and tumor grade. In whole 87 CpG dinucleotides analyzed, three distinct CpG regions covering 28 CpG dinucleotides were significantly correlated with MGMT gene expression; 10 CpG dinucleotides were significantly correlated with glioma classification (p < 0.05). Isocitrate dehydrogenase 1 (IDH1) mutation and MGMT gene hypermethylation significantly co-existed, but not for MGMT gene expression. The validation cohort of gliomas treated with standard of care and comparison of the CpGs we identified with the current CpGs used in clinical setting will be very important for gliomas individual medicine in the future.

  2. BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

    PubMed Central

    2013-01-01

    Background Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have been found, the mechanisms by which these associations are established are still unclear. We studied genome-wide DNA methylation differences between colorectal tumors carrying a BRAF mutation and BRAF wildtype tumors. Results Using differential methylation hybridization on oligonucleotide microarrays representing 32,171 CpG-rich regions, we identified 1,770 regions with differential methylation between colorectal tumor and paired normal colon. Next, we compared the tumor/normal methylation ratios between different groups of patients. Related to CIMP, we identified 749 differentially methylated regions, of which 86% had a higher tumor/normal methylation ratio in the CIMP-positive group. We identified 758 regions with a BRAF mutation-specific methylation change, of which 96% had a higher tumor/normal methylation ratio in the BRAF mutant group. Among the genes affected by BRAF mutation-specific methylation changes, we found enrichment of several cancer-related pathways, including the PI3 kinase and Wnt signaling pathways. To focus on genes that are silenced in a tumor-specific rather than a lineage-specific manner, we used information on the epigenetic silencing mark H3K27me3 in embryonic stem (ES) cells. Among the genes showing BRAF mutation-specific promoter methylation but no H3K27me3 mark in ES cells were forkhead box (FOX) transcription factors associated with the PI3 kinase pathway, as well as MLH1 and SMO. Repression of FOXD3 gene expression in tumors could be related to its promoter hypermethylation. Conclusions We identified new BRAF mutation-specific methylation changes in colorectal cancer. Epigenetic downregulation of these targets may contribute to mutationally active BRAF

  3. 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

  4. Methylation of tumour suppressor gene promoters in the presence and absence of transcriptional silencing in high hyperdiploid acute lymphoblastic leukaemia.

    PubMed

    Paulsson, Kajsa; An, Qian; Moorman, Anthony V; Parker, Helen; Molloy, Gael; Davies, Teresa; Griffiths, Mike; Ross, Fiona M; Irving, Julie; Harrison, Christine J; Young, Bryan D; Strefford, Jon C

    2009-03-01

    Promoter methylation is a common phenomenon in tumours, including haematological malignancies. In the present study, we investigated 36 cases of high hyperdiploid (>50 chromosomes) acute lymphoblastic leukaemia (ALL) with methylation-specific multiplex ligase-dependent probe amplification to determine the extent of aberrant methylation in this subgroup. The analysis, which comprised the promoters of 35 known tumour suppressor genes, showed that 16 genes displayed abnormal methylation in at least one case each. The highest number of methylated gene promoters seen in a single case was thirteen, with all but one case displaying methylation for at least one gene. The most common targets were ESR1 (29/36 cases; 81%), CADM1 (IGSF4, TSLC1; 25/36 cases; 69%), FHIT (24/36 cases; 67%) and RARB (22/36 cases; 61%). Interestingly, quantitative reverse transcription-polymerase chain reaction showed that although methylation of the CADM1 and RARB promoters resulted in the expected pattern of downregulation of the respective genes, no difference could be detected in FHIT expression between methylation-positive and -negative cases. Furthermore, TIMP3 was not expressed regardless of methylation status, showing that aberrant methylation does not always lead to gene expression changes. Taken together, our findings suggest that aberrant methylation of tumour suppressor gene promoters is a common phenomenon in high hyperdiploid ALL.

  5. Gene promoter methylation assayed in exhaled breath, with differences in smokers and lung cancer patients

    PubMed Central

    2009-01-01

    Background There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs. Methods To investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5β promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)]. Results We found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5β and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5β, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5β promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (padj = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1)~-215~-113 and (2) -84 ~+26); while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the

  6. Core promoter analysis of porcine Six1 gene and its regulation of the promoter activity by CpG methylation.

    PubMed

    Wu, Wangjun; Ren, Zhuqing; Liu, Honglin; Wang, Linjie; Huang, Ruihua; Chen, Jie; Zhang, Lin; Li, Pinghua; Xiong, Yuanzhu

    2013-10-25

    Six1, an evolutionary conserved transcription factor, has been shown to play an important role in organogenesis and diseases. However, no reports were shown to investigate its transcriptional regulatory mechanisms. In the present study, we first identified porcine Six1 gene core promoter region (+170/-360) using luciferase reporter assay system and found that promoter activities were significantly higher in the mouse myoblast C2C12 cells than that in the mouse fibroblast C3H10T1/2 cells, implying that Six1 promoter could possess muscle-specific characteristics. Moreover, our results showed that promoter activities of Six1 were decreased as induction of differentiation of C2C12 cells, which was accompanied by the down-regulation of mRNA expression of Six1 gene. In addition, we found that the DNA methylation of Six1 promoters in vitro obviously influences the promoter activities and the DNA methylation level of Six1 promoter core region was negatively correlated to Six1 gene expression in vivo. Taken together, we preliminarily clarified transcriptional regulatory mechanisms of Six1 gene, which should be useful for investigating its subtle transcriptional regulatory mechanisms in the future. On the other hand, based on Six1 involved in tumorigenesis, our data also provide a genetic foundation to control the generation of diseases via pursuing Six1 as therapeutic target gene.

  7. Aberrant promoter methylation of cancer-related genes in human breast cancer

    PubMed Central

    Wu, Liang; Shen, Ye; Peng, Xianzhen; Zhang, Simin; Wang, Ming; Xu, Guisheng; Zheng, Xianzhi; Wang, Jianming; Lu, Cheng

    2016-01-01

    The clinical relevance of aberrant DNA methylation is being increasingly recognized in breast cancer. The present study aimed to evaluate the promoter methylation status of seven candidate genes and to explore their potential use as a biomarker for the diagnosis of breast cancer. A total of 70 Chinese patients with breast cancer were recruited, and matched with 20 patients with benign breast disease (BBD). Methylation-specific polymerase chain reaction was performed to measure the methylation status of selected genes. The protein expression of candidate genes was determined by immunohistochemistry. Hypermethylation of Breast cancer 1, early onset; DNA repair associated (BRCA1), glutathione S-transferase pi 1 (GSTP1), cyclin dependent kinase inhibitor 2A, O-6-methylguanine-DNA methyltransferase, phosphatase and tensin homolog, retinoic acid receptor beta 2 and cyclin D2 was observed to be more common in cancerous tissues (24.3, 31.4, 40.0, 27.1, 48.6, 55.7 and 67.1%, respectively) as compared with BBD controls (0.0, 0.0, 20.0, 25.0, 40.0, 40.0 and 45.0%, respectively). Immunohistochemical analysis demonstrated a correlation between the methylation of the target gene and downregulation of protein expression. When BRCA1 and GSTP1 were combined as the biomarker, the area under the receiver operating characteristic curve reached 0.721 (95% confidence interval, 0.616–0.827). The present findings indicated that promoter methylation of cancer-related genes was frequently observed in patients with breast cancer and was associated with various clinical features. Hypermethylation of BRCA1 and GSTP1 may be used as promising biomarkers for breast cancer. PMID:28105221

  8. Interleukin 6 supports the maintenance of p53 tumor suppressor gene promoter methylation.

    PubMed

    Hodge, David R; Peng, Benjamin; Cherry, James C; Hurt, Elaine M; Fox, Stephen D; Kelley, James A; Munroe, David J; Farrar, William L

    2005-06-01

    A strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. Interleukin 6 (IL-6) is an inflammatory cytokine known to play a role in the growth and survival of many types of tumors, yet the mechanisms employed by this pleomorphic cytokine to accomplish this feat are still poorly understood. Another important factor in tumor development seems to be the hypermethylation of CpG islands located within the promoter regions of tumor suppressor genes. This common epigenetic alteration enables tumor cells to reduce or inactivate the expression of important tumor suppressor and cell cycle regulatory genes. Here we show that in the IL-6-responsive human multiple myeloma cell line KAS 6/1, the promoter region of p53 is epigenetically modified by methyltransferases, resulting in decreased levels of expression. Furthermore, cells treated with IL-6 exhibit an increase in the expression of the DNA maintenance methylation enzyme, DNMT-1. The DNA methyltransferase inhibitor zebularine reverses the methylation of the p53 promoter, allowing the resumption of its expression. However, when zebularine is withdrawn from the cells, the reestablishment of the original CpG island methylation within the p53 promoter does not occur in the absence of IL-6, and cells which do not receive IL-6 eventually die, as p53 expression continues unchecked by remethylation. Interestingly, this loss of viability seems to involve not the withdrawal of cytokine, but the inability of the cell to resilence the promoter. Consistent with this model, when cells that express IL-6 in an autocrine fashion are subjected to identical treatment, p53 expression is reduced shortly after withdrawal of zebularine. Therefore, it seems IL-6 is capable of maintaining promoter methylation thus representing one of the possible mechanisms used by inflammatory mediators in the growth and survival of tumors.

  9. Gene promoter methylation and expression of Pin1 differ between patients with frontotemporal dementia and Alzheimer's disease.

    PubMed

    Ferri, Evelyn; Arosio, Beatrice; D'Addario, Claudio; Galimberti, Daniela; Gussago, Cristina; Pucci, Mariangela; Casati, Martina; Fenoglio, Chiara; Abbate, Carlo; Rossi, Paolo Dionigi; Scarpini, Elio; Maccarrone, Mauro; Mari, Daniela

    2016-03-15

    Frontotemporal Dementia (FTD) and Alzheimer's Disease (AD) share the accumulation of fibrillar aggregates of misfolded proteins. To better understand these neurodegenerative diseases and identify biomarkers in easily accessible cells, we investigated DNA methylation at Pin1 gene promoter and its expression in peripheral blood mononuclear cells of FTD patients. We found a lower gene expression of Pin1 with a higher DNA methylation in three CpG sites at Pin1 gene promoter analysed in FTD subjects, in contrast to a higher gene expression with a lower methylation in AD subjects and controls. These data suggest an important and distinct involvement of Pin1 in these two types of dementia.

  10. 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

  11. Promoter Methylation Pattern Controls Corticotropin Releasing Hormone Gene Activity in Human Trophoblasts

    PubMed Central

    Pan, Xin; Bowman, Maria; Scott, Rodney J.; Fitter, John; Smith, Roger

    2017-01-01

    Placental CRH production increases with advancing pregnancy in women and its course predicts gestational length. We hypothesized that CRH gene expression in the placenta is epigenetically controlled setting gestational trajectories characteristic of normal and pathological pregnancies. Here we determined histone modification and DNA methylation levels and DNA methylation patterns at the CRH promoter in primary trophoblast cultures by chromatin immunoprecipitation combined with clonal bisulfite sequencing and identified the transcriptionally active epialleles that associate with particular histone modifications and transcription factors during syncytialisation and cAMP-stimulation. CRH gene expression increased during syncytial differentiation and cAMP stimulation, which was associated with increased activating and decreased repressive histone modification levels at the promoter. DNA methylation levels remained unchanged. The nine CpGs of the CRH proximal promoter were partially and allele-independently methylated displaying many (>100) epialleles. RNA-polymerase-II (Pol-II) bound only to three particular epialleles in cAMP-stimulated cells, while phospho-cAMP response element-binding protein (pCREB) bound to only one epiallele, which was different from those selected by Pol-II. Binding of TATA-binding protein increased during syncytial differentiation preferentially at epialleles compatible with Pol-II and pCREB binding. Histone-3 acetylation was detected only at epialleles targeted by Pol-II and pCREB, while gene activating histone-4 acetylation and histone-3-lysine-4 trimethylation occurred at CRH epialleles not associated with Pol-II or pCREB. The suppressive histone-3-lysine-27 trimethyl and–lysine-9 trimethyl modifications showed little or no epiallele preference. The epiallele selectivity of activating histone modifications and transcription factor binding demonstrates the epigenetic and functional diversity of the CRH gene in trophoblasts, which is

  12. A three-gene signature for prognosis in patients with MGMT promoter-methylated glioblastoma.

    PubMed

    Wang, Wen; Zhang, Lu; Wang, Zheng; Yang, Fan; Wang, Haoyuan; Liang, Tingyu; Wu, Fan; Lan, Qing; Wang, Jiangfei; Zhao, Jizong

    2016-10-25

    Glioblastoma is the most malignant tumor and has high mortality rate. The methylated prompter of MGMT results in chemotherapy sensitivity for these patients. However, there are still other factors that affected the prognosis for the glioblastoma patients with similar MGMT methylation status. We developed a signature with three genes screened from the whole genome mRNA expression profile from Chinese Glioma Genome Atlas (CGGA) and RNAseq data from The Cancer Genome Atlas (TCGA). Patients with MGMT methylation in low risk group had longer survival than those in high risk group (median overall survival 1074 vs. 372 days; P = 0.0033). Moreover, the prognostic value of the signature was significant difference in cohorts stratified by MGMT methylation and chemotherapy (P=0.0473), while there is no significant difference between low and high risk group or unmethylated MGMT patients without chemotherapy. Multivariate analysis indicated that the risk score was an independent prognosis factor (P = 0.004). In conclusion, our results showed that the signature has prognostic value for patients with MGMT promoter-methylated glioblastomas based on bioinformatics analysis.

  13. The influence of one-carbon metabolism on gene promoter methylation in a population-based breast cancer study

    PubMed Central

    Gammon, Marilie D; Jefferson, Elizabeth; Zhang, Yujing; Cho, Yoon Hee; Wetmur, James G; Teitelbaum, Susan L; Bradshaw, Patrick T; Terry, Mary Beth; Garbowski, Gail; Hibshoosh, Hanina; Neugut, Alfred I; Santella, Regina M

    2011-01-01

    Abnormal methylation in gene promoters is a hallmark of the cancer genome; however, factors that may influence promoter methylation have not been well elucidated. As the one-carbon metabolism pathway provides the universal methyl donor for methylation reactions, perturbation of this pathway might influence DNA methylation and, ultimately, affect gene functions. Utilizing approximately 800 breast cancer tumor tissues from a large population-based study, we investigated the relationships between dietary and genetic factors involved in the one-carbon metabolism pathway and promoter methylation of a panel of 13 breast cancer-related genes. We found that CCND2, HIN1 and CHD1 were the most “dietary sensitive” genes, as methylation of their promoters was associated with intakes of at least two out of the eight dietary methyl factors examined. On the other hand, some micronutrients (i.e., B2 and B6) were more “epigenetically active” as their intake levels correlated with promoter methylation status in 3 out of the 13 breast cancer genes evaluated. Both positive (hypermethylation) and inverse (hypomethylation) associations with high micronutrient intake were observed. Unlike what we saw for dietary factors, we did not observe any clear patterns between one-carbon genetic polymorphisms and the promoter methylation status of the genes examined. Our results provide preliminary evidence that one-carbon metabolism may have the capacity to influence the breast cancer epigenome. Given that epigenetic alterations are thought to occur early in cancer development and are potentially reversible, dietary modifications may offer promising venues for cancer intervention and prevention. PMID:22048254

  14. Promoter methylation and histone modifications affect the expression of the exogenous DsRed gene in transgenic goats.

    PubMed

    Nuo, M T; Yuan, J L; Yang, W L; Gao, X Y; He, N; Liang, H; Cang, M; Liu, D J

    2016-08-29

    Transgene silencing, which is common in transgenic plants and animals, limits the generation and application of genetically modified organisms, and is associated with the exogenous gene copy number, the methylation status of its promoters, and histone modification abnormalities. Here, we analyzed the expression of the exogenous gene DsRed and the methylation status of its cytomegalovirus (CMV) promoter in six healthy transgenic cashmere goats and transgenic nuclear donor cells. The CMV promoter exhibited high methylation levels (74.4-88.2%) in four of the goats, a moderate methylation level (58.7%) in one, and a low methylation level (21.2%) in one, while the methylation level of the transgenic nuclear donor cells was comparatively low (14.3%). DsRed expression was negatively correlated with promoter methylation status. Transgenic cashmere goats carried one to three copies of the CMV promoter fragment and one to six copies of the DsRed fragment, but copy number showed no obvious correlation with DsRed expression. After treatment with the methylation inhibitor 5-azacytidine, DsRed expression in transgenic goat cells significantly increased and CMV promoter methylation significantly decreased; this indicated an inverse correlation between promoter methylation status and DsRed expression. After treatment with the histone deacetylase inhibitor trichostatin A, DsRed expression increased, indicating that an abnormal histone modification in transgenic goats is also involved in exogenous gene silencing. These findings indicate the potential of trichostatin A and 5-azacytidine to rescue the biological activity of silenced exogenous transgenes in adult-derived transgenic cells under culture conditions.

  15. In vitro methylation of nuclear respiratory factor-2 binding sites suppresses the promoter activity of the human TOMM70 gene.

    PubMed

    Blesa, José R; Hegde, Anita A; Hernández-Yago, José

    2008-12-31

    TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported that two binding sites for transcription factor NRF-2 in the promoter region of the human TOMM70 gene are essential in activating transcription (Blesa et al., Mitochondrion 2004; 3:251-59. Blesa et al., Biochem Cell Biol 2006; 84:813-22). This region contains thirteen CpG methylation sites, three of which occur in the sequence 5'-CCGG-3' that is specifically recognized by HpaII methylase which modifies the internal cytosine residue. Interestingly, each NRF-2 site contains one CCGG sequence, allowing specific methylation of the NRF-2 sites and, therefore, providing an ideal model to study how methylation of these sites affects promoter activity. In this paper we report that site-specific methylation of the NRF-2 binding sites in the TOMM70 promoter down-regulated expression of a luciferase reporter in HeLa S3 cells. Electrophoretic mobility shift assays confirmed abrogation of NRF-2 binding at the methylated sites. These results suggest that methylation of the TOMM70 promoter in mammalian cells may silence TOMM70 expression. However, studies of methylation degree on DNAs from different sources found no methylation in the promoter regions of TOMM70 and other TOMM/TIMM family genes. Thus, although in vitro methylation inactivates the expression of TOMM70, our results suggest that this is not the mechanism modulating its expression in vivo. Since a number of nuclear genes encoding mitochondrial translocases have NRF-2 binding sequences containing CpG methylation sites, a possible role of methylation as a regulatory mechanism of mitochondrial biogenesis can be ruled out.

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

    PubMed

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

    2016-08-01

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

  17. Construction and Analysis of an Adipose Tissue-Specific and Methylation-Sensitive Promoter of Leptin Gene.

    PubMed

    Zhang, Qinkai; Xu, Denggao; Zhang, Min; Dong, Xiao; Dong, Huansheng; Pan, Qingjie

    2016-11-01

    DNA methylation plays a very important role in the regulation of gene expression. Under general situations, methylation in a gene promoter region is frequently accompanied by transcriptional suppression, and those genes that are highly methylated display the phenomenon of low expression. In contrast, those genes whose methylation level is low display the phenomenon of active expression. In this study, we conducted DNA methylation analysis on the CpG sites within the promoter regions of five adipose tissue-specific transcriptional factors-Adiponectin, Chemerin, Leptin, Smaf-1, and Vaspin-and examined their messenger RNA (mRNA) expression levels in different mouse tissues. We also performed analyses on the correlation between the DNA methylation levels of these genes and their mRNA expression levels in these tissues. The correlation coefficient for Leptin was the highest, and it displayed a high expression in an adipose tissue-specific manner. Thus, we cloned the regulatory region of Leptin gene and incorporated its promoter into the eukaryotic expression vector pEGFP-N1 and constructed a recombinant plasmid named pEGFP-N1-(p-Lep). This recombinant plasmid was first verified by DNA sequencing and then transfected into mouse pre-adipocytes via electroporation. Measurement of the activity of luciferase (reporter) indicated that p-Lep was capable of driving the expression of the reporter gene. This study has paved a solid basis for subsequent studies on generating transgenic animals.

  18. CDH1 promoter methylation correlates with decreased gene expression and poor prognosis in patients with breast cancer.

    PubMed

    Liu, Jian; Sun, Xin; Qin, Sida; Wang, Huangzhen; DU, Ning; Li, Yanbo; Pang, Yamei; Wang, Cuicui; Xu, Chongwen; Ren, Hong

    2016-04-01

    The E-cadherin gene (CDH1) is associated with poor prognosis and metastasis in patients with breast cancer, and methylation of its promoter is correlated with decreased gene expression. However, there is currently no direct evidence that CDH1 promoter methylation indicates poor prognosis in patients with breast cancer. In the present study, methylation-specific polymerase chain reaction (PCR) was applied to detect the methylation status of the CDH1 promoter in 137 primary breast cancer, 85 matched normal breast tissue and 13 lung metastasis specimens. Reverse transcription-quantitative PCR was used to assess the relative expression levels of CDH1 mRNA, and correlation analysis between CDH1 methylation status, and gene expression, clinicopathological characteristics and patient survival was performed. Methylation of CDH1 was identified in 40.9% (56/137) of primary breast cancer specimens, 61.5% (8/13) of lung metastasis specimens and none of the matched normal breast specimens. The downregulation of CDH1 mRNA and E-cadherin protein expression were identified to be significantly correlated with CDH1 methylation (P<0.05). In addition, CDH1 methylation was significantly associated with lymph node metastasis and estrogen receptor status of patients (P<0.05). In univariate analyses, patients with CDH1 methylation exhibited poor overall survival (OS) and disease-free survival (DFS; P<0.05). Furthermore, multivariate analyses revealed that CDH1 methylation was an independent prognostic factor predicting poor OS (HR, 1.737; 95% CI, 0.957-3.766; P=0.041) and DFS (HR, 2.018; 95% CI, 2.057-3.845; P=0.033) in patients with breast cancer. Therefore, the present study suggests that CDH1 promoter methylation may be correlated with breast carcinogenesis and indicates poor prognosis in patients with breast cancer.

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

    PubMed

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

    2016-05-01

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

  20. DNA methylation abnormalities at gene promoters are extensive and variable in the elderly and phenocopy cancer cells.

    PubMed

    Gautrey, Hannah E; van Otterdijk, Sanne D; Cordell, Heather J; Mathers, John C; Strathdee, Gordon

    2014-07-01

    Abnormal patterns of DNA methylation are one of the hallmarks of cancer cells. The process of aging has also been associated with similar, albeit less dramatic, changes in methylation patterns, leading to the hypothesis that age-related changes in DNA methylation may partially underlie the increased risk of cancer in the elderly. Here we studied 377 participants aged 85 yr from the Newcastle 85+ Study to investigate the extent of, and interindividual variation in, age-related changes in DNA methylation at specific CpG islands. Using highly quantitative pyrosequencing analysis, we found extensive and highly variable methylation of promoter-associated CpG islands with levels ranging from 4% to 35%, even at known tumor suppressor genes such as TWIST2. Furthermore, the interindividual differences in methylation seen across this elderly population phenocopies multiple features of the altered methylation patterns seen in cancer cells. Both aging- and cancer-related methylation can occur at similar sets of genes, both result in the formation of densely methylated, and likely transcriptionally repressed, alleles, and both exhibit coordinate methylation across multiple loci. In addition, high methylation levels were associated with subsequent diagnosis of leukemia or lymphoma during a 3-yr follow-up period (P=0.00008). These data suggest that the accumulation of age-related changes in promoter-associated CpG islands may contribute to the increased cancer risk seen during aging.-Gautrey, H. E., van Otterdijk, S. D., Cordell, H. J., Newcastle 85+ study core team, Mathers, J. C., Strathdee, G. DNA methylation abnormalities at gene promoters are extensive and variable in the elderly and phenocopy cancer cells.

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

    PubMed

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

    2015-12-01

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

  2. Methyl-CpG-Binding Protein MeCP2 Represses Sp1-Activated Transcription of the Human Leukosialin Gene When the Promoter Is Methylated

    PubMed Central

    Kudo, Shinichi

    1998-01-01

    Human leukosialin (CD43) is expressed in a cell lineage-specific as well as a differentiation stage-specific fashion. The leukosialin promoter, made up of an Sp1 binding site and a sequence similar to that of an initiator, possesses high transcriptional potential. Previous data have demonstrated that the leukosialin gene is down-regulated in nonproducing cells by DNA methylation. In this paper the repressive mechanism of DNA methylation in expression systems is reported. In vitro DNA methylation with SssI (CpG) methylase of leukosialin-chloramphenicol acetyltransferase (CAT) constructs drastically reduced transcriptional activities in stable transfection systems with the human HeLa and Jurkat cell lines. On the other hand, the transcriptional repression by in vitro methylation was less pronounced in Drosophila melanogaster cells, which lack genomic methylation. In these cells, Sp1 could transactivate equally well both the unmethylated and methylated leukosialin promoter. In order to test whether one of the methyl-CpG-binding proteins, MeCP2, is responsible for transcriptional repression of the leukosialin gene, I isolated the human MeCP2 cDNA (encoding 486 amino acid residues) and expressed it in Drosophila cells. I found that MeCP2 substantially inhibited Sp1-activated transcription when the leukosialin promoter was methylated. The level of repression was directly proportional to the amount of MeCP2 expression vector transfected. Analysis of C-terminal deletion mutants of MeCP2 showed that repressive activity of Sp1 transactivation is localized to the N-terminal region consisting of amino acid residues 1 to 193, which encompass the methyl-binding domain. These results suggest that interference with Sp1 transactivation by MeCP2 is an important factor in the down-regulation of leukosialin gene expression by DNA methylation. PMID:9710633

  3. [DNA methylation in the promoter regions of the laminin family genes in normal and breast carcinoma tissues].

    PubMed

    Simonova, O A; Kuznetsova, E B; Poddubskaya, E V; Kekeeva, T V; Kerimov, R A; Trotsenko, I D; Tanas, A S; Rudenko, V V; Alekseeva, E A; Zaletayev, D V; Strelnikov, V V

    2015-01-01

    Extracellular glycoproteins of the laminin family are essential components of basement membranes involved in a number of biological processes, including tissue differentiation, wound healing, and tumorigenesis. We present the first comprehensive study of promoter methylation status of the genes encoding laminin chains in normal tissues (peripheral blood leucocytes, buccal epithelial cells, autopsy breast tissue samples) and in breast carcinoma samples. Based on the results of this study, we divide laminin genes into three categories. Genes, constitutively methylated in breast tissues include LAMA3A, LAMB2, LAMB3, and LAMC2. Genes prone to abnormal methylation in breast carcinoma include LAMA1, LAMA2, LAMA3B, LAMA4, LAMB1, and LAMC3. Genes that are rarely if ever methylated in breast carcinoma include LAMA5 and LAMC1. The constitutively methylated group includes all of the genes that encode subunits of laminin-5 (the historical name of laminin 332), the promoters of which were previously considered unmethylated in normal tissues and prone to abnormal methylation in breast cancer.

  4. Differential Promoter Methylation and Histone Modification Contribute to the Brain Specific Expression of the Mouse Mbu-1 Gene

    PubMed Central

    Kim, Byungtak; Kang, Seongeun; Kim, Sun Jung

    2012-01-01

    Mbu-1 (Csrnp-3) is a mouse gene that was identified in our previous study as showing highly restricted expression to the central nervous system. In this study, to elucidate the regulatory mechanism for tissue specificity of the gene, epigenetic approaches that identify the profiles of CpG methylation, as well as histone modifications at the promoter region were conducted. Methylation-specific PCR revealed that the CpG sites in brain tissues from embryo to adult stages showed virtually no methylation (0.052–0.67%). Lung (9.0%) and pancreas (3.0%) also showed lower levels. Other tissues such as liver, kidney, and heart showed much higher methylation levels ranging from approximately 39–93%. Treatment of 5-aza-2′-deoxycytidine (5-Aza-dC) significantly decreased promoter methylation, reactivating Mbu-1 expression in NG108-15 and Neuro-2a neuronal cells. Chromatin immunoprecipitation assay revealed that 5-Aza-dC decreased levels of acetylated H3K9 and methylated H3K4, and increased methylated H3K9. This result indicates that CpG methylation converses with histone modifications in an opposing sense of regulating Mbu-1 expression. PMID:23076708

  5. 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…

  6. 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

  7. 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.

  8. 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.

  9. Altered expression of PTCH and HHIP in gastric cancer through their gene promoter methylation: novel targets for gastric cancer.

    PubMed

    Song, Yu; Tian, Ye; Zuo, Yun; Tu, Jian-Cheng; Feng, Yu-Fang; Qu, Chen-Jiang

    2013-04-01

    Human hedgehog-interacting protein (HHIP) and protein patched homolog (PTCH) are two negative regulators of the hedgehog signal, however, the mechanism of action in gastric cancer is unknown. Methylation of TSG promoters has been considered as a causative mechanism of tumorigenesis. In the present study, we first determined the expression of PTCH and HHIP mRNA and protein in gastric cancer tissues and adjacent normal tissues, and then detected methylation of the two genes to associate their expression and gene promoter methylation in gastric cancer. Expression in gastric cancer tissues and the cancer cells (AGS) were evaluated by reverse transcription-PCR (RT-PCR), qRT-PCR and IHC, while the methylation expression was valued by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). Cell viability and apoptosis were analyzed by MTT assay and flow cytometry following treatment with 5-aza-dc. Results showed that PTCH and HHIP expression was reduced in gastric cancer tissues that were not associated with clinical features. Moreover, methylation of the promoters was reversely correlated with the expression. Following treatment with 5-aza-dc, AGS reduced cell viability and induced apoptosis, which is associated with upregulation of HHIP expression. The data demonstrated that loss of expression of HHIP and PTCH is associated with the methylation of gene promoters. In addition, 5-aza-dc-induced apoptosis correlated with the upregulation of HHIP expression in AGS. The findings demonstrated that the PTCH and HHIP genes may be novel targets for the control of gastric cancer.

  10. Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer.

    PubMed

    Kuhmann, Christine; Li, Carmen; Kloor, Matthias; Salou, Mariam; Weigel, Christoph; Schmidt, Christopher R; Ng, Linda W C; Tsui, Wendy W Y; Leung, Suet Y; Yuen, Siu T; Becker, Natalia; Weichenhan, Dieter; Plass, Christoph; Schmezer, Peter; Chan, Tsun L; Popanda, Odilia

    2014-04-15

    Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

  11. Promoter methylation of yes-associated protein (YAP1) gene in polycystic ovary syndrome

    PubMed Central

    Jiang, Li-Le; Xie, Juan-Ke; Cui, Jin-Quan; Wei, Duo; Yin, Bao-Li; Zhang, Ya-Nan; Chen, Yuan-Hui; Han, Xiao; Wang, Qian; Zhang, Cui-Lian

    2017-01-01

    Abstract Background: DNA methylation modification has been proved to influence the phenotype of polycystic ovary syndrome (PCOS). Genome-wide association studies (GWAS) demonstrate that yes-associated protein (YAP1) genetic sites are associated with PCOS. The study aims to detect the methylation status of YAP1 promoter in ovary granulosa cells (GCs) of PCOS patients and explore novel therapeutic targets for PCOS. Methods: Randomized controlled trial was applied and a total of 72 women were included in the study, including 36 cases of PCOS patients and 36 cases of health controls. Ovary GCs were extracted from in vitro fertilization embryo transfer. Methylation status of YAP1 promoter was detected by bisulfite sequencing PCR (BSP). Protein and mRNA expression of YAP1 were measured by western blotting and real-time quantitate PCR. Results: Overall methylation level of YAP1 promoter region from PCOS group was significantly lower than that from control group. CpG sites analysis revealed that 12 sites (−443, −431, −403, −371, −331, −120, −49, −5, +1, +9, +15, +22) were significantly hypomethylated in women with PCOS (P < 0.05). A significant upregulation of YAP1 mRNA and protein expression levels was observed. Testosterone concentration could alleviate the methylation status and demonstrate obvious dose–dependent relation. Conclusion: Our research achievements manifest that hypomethylation of YAP1 promoter promotes the YAP1 expression, which plays a key role in the pathogenesis and accelerate PCOS. PMID:28079802

  12. Promoter analysis of mouse Scn3a gene and regulation of the promoter activity by GC box and CpG methylation.

    PubMed

    Deng, Guang-Fei; Qin, Jia-Ming; Sun, Xun-Sha; Kuang, Zu-Ying; Su, Tao; Zhao, Qi-Hua; Shi, Yi-Wu; Liu, Xiao-Rong; Yu, Mei-Juan; Yi, Yong-Hong; Liao, Wei-Ping; Long, Yue-Sheng

    2011-06-01

    Voltage-gated sodium channel α-subunit type III (Na(v)1.3) is mainly expressed in the central nervous system and is associated with neurological disorders. The expression of mouse Scn3a product (Na(v)1.3) mainly occurs in embryonic and early postnatal brain but not in adult brain. Here, we report for the first time the identification and characterization of the mouse Scn3a gene promoter region and regulation of the promoter activity by GC box and CpG methylation. Luciferase assay showed that the promoter region F1.2 (nt -1,049 to +157) had significantly higher activity in PC12 cells, comparing with that in SH-SY5Y cells and HEK293 cells. A stepwise 5' truncation of the promoter region found that the minimal functional promoter located within the region nt -168 to +157. Deletion of a GC box (nt -254 to -258) in the mouse Scn3a promoter decreased the promoter activity. CpG methylation of the F1.2 without the GC box completely repressed the promoter activity, suggesting that the GC box is a critical element in the CpG-methylated Scn3a promoter. These results suggest that the GC box and CpG methylation might play important roles in regulating mouse Scn3a gene expression.

  13. A novel bisulfite-microfluidic temperature gradient capillary electrophoresis platform for highly sensitive detection of gene promoter methylation.

    PubMed

    Zhang, Huidan; Shan, Lianfeng; Wang, Xiaonan; Ma, Qian; Fang, Jin

    2013-04-15

    The hypermethylated tumor suppressor gene promoters are widely recognized as promising markers for cancer screening and ideal targets for cancer therapy, however, a major obstacle in their clinical study is highly sensitive screening. To address this limitation, we developed a novel bisulfite-microfluidic temperature gradient capillary electrophoresis (bisulfite-μTGCE) platform for gene methylation analysis by combining bisulfite treatment and slantwise radiative heating system-based μTGCE. Bisulfite-treated genomic DNA (gDNA) was amplified with universal primers for both methylated and unmethylated sequences, and introduced into glass microfluidic chip to perform electrophorectic separation under a continuous temperature gradient based on the formation of heteroduplexes. Eight CDKN2A promoter model fragments with different number and location of methylation sites were prepared and successfully analyzed according to their electrophoretic peak patterns, with high stability, picoliter-scale sample consumption, and significantly increased detection speed. The bisulfite-μTGCE could detect methylated gDNA with a detection limit of 7.5pg, and could distinguish as low as 0.1% methylation level in CDKN2A in an unmethylated background. Detection of seven colorectal cancer (CRC) cell lines with known and unknown methylation statuses of CDKN2A promoter and 20 tumor tissues derived from CRC patients demonstrated the capability of detecting hypermethylation in real-world samples. The wider adaptation of this platform was further supported by the detection of the CDKN2A and MLH1 promoters' methylation statuses in combination. This highly sensitive, fast, and low-consumption platform for methylation detection shows great potential for future clinical applications.

  14. 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

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

    PubMed

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

    2016-10-01

    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 in

  16. Integrated analysis of promoter mutation, methylation and expression of AKT1 gene in Chinese breast cancer patients

    PubMed Central

    Heng, Jianfu; Guo, Xinwu; Wu, Wenhan; Wang, Yue; Li, Guoli; Chen, Ming; Peng, Limin; Wang, Shouman; Dai, Lizhong; Tang, Lili; Wang, Jun

    2017-01-01

    Background As downstream mediators of PI3K /PTEN /AKT /mTORC1 pathway, the AKT isoforms play critical roles in tumorgenesis. Although the pleiotropic effects of AKT1 in breast cancer have been reported, the genetic and epigenetic characteristics of AKT1 promoter region in breast cancer remains to be identified. In this study we aimed to investigate the promoter mutation spectrum, methylation and gene expression pattern of AKT1 and their relationship with breast cancer. Methods By using PCR target sequence enrichment and next-generation sequencing technology, we sequenced AKT1 promoter region in pairs of breast tumor and normal tissues from 95 unselected Chinese breast cancer patients. The methylation of the promoter region and the expression profile of AKT1 in the same cohort were detected with bisulfite next-generation sequencing and qPCR, respectively. Results We identified 28 somatic mutations in 23 of the 95 (24.2%) breast cancer samples. And 19 of the 28 mutations were located in transcription factor (TF) binding sites. In the 23 patients with somatic mutations, no significant change of methylation or expression was found comparing with other patients. AKT1 promoter region was significantly hypo-methylated in tumor compared with matched normal tissue (P = 0.0014) in the 95 patients. The expression of AKT1 was significantly suppressed in tumor tissue (P = 0.0375). In clinicopathological factor analysis, AKT1 showed significant hypo-methylation (P = 0.0249) and suppressed expression (P = 0.0375) in HER2 negative subtype. And a trend of decrease in expression level (P = 0.0624) of AKT1 in the ER negative subtype was observed, which is significantly decreased in basal-like breast tumor (P = 0.0328). Conclusions Hypo-methylation and suppressed expression of AKT1 was observed to be associated with breast cancer in our cohort. The methylation and expression of AKT1 were both significantly associated with HER2 status. The promoter mutation of AKT1 did not show

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

    PubMed Central

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

    2015-01-01

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

  18. Association study of polymorphism in the serotonin transporter gene promoter, methylation profiles, and expression in patients with major depressive disorder.

    PubMed

    Iga, Jun-Ichi; Watanabe, Shin-Ya; Numata, Shusuke; Umehara, Hidehiro; Nishi, Akira; Kinoshita, Makoto; Inoshita, Masatoshi; Shimodera, Shinji; Fujita, Hirokazu; Ohmori, Tetsuro

    2016-05-01

    The serotonin transporter (5HTT) may be associated with the pathogenesis of major depressive disorder (MDD). The 5HTT-linked polymorphic region (5HTTLPR) genotype may determine how levels of 5HTT mRNA are influenced by promoter methylation. We examined the association of 5HTT gene methylation, which influences gene expression, and the 5HTTLPR genotype before antidepressant treatment and expression before and after treatment. The aims of this study were (1) to investigate the association between 5HTT methylation or expression in leukocytes and depression and (2) to investigate a possible effect of 5HTT methylation, expression, and genotype on clinical symptoms in MDD. The 5HTTLPR genotype was significantly associated with mean methylation levels in patients only (patients: r = 0.40, p = 0.035, controls: p = 0.96). The mean methylation level was significantly increased in patients compared with controls (patients: 5.30 ± 0.24, controls: 4.70 ± 0.19, unpaired t-test, p = 0.04). 5HTT expression using real-time PCR and Taqman probes was increased in unmedicated patients compared with controls and then decreased 8 weeks after antidepressant treatment. The mean 5HTT expression level was not associated with the 5HTTLPR genotype in patients or controls. Increased depressive symptoms were related to decreased levels of methylation. Copyright © 2016 John Wiley & Sons, Ltd.

  19. 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.

  20. Methylation level of CpG islands in GGH gene promoter in pediatric acute leukemia

    PubMed Central

    Wang, Huihui; Mai, Huirong; Yuan, Xiuli; Li, Changgang; Wen, Feiqiu

    2017-01-01

    Background γ-Glutamyl hydrolase (GGH) regulates intracellular folates and antifolates such as methotrexate (MTX) for proper nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. In addition to genetic polymorphism and karyotypic abnormalities, methylation of CpG island 1 (CpG1) in the promoter region is found to modulate GGH activity by reducing GGH mRNA expression in acute lymphoblastic leukemia (ALL) cells. We aim to investigate methylation status of two CpG islands (CpG1 and CpG2) in the GGH promoter region in pediatric patients with ALL and acute myelogenous leukemia (AML). Methods 70B-ALL, 29 AML, 10 ITP (idiopathic thrombocytopenic purpura) and 40 healthy children are recruited in the present study. MS-HRM (methylation-sensitive high-resolution melting) and bisulfite sequencing PCR (BSP) are used to detect methylation change and its level in CpG1 and CpG2 in the GGH promoter region. GGH mRNA expression is quantified by real-time PCR. Correlation between CpG island methylation and GGH mRNA expression is assessed by statistical software. Results Methylations of CpG1 are detected in leukemia cells samples obtained from 30.9% (21/68) of patients with ALL and 20.7% (6/29) of patients with AML. These methylations are not detected in the controls. Methylations of CpG2 are detected in leukemia cell samples obtained from 44.1% (30/68) of the ALL patients and 37.9% (11/29) of the AML patients. These percentages are significantly higher than that observed in the control cell samples: 6.0% (3/50) (Fisher's exact test, P = 0.000). The abundance of CpG1 methylation in all leukemia cell samples is classified as Grade I (methylation level is less than 10%) and the abundance of CpG2 methylation in leukemia cell samples is classified in separate grades. Our results indicate that methylation of CpG1 or hypermethylation (the methylation level is greater than 10%) of CpG2 could significantly reduce GGH mRNA expression in leukemia cells from the ALL and AML

  1. Promoter methylation in the PTCH gene in cervical epithelial cancer and ovarian cancer tissue as studied by eight novel Pyrosequencing® assays.

    PubMed

    Löf-Öhlin, Zarah M; Levanat, Sonja; Sabol, Maja; Sorbe, Bengt; Nilsson, Torbjörn K

    2011-03-01

    DNA methylation status in the CpG sites of promoter regions in cancer-related genes, such as PTCH, has traditionally been investigated using either dye-terminator sequencing or methylation-specific PCR. We aimed to study the PTCH gene promoter methylation in gynecological cancers, with a method that gives a quantitative measure of the methylation status of the promoter region of the studied gene, and for this purpose, we designed novel Pyrosequencing-based assays. Bisulfite-treated genomic DNA (bsDNA) was amplified by standard PCR and applied to novel Pyrosequencing® assays, in order to measure the methylated fraction (%) at each CpG site of the PTCH gene promoter. We analyzed 22 squamous cell cervical cancer tissue specimens (11 with good and 11 with poor outcomes after radiotherapy) and 5 ovarian cancer tissue specimens matched with 5 normal ovarian tissue specimens. Six optimized PCR protocols which generated 8 Pyrosequencing assays covering 63 CpG sites in the promoter regions 1 and 2 as well as the previously unanalyzed promoter region 3 in the PTCH gene were developed. The 27 tumor tissue specimens and 5 normal tissues did not show any methylation within any of the 63 CpG sites. Our data suggest that methylation of the PTCH promoter is not a high-prevalence feature of squamous cell cervical cancer or ovarian cancer, but Pyrosequencing assays are a good method for studying promoter methylation.

  2. Correlation of CTGF gene promoter methylation with CTGF expression in type 2 diabetes mellitus with or without nephropathy.

    PubMed

    Zhang, Hao; Cai, Xu; Yi, Bin; Huang, Jing; Wang, Jianwen; Sun, Jian

    2014-06-01

    Increasing evidence shows that DNA methylation is involved in the development and progression of diabetes mellitus (DM) and its complications. Previous studies conducted by our group have indicated that high glucose levels may induce the demethylation process of the connective tissue growth factor (CTGF) gene promoter and increase the expression of CTGF in human glomerular mesangial cells. Based on these findings, the aim of the present study was to investigate the methylation level of genomic DNA and the CTGF promoter in patients with type 2 DM and to analyze its possible correlation with CTGF expression. Methylation levels of the whole genomic DNA were detected by high-performance liquid chromatography in a non-diabetes control (NDM) group (n=29), a diabetes without nephropathy (NDN) group (n=37) and a diabetes with nephropathy (DN) group (n=38). CTGF promoter methylation levels were detected by methylation-specific polymerase chain reaction and bisulfite sequencing. The levels of serum CTGF were assessed using the enzyme-linked immunosorbent assay. The methylation levels of the whole genomic DNA were not significantly different among the three groups. However, the CTGF methylation levels in the two diabetes groups were significantly lower than those in the NDM group (P<0.05), with the lowest methylation level in the DN group (P<0.05). The CTGF protein levels in the DN group were significantly higher than those in the NDM and NDN groups (P<0.05). Levels of CTGF were negatively correlated with the estimated glomerular filtration rate (eGFR) and the methylation level of the promoter, while they were positively correlated with age, urinary albumin-to-creatinine ratio (UACR), blood urea nitrogen, creatinine, fasting blood sugar and postprandial blood glucose. Multiple stepwise regression analysis showed that CTGF expression was associated with the UACR, CTGF methylation level and eGFR. DNA methylation is a regulatory mechanism of CTGF expression, which is decreased

  3. 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.

  4. 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

  5. Alcohol and nicotine codependence-associated DNA methylation changes in promoter regions of addiction-related genes

    PubMed Central

    Xu, Hongqin; Wang, Fan; Kranzler, Henry R.; Gelernter, Joel; Zhang, Huiping

    2017-01-01

    Altered DNA methylation in addiction-related genes may modify the susceptibility to alcohol or drug dependence (AD or ND). We profiled peripheral blood DNA methylation levels of 384 CpGs in promoter regions of 82 addiction-related genes in 256 African Americans (AAs) (117 cases with AD-ND codependence and 139 controls) and 196 European Americans (103 cases with AD-ND codependence and 93 controls) using Illumina’s GoldenGate DNA methylation array assays. AD-ND codependence-associated DNA methylation changes were analyzed using linear mixed-effects models with consideration of batch effects and covariates age, sex, and ancestry proportions. Seventy CpGs (in 41 genes) showed nominally significant associations (P < 0.05) with AD-ND codependence in both AAs and EAs. One CpG (HTR2B cg27531267) was hypomethylated in AA cases (P = 7.2 × 10−5), while 17 CpGs in 16 genes (including HTR2B cg27531267) were hypermethylated in EA cases (5.6 × 10−9 ≤ P ≤ 9.5 × 10−5). Nevertheless, 13 single nucleotide polymorphisms (SNPs) nearby HTR2B cg27531267 and the interaction of these SNPs and cg27531267 did not show significant effects on AD-ND codependence in either AAs or EAs. Our study demonstrated that DNA methylation changes in addiction-related genes could be potential biomarkers for AD-ND co-dependence. Future studies need to explore whether DNA methylation alterations influence the risk of AD-ND codependence or the other way around. PMID:28165486

  6. Loss of heterozygosity of the Mutated in Colorectal Cancer gene is not associated with promoter methylation in non-small cell lung cancer.

    PubMed

    Poursoltan, Pirooz; Currey, Nicola; Pangon, Laurent; van Kralingen, Christa; Selinger, Christina I; Mahar, Annabelle; Cooper, Wendy A; Kennedy, Catherine W; McCaughan, Brian C; Trent, Ronald; Kohonen-Corish, Maija R J

    2012-08-01

    'Mutated in Colorectal Cancer' (MCC) is emerging as a multifunctional protein that affects several cellular processes and pathways. Although the MCC gene is rarely mutated in colorectal cancer, it is frequently silenced through promoter methylation. Previous studies have reported loss of heterozygosity (LOH) of the closely linked MCC and APC loci in both colorectal and lung cancers. APC promoter methylation is a marker of poor survival in non-small cell lung cancer (NSCLC). However, MCC methylation has not been previously studied in lung cancer. Therefore, we wanted to determine if MCC is silenced through promoter methylation in lung cancer and whether this methylation is associated with LOH of the MCC locus or methylation of the APC gene. Three polymorphic markers for the APC/MCC locus were analysed for LOH in 64 NSCLC specimens and matching normal tissues. Promoter methylation of both genes was determined using methylation specific PCR in primary tumours. LOH of the three markers was found in 41-49% of the specimens. LOH within the MCC locus was less common in adenocarcinoma (ADC) (29%) than in squamous cell carcinoma (SCC) (72%; P=0.006) or large cell carcinoma (LCC) (75%; P=0.014). However, this LOH was not accompanied by MCC promoter methylation, which was found in only two cancers (3%). In contrast, 39% of the specimens showed APC methylation, which was more common in ADC (58%) than in SCC (13%). Western blotting revealed that MCC was expressed in a subset of lung tissue specimens but there was marked variation between patients rather than between cancer and matching non-cancer tissue specimens. In conclusion, we have shown that promoter methylation of the APC gene does not extend to the neighbouring MCC gene in lung cancer, but LOH is found at both loci. The variable levels of MCC expression were not associated with promoter methylation and may be regulated through other cellular mechanisms.

  7. Polymorphism and DNA methylation in the promoter modulate KISS1 gene expression and are associated with litter size in goats.

    PubMed

    An, X P; Hou, J X; Lei, Y N; Gao, T Y; Cao, B Y

    2015-04-01

    Polymorphisms in the promoter region are likely to impact KISS1 gene transcription and reproductive traits. In this study, Guanzhong (GZ, n=350) and Boer (BE, n=196) goats were used to detect polymorphism in the promoter of the goat KISS1 gene by DNA sequencing. In the GZ goats, the g.1384G>A mutation was identified in the promoter of the goat KISS1 gene. Guanzhong goats were in Hardy-Weinberg disequilibrium at g.1384G>A locus (P<0.05). The 1384A allele was predicted to eliminate methylation, AHR-arnt heterodimers and AHR-related factors (AHRR) and myoblast determining factors (MYOD) transcription factor-binding sites. Statistical results indicated that the g.1384G>A SNP was associated with litter size in the GZ goats (P<0.05). Luciferase assay analysis suggested that the 1384A allele increased luciferase activity when compared to the 1384G allele. The RT-qPCR assay also demonstrated that the 1384A allele had greater amounts of KISS1 mRNA than the 1384G allele in homozygous individuals. Functional analysis suggested that this g.1384G>A SNP may be an important genetic regulator of KISS1 gene expression with effects on downstream processes that are modulated by KISS1 gene because of the changes of methylation and transcription factor-binding sites. Therefore, the current study provides evidence in goats for genetic markers that might be used in breeding programs.

  8. H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation

    PubMed Central

    Guillemette, Benoit; Drogaris, Paul; Lin, Hsiu-Hsu Sophia; Armstrong, Harry; Hiragami-Hamada, Kyoko; Imhof, Axel; Bonneil, Éric; Thibault, Pierre; Verreault, Alain; Festenstein, Richard J.

    2011-01-01

    Methylation of histone H3 lysine 4 (H3K4me) is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac) has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs) Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC) Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP), we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3), a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS), which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me). PMID:21483810

  9. Early life adversity and serotonin transporter gene variation interact to affect DNA methylation of the corticotropin-releasing factor gene promoter region in the adult rat brain.

    PubMed

    van der Doelen, Rick H A; Arnoldussen, Ilse A; Ghareh, Hussein; van Och, Liselot; Homberg, Judith R; Kozicz, Tamás

    2015-02-01

    The interaction between childhood maltreatment and the serotonin transporter (5-HTT) gene linked polymorphic region has been associated with increased risk to develop major depression. This Gene × Environment interaction has furthermore been linked with increased levels of anxiety and glucocorticoid release upon exposure to stress. Both endophenotypes are regulated by the neuropeptide corticotropin-releasing factor (CRF) or hormone, which is expressed by the paraventricular nucleus of the hypothalamus, the bed nucleus of the stria terminalis, and the central amygdala (CeA). Therefore, we hypothesized that altered regulation of the expression of CRF in these areas represents a major neurobiological mechanism underlying the interaction of early life stress and 5-HTT gene variation. The programming of gene transcription by Gene × Environment interactions has been proposed to involve epigenetic mechanisms such as DNA methylation. In this study, we report that early life stress and 5-HTT genotype interact to affect DNA methylation of the Crf gene promoter in the CeA of adult male rats. Furthermore, we found that DNA methylation of a specific site in the Crf promoter significantly correlated with CRF mRNA levels in the CeA. Moreover, CeA CRF mRNA levels correlated with stress coping behavior in a learned helplessness paradigm. Together, our findings warrant further investigation of the link of Crf promoter methylation and CRF expression in the CeA with behavioral changes that are relevant for psychopathology.

  10. Unmasking of epigenetically silenced genes reveals DNA promoter methylation and reduced expression of PTCH in breast cancer.

    PubMed

    Wolf, Ido; Bose, Shikha; Desmond, Julian C; Lin, Bryan T; Williamson, Elizabeth A; Karlan, Beth Y; Koeffler, H Phillip

    2007-10-01

    A pharmacological-based global screen for epigenetically silenced tumor suppressor genes was performed in MCF-7 and MDA-MB-231 breast cancer cells. Eighty-one genes in MCF-7 cells and 131 in MDA-MB-231 cells were identified, that had low basal expression and were significantly upregulated following treatment. Eighteen genes were studied for methylation and/or expression in breast cancer; PTCH, the receptor for the hedgehog (Hh) pathway and a known tumor suppressor gene, was selected for further analysis. Methylation of the PTCH promoter was found in MCF-7 cells and in breast cancer samples, and correlated with low PTCH expression. Immunohistochemical analysis of breast tissue arrays revealed high expression of PTCH in normal breast compared to ductal carcinomas in situ (DCIS) and invasive ductal carcinomas; furthermore, association was found between PTCH expression and favorable prognostic factors. PTCH is an inhibitor of the Hh pathway, and its silencing activates the pathway and promotes growth. Indeed, high activity of the Hh pathway was identified in MCF-7 cells and overexpression of PTCH inhibited the pathway. Moreover, treatment with cyclopamine, an inhibitor of the pathway, reduced cell growth and slowed the cell cycle in these cells. Thus, unmasking of epigenetic silencing in breast cancer enabled us to discover a large number of candidate tumor suppressor genes. Further analysis suggested a role of one of these genes, PTCH, in breast cancer tumorigenesis.

  11. 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.

  12. Protective vaccination and blood-stage malaria modify DNA methylation of gene promoters in the liver of Balb/c mice.

    PubMed

    Al-Quraishy, Saleh; Dkhil, Mohamed A; Abdel-Baki, Abdel-Azeem S; Ghanjati, Foued; Erichsen, Lars; Santourlidis, Simeon; Wunderlich, Frank; Araúzo-Bravo, Marcos J

    2017-03-18

    Epigenetic mechanisms such as DNA methylation are increasingly recognized to be critical for vaccination efficacy and outcome of different infectious diseases, but corresponding information is scarcely available for host defense against malaria. In the experimental blood-stage malaria Plasmodium chabaudi, we investigate the possible effects of a blood-stage vaccine on DNA methylation of gene promoters in the liver, known as effector against blood-stage malaria, using DNA methylation microarrays. Naturally susceptible Balb/c mice acquire, by protective vaccination, the potency to survive P. chabaudi malaria and, concomitantly, modifications of constitutive DNA methylation of promoters of numerous genes in the liver; specifically, promoters of 256 genes are hyper(=up)- and 345 genes are hypo(=down)-methylated (p < 0.05). Protective vaccination also leads to changes in promoter DNA methylation upon challenge with P. chabaudi at peak parasitemia on day 8 post infection (p.i.), when 571 and 1013 gene promoters are up- and down-methylated, respectively, in relation to constitutive DNA methylation (p < 0.05). Gene set enrichment analyses reveal that both vaccination and P. chabaudi infections mainly modify promoters of those genes which are most statistically enriched with functions relating to regulation of transcription. Genes with down-methylated promoters encompass those encoding CX3CL1, GP130, and GATA2, known to be involved in monocyte recruitment, IL-6 trans-signaling, and onset of erythropoiesis, respectively. Our data suggest that vaccination may epigenetically improve parts of several effector functions of the liver against blood-stage malaria, as, e.g., recruitment of monocyte/macrophage to the liver accelerated liver regeneration and extramedullary hepatic erythropoiesis, thus leading to self-healing of otherwise lethal P. chabaudi blood-stage malaria.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: correlation with physiological and pathological characteristics, and with biomarkers of one-carbon metabolism.

    PubMed

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

    2014-04-01

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

  18. 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

  19. Usefulness of DNA Methylation Levels in COASY and SPINT1 Gene Promoter Regions as Biomarkers in Diagnosis of Alzheimer’s Disease and Amnestic Mild Cognitive Impairment

    PubMed Central

    Shinagawa, Shunichiro; Nagata, Tomoyuki; Shimada, Kazuya; Shibata, Nobuto; Ohnuma, Tohru; Kasanuki, Koji; Arai, Heii; Yamada, Hisashi; Nakayama, Kazuhiko; Kondo, Kazuhiro

    2016-01-01

    In order to conduct early therapeutic interventions for Alzheimer’s disease (AD), convenient, early diagnosis markers are required. We previously reported that changes in DNA methylation levels were associated with amnestic mild cognitive impairment (aMCI) and AD. As the results suggested changes in DNA methylation levels in the COASY and SPINT1 gene promoter regions, in the present study we examined DNA methylation in these regions in normal controls (NCs, n = 30), aMCI subjects (n = 28) and AD subjects (n = 30) using methylation-sensitive high resolution melting (MS-HRM) analysis. The results indicated that DNA methylation in the two regions was significantly increased in AD and aMCI as compared to NCs (P < 0.0001, P < 0.0001, ANOVA). Further analysis suggested that DNA methylation in the COASY gene promoter region in particular could be a high sensitivity, high specificity diagnosis biomarker (COASY: sensitivity 96.6%, specificity 96.7%; SPINT1: sensitivity 63.8%, specificity 83.3%). DNA methylation in the COASY promoter region was associated with CDR Scale Sum of Boxes (CDR-SB), an indicator of dementia severity. In the SPINT1 promoter region, DNA methylation was negatively associated with age in NCs and elevated in aMCI and AD subjects positive for antibodies to Herpes simplex virus type 1 (HSV-1). These findings suggested that changes in DNA methylation in the COASY and SPINT1 promoter regions are influenced by various factors. In conclusion, DNA methylation levels in the COASY and SPINT1 promoter regions were considered to potentially be a convenient and useful biomarker for diagnosis of AD and aMCI. PMID:27992572

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

    PubMed Central

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

    2016-01-01

    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

  1. 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.

  2. 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...

  3. DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes.

    PubMed

    Tran, Robert K; Henikoff, Jorja G; Zilberman, Daniel; Ditt, Renata F; Jacobsen, Steven E; Henikoff, Steven

    2005-01-26

    Cytosine DNA methylation in vertebrates is widespread, but methylation in plants is found almost exclusively at transposable elements and repetitive DNA. Within regions of methylation, methylcytosines are typically found in CG, CNG, and asymmetric contexts. CG sites are maintained by a plant homolog of mammalian Dnmt1 acting on hemi-methylated DNA after replication. Methylation of CNG and asymmetric sites appears to be maintained at each cell cycle by other mechanisms. We report a new type of DNA methylation in Arabidopsis, dense CG methylation clusters found at scattered sites throughout the genome. These clusters lack non-CG methylation and are preferentially found in genes, although they are relatively deficient toward the 5' end. CG methylation clusters are present in lines derived from different accessions and in mutants that eliminate de novo methylation, indicating that CG methylation clusters are stably maintained at specific sites. Because 5-methylcytosine is mutagenic, the appearance of CG methylation clusters over evolutionary time predicts a genome-wide deficiency of CG dinucleotides and an excess of C(A/T)G trinucleotides within transcribed regions. This is exactly what we find, implying that CG methylation clusters have contributed profoundly to plant gene evolution. We suggest that CG methylation clusters silence cryptic promoters that arise sporadically within transcription units.

  4. Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.

    PubMed

    Goedecke, Simon; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

    2015-12-01

    Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples.

  5. 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

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

    PubMed

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

    2016-07-05

    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.

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

    PubMed Central

    2015-01-01

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

  8. Investigation into the promoter DNA methylation of three genes (CAMK1D, CRY2 and CALM2) in the peripheral blood of patients with type 2 diabetes.

    PubMed

    Cheng, Jia; Tang, Linlin; Hong, Qingxiao; Ye, Huadan; Xu, Xuting; Xu, Leiting; Bu, Shizhong; Wang, Qinwen; Dai, Dongjun; Jiang, Danjie; Duan, Shiwei

    2014-08-01

    Promoter DNA methylation may reflect the interaction between genetic backgrounds and environmental factors in the development of metabolic disorders, including type 2 diabetes (T2D). Calcium/calmodulin-dependent protein kinase 1D (CAMK1D), cryptochrome 2 (CRY2) and calmodulin 2 (CALM2) genes have been identified to be associated with a risk of T2D. Therefore, the aim of the present study was to investigate the contribution of promoter DNA methylation of these genes to the risk of T2D. Using bisulfite pyrosequencing technology, the DNA methylation levels of the CpG dinucleotides within the CAMK1D, CRY2 and CALM2 gene promoters were measured in 48 patients with T2D and 48 age- and gender-matched healthy controls. The results demonstrated that the promoters of these three genes were hypomethylated in the peripheral blood of all the subjects, and DNA methylation of these three genes did not contribute to the risk of T2D.

  9. Litter and Sex Effects on Maternal Behavior and DNA Methylation of the Nr3c1 Exon17 Promoter Gene in Hippocampus and Cerebellum

    PubMed Central

    Kosten, Therese A; Nielsen, David A

    2014-01-01

    Early life events can alter gene expression through DNA methylation. The methylation status of the exon 17 promoter of the glucocorticoid receptor (Nr3c1 gene) in hippocampus associates with frequency of pup licking. Much of this work was conducted with male rats. Because dams more frequently lick male pups, this may contribute to sex differences in phenotypes through DNA methylation. Modifying litter gender composition (LGC), in which offspring of single-sex litters are compared to mixed-sex litters, alters maternal behavior. Previously, we demonstrated that LGC and sex affected pup licking times as well as anxiety and hippocampal DNA methylation of the Nr3c1 exon 17 promoter gene in adolescence. Now, we expand upon this work by examining effects in cerebellum and measuring mRNA levels. We also re-assessed DNA methylation in hippocampus using pyrosequencing and re-analyzed pup licking with the more commonly used frequency measure. Litters, culled to 8 pups on postnatal day 1 (PN1), were assigned to one of three conditions: all male, (n=10), all female, (n=12), or half of each sex (n=20). Licking was rated on PN4, 7, and 10. On PN35, hippocampal and cerebellar samples were obtained. Single-sex males were licked the least and mixed-sex males, the most. Hippocampal Nr3c1 mRNA levels were lowest in mixed females with no LGC or Sex effects in DNA methylation. Cerebellar DNA methylation levels were lowest in mixed males with no effect on mRNA levels. Maternal pup licking associated with DNA methylation of the Nr3c1 exon 17 promoter gene in cerebellum and with hippocampal mRNA. PMID:24721039

  10. Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene.

    PubMed

    Moon, Dong Chan; Choi, Chul Hee; Lee, Su Man; Lee, Jung Hwa; Kim, Seung Il; Kim, Dong Sun; Lee, Je Chul

    2012-01-01

    Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.

  11. 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

  12. 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

  13. Gene methylation in gastric cancer.

    PubMed

    Qu, Yiping; Dang, Siwen; Hou, Peng

    2013-09-23

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  15. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  18. The relationship between RASSF1A gene promoter methylation and the susceptibility and prognosis of melanoma: A meta-analysis and bioinformatics

    PubMed Central

    Li, Haili; Tang, Wenru; Jia, Shuting; Wu, Xiaoming; Luo, Ying

    2017-01-01

    Background The function of the tumor suppressor gene RASSF1A in cancer cells has been detailed in many studies. However, due to the methylation of its promoter, the expression of RASSF1A is missing in most cancers. In the literature, we found that the conclusion regarding the relationship between RASSF1A gene promoter methylation and the susceptibility and prognosis of melanoma was not unified. This study adopts the use of a meta-analysis and bioinformatics to explore the relationship between RASSF1A gene promoter methylation and the susceptibility and prognosis of melanoma. Methods Data on melanoma susceptibility were downloaded from the PubMed, Cochrane Library, Web of Science and Google Scholar databases, which were analyzed via a meta-analysis. The effect sizes were estimated by measuring an odds ratio (OR) with a 95% confidence interval (CI). We also used a chi-squared-based Q test to examine the between-study heterogeneity, and used funnel plots to evaluate publication bias. The data on melanoma prognosis, which were analyzed by bioinformatics methods, were downloaded from The Cancer Genome Atlas (TCGA) project. The effect sizes were estimated by measuring the hazard ratios (HRs) with a 95% confidence interval (CI). Results Our meta-analysis included 10 articles. We found that RASSF1A gene promoter methylation was closely related to melanoma susceptibility (OR = 12.67, 95% CI: 6.16 ∼ 26.05, z = 6.90, P<0.0001 according to a fixed effects model and OR = 9.25, 95% CI: 4.37 ∼ 19.54, z = 5.82, P<0.0001 according to a random effects model). The results of the meta-analysis did not reveal any heterogeneity (tau2 = 0.00; H = 1 [1; 1.55]; I2 = 0% [0%; 58.6%], P = 0.5158) or publication bias (t = 0.87, P = 0.4073 by Egger’s test; Z = 0.45, P = 0.6547 by Begg’s test); therefore, we believe that the results of our meta-analysis were more reliable. To explore the relationship between RASSF1A gene methylation, the prognosis of melanoma and the clinical features of

  19. Characteristics of fads2 gene expression and putative promoter in European sea bass (Dicentrarchus labrax): comparison with salmonid species and analysis of CpG methylation.

    PubMed

    Geay, Florian; Zambonino-Infante, José; Reinhardt, Richard; Kuhl, Heiner; Santigosa, Ester; Cahu, Chantal; Mazurais, David

    2012-03-01

    Marine fish species exhibit low capacity to biosynthesise highly unsaturated fatty acid (HUFA) in comparison to strict freshwater and anadromous species. It is admitted that the Delta(6) desaturase (FADS2) is a key enzyme in the HUFA biosynthetic pathway. We investigated by quantitative PCR the relative amounts of FADS2 mRNA in European sea bass (Dicentrarchus labrax) in comparison with a salmonid species, the rainbow trout (Oncorhynchus mykiss L.). The analysis of the expression data was performed regarding the difference of the characteristics of a critical fragment of the fads2 gene promoter between sea bass and Atlantic salmon. The lower level of fads2 gene expression observed in sea bass suggested that fads2 gene putative promoter, which exhibited an E-box like Sterol Regulatory Element (SRE) site but lacked a Sp1 site, is less active in this marine species. The cytosine methylation of CpG sites in the putative promoter region including E-box like SRE and NF-Y binding sites of sea bass fads2 gene was also investigated following a nutritional conditioning of larvae. However, no significant difference of CpG methylation could be found for any of the 28 CpGs analysed between larvae fed diet with high or low HUFA contents. In conclusion, the present data revealed lower constitutive expression of the fads2 gene possibly related to different characteristics of gene promoter in sea bass in comparison with salmonid species, and indicated that long-term conditioning of fads2 gene expression did not influence the methylation of the gene promoter at potential SRE binding site.

  20. HNF1B variants associate with promoter methylation and regulate gene networks activated in prostate and ovarian cancer

    PubMed Central

    Ross-Adams, Helen; Ball, Stephen; Lawrenson, Kate; Halim, Silvia; Russell, Roslin; Wells, Claire; Strand, Siri H.; Ørntoft, Torben F.; Larson, Melissa; Armasu, Sebastian; Massie, Charles E.; Asim, Mohammad; Mortensen, Martin M.; Borre, Michael; Woodfine, Kathryn; Warren, Anne Y.; Lamb, Alastair D.; Kay, Jonathan; Whitaker, Hayley; Ramos-Montoya, Antonio; Murrell, Adele; Sørensen, Karina D.; Fridley, Brooke L.; Goode, Ellen L.; Gayther, Simon A.; Masters, John

    2016-01-01

    Two independent regions within HNF1B are consistently identified in prostate and ovarian cancer genome-wide association studies (GWAS); their functional roles are unclear. We link prostate cancer (PC) risk SNPs rs11649743 and rs3760511 with elevated HNF1B gene expression and allele-specific epigenetic silencing, and outline a mechanism by which common risk variants could effect functional changes that increase disease risk: functional assays suggest that HNF1B is a pro-differentiation factor that suppresses epithelial-to-mesenchymal transition (EMT) in unmethylated, healthy tissues. This tumor-suppressor activity is lost when HNF1B is silenced by promoter methylation in the progression to PC. Epigenetic inactivation of HNF1B in ovarian cancer also associates with known risk SNPs, with a similar impact on EMT. This represents one of the first comprehensive studies into the pleiotropic role of a GWAS-associated transcription factor across distinct cancer types, and is the first to describe a conserved role for a multi-cancer genetic risk factor. PMID:27732966

  1. Dietary methyl donors, methyl metabolizing enzymes, and epigenetic regulators: diet-gene interactions and promoter CpG island hypermethylation in colorectal cancer.

    PubMed

    de Vogel, Stefan; Wouters, Kim A D; Gottschalk, Ralph W H; van Schooten, Frederik J; de Goeij, Anton F P M; de Bruïne, Adriaan P; Goldbohm, R Alexandra; van den Brandt, Piet A; van Engeland, Manon; Weijenberg, Matty P

    2011-01-01

    Dietary methyl donors might influence DNA methylation during carcinogenesis of colorectal cancer (CRC). Among 609 CRC cases and 1,663 subcohort members of the Netherlands Cohort Study on diet and cancer (n = 120,852), we estimated CRC risk according to methyl donor intake across genotypes of folate metabolizing enzymes and methyltransferases.Although diet-gene interactions were not statistically significant, methionine intake was inversely associated with CRC among subjects having both common rs2424913 and rs406193 DNMT3B C > T genotypes (highest versus lowest tertile: RR = 0.44; p (trend) = 0.05). Likewise, vitamin B2 was modestly inversely associated among individuals with the MTHFR c.665CC (rs1801133) genotype (RR = 0.66; p (trend) = 0.08), but with a significant reduced risk when ≤ 1 rare allele occurred in the combination of folate metabolizing enzymes MTHFR, MTRR and MTR (RR = 0.30; p (trend) = 0.005). Folate or vitamin B6 were neither inversely associated with CRC nor was methyl donor intake associated with the CpG island methylator phenotype (CIMP).Despite the absence of heterogeneity across genotypes, might an effect of methyl donors on CRC be more pronounced among individuals carrying common variants of folate metabolizing enzymes or DNA methyltransferases. Combining genotypes may assist to reveal diet associations with CRC, possibly because rare variants of related genes may collectively affect specific metabolic pathways or enzymatic functions.

  2. Endometrial tumour BRAF mutations and MLH1 promoter methylation as predictors of germline mismatch repair gene mutation status: a literature review.

    PubMed

    Metcalf, Alexander M; Spurdle, Amanda B

    2014-03-01

    Colorectal cancer (CRC) that displays high microsatellite instability (MSI-H) can be caused by either germline mutations in mismatch repair (MMR) genes, or non-inherited transcriptional silencing of the MLH1 promoter. A correlation between MLH1 promoter methylation, specifically the 'C' region, and BRAF V600E status has been reported in CRC studies. Germline MMR mutations also greatly increase risk of endometrial cancer (EC), but no systematic review has been undertaken to determine if these tumour markers may be useful predictors of MMR mutation status in EC patients. Endometrial cancer cohorts meeting review inclusion criteria encompassed 2675 tumours from 20 studies for BRAF V600E, and 447 tumours from 11 studies for MLH1 methylation testing. BRAF V600E mutations were reported in 4/2675 (0.1%) endometrial tumours of unknown MMR mutation status, and there were 7/823 (0.9%) total sequence variants in exon 11 and 27/1012 (2.7%) in exon 15. Promoter MLH1 methylation was not observed in tumours from 32 MLH1 mutation carriers, or for 13 MSH2 or MSH6 mutation carriers. MMR mutation-negative individuals with tumour MLH1 and PMS2 IHC loss displayed MLH1 methylation in 48/51 (94%) of tumours. We have also detailed specific examples that show the importance of MLH1 promoter region, assay design, and quantification of methylation. This review shows that BRAF mutations occurs so infrequently in endometrial tumours they can be discounted as a useful marker for predicting MMR-negative mutation status, and further studies of endometrial cohorts with known MMR mutation status are necessary to quantify the utility of tumour MLH1 promoter methylation as a marker of negative germline MMR mutation status in EC patients.

  3. 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

  4. Downregulation of N-myc downstream regulated gene 1 caused by the methylation of CpG islands of NDRG1 promoter promotes proliferation and invasion of prostate cancer cells.

    PubMed

    Li, Yalin; Pan, Pan; Qiao, Pengfei; Liu, Ranlu

    2015-09-01

    Current studies tend to consider N-myc downstream regulated gene 1 (NDRG1) as a tumor suppressor gene, inhibiting cell proliferation and invasion. NDRG1 expression in cancer cells is generally low, but the molecular mechanism is unclear. Aberrant methylation of CpG islands (CGIs) in gene promoter was able to inactivate tumor suppressor genes and activate oncogenes, disordering cell proliferation and apoptosis, playing a promotion role in tumor occurrence and progression. The present study was performed to investigate the effect of epigenetic modification of NDRG1 on prostate cancer (PCa) cells. The protein expression in human specimens was measured by immunohistochemical staining. The expression level of NDRG1 was changed by plasmid vectors in PCa cells. These cells were used to study proliferation and invasiveness. NDRG1 expression in normal prostate cells was higher than that in PCa cells. Downregulation of NDRG1 expression enhanced cell proliferation and invasiveness. In contrast, its upregulation could reduce cell proliferation and invasiveness. In PCa cells, the methylation rate of CGIs in the promoter region of NDRG1 was higher than that in normal prostate cells. 5-Aza-CdR, a methylation inhibitor, was able to effectively reverse the aberrant methylation of NDRG1, enhancing its expression, inhibiting cell growth. NDRG1 can inhibit the cell proliferation and invasion of PCa, but its expression level is low. The aberrant methylation of NDRG1 promoter is an important mechanism for gene silencing, playing an important role in tumor occurrence and progression. Therefore, reversing the aberrant methylation of NDRG1 may be used for PCa treatment.

  5. The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

    PubMed Central

    Santana-Lemos, Bárbara Amélia; de Lima Lange, Ana Paula Alencar; de Lira Benício, Mariana Tereza; da Silva José, Thiago Donizete; Lucena-Araújo, Antônio Roberto; Krause, Alexandre; Thomé, Carolina Hassibe; Rego, Eduardo Magalhães

    2011-01-01

    Impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n=18) presented lower levels of CEBPA expression compared to healthy controls (n=5), but higher levels than those in acute myeloid leukemia with t(8;21) (n=9) and with inv(16) (n=5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved. PMID:21134977

  6. 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

  7. DNA Methylation of BDNF Gene in Schizophrenia.

    PubMed

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

    2016-02-06

    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 AND 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.

  8. 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

  9. 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

  10. 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

  11. Clinical effect of DAPK promoter methylation in gastric cancer

    PubMed Central

    Jia, Wenzhuo; Yu, Tao; Cao, Xianglong; An, Qi; Yang, Hua

    2016-01-01

    Abstract Background: The loss of death-associated protein kinase (DAPK) gene expression through promoter methylation is involved in many tumors. However, the relationship between DAPK promoter methylation and clinicopathological features of gastric cancer (GC) remains to be done. Therefore, we performed a meta-analysis to assess the role of DAPK promoter methylation in GC. Methods: Literature databases were searched to retrieve eligible studies. The pooled odds ratios (ORs) with its 95% confidence intervals (CIs) were calculated using the Stata 12.0 software. Results: Final 22 available studies with 1606 GC patients and 1508 nonmalignant controls were analyzed. A significant correlation was found between DAPK promoter methylation and GC (OR = 3.23, 95% CI = 1.70–6.14, P < 0.001), but we did not find any significant association in Caucasian population, and in blood samples in subgroup analyses. DAPK promoter methylation was associated with tumor stage and lymph node status (OR = 0.69, 95% CI = 0.49–0.96, P = 0.03; OR = 1.50, 95% CI = 1.12–2.01, P = 0.007; respectively). However, we did not find that DAPK promoter methylation was associated with gender status and tumor histology. Conclusion: Our findings suggested that DAPK promoter methylation may play a key role in the carcinogenesis and progression of GC. In addition, methylated DAPK was a susceptible gene for Asian population. However, more studies with larger subjects should be done to further evaluate the effect of DAPK promoter methylation in GC patients, especially in blood and Caucasian population subgroup. PMID:27787359

  12. 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

  13. Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers.

    PubMed

    Dallol, Ashraf; Forgacs, Eva; Martinez, Alonso; Sekido, Yoshitaka; Walker, Rosemary; Kishida, Takeshi; Rabbitts, Pamela; Maher, Eamonn R; Minna, John D; Latif, Farida

    2002-05-02

    The human homologue of the Drosophila Roundabout gene DUTT1 (Deleted in U Twenty Twenty) or ROBO1 (Locus Link ID 6091), a member of the NCAM family of receptors, was recently cloned from the lung cancer tumour suppressor gene region 2 (LCTSGR2 or U2020 region) at 3p12. DUTT1 maps within a region of overlapping homozygous deletions characterized in both small cell lung cancer lines (SCLC) and in a breast cancer line. In this report we (a) defined the genomic organization of the DUTT1 gene, (b) performed mutation and expression analysis of DUTT1 in lung, breast and kidney cancers, (c) identified tumour specific promoter region methylation of DUTT1 in human cancers. The gene was found to contain 29 exons and spans at least 240 kb of genomic sequence. The 5' region contains a CpG island, and the poly(A)(+) tail has an atypical 5'-GATAAA-3' signal. We analysed DUTT1 for mutations in lung, breast and kidney cancers, no inactivating mutations were detected by PCR-SSCP. However, seven germline missense changes were found and characterized. DUTT1 expression was not detectable in one out of 18 breast tumour lines analysed by RT-PCR. Bisulfite sequencing of the promoter region of DUTT1 gene in the HTB-19 breast tumour cell line (not expressing DUTT1) showed complete hypermethylation of CpG sites within the promoter region of the DUTT1 gene (-244 to +27 relative to the translation start site). The expression of DUTT1 gene was reactivated in HTB-19 after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The same region was also found to be hypermethylated in six out of 32 (19%) primary invasive breast carcinomas and eight out of 44 (18%) primary clear cell renal cell carcinomas (CC-RCC) and in one out of 26 (4%) primary NSCLC tumours. Furthermore 80% of breast and 75% of CC-RCC tumours showing DUTT1 methylation had allelic losses for 3p12 markers hence obeying Knudson's two hit hypothesis. Our findings suggest that DUTT1 warrants further analysis as a candidate for

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

    PubMed Central

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

    2015-01-01

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

  15. Regulated transcription of human matrix metalloproteinase 13 (MMP13) and interleukin-1β (IL1B) genes in chondrocytes depends on methylation of specific proximal promoter CpG sites.

    PubMed

    Hashimoto, Ko; Otero, Miguel; Imagawa, Kei; de Andrés, María C; Coico, Jonathan M; Roach, Helmtrud I; Oreffo, Richard O C; Marcu, Kenneth B; Goldring, Mary B

    2013-04-05

    The role of DNA methylation in the regulation of catabolic genes such as MMP13 and IL1B, which have sparse CpG islands, is poorly understood in the context of musculoskeletal diseases. We report that demethylation of specific CpG sites at -110 bp and -299 bp of the proximal MMP13 and IL1B promoters, respectively, detected by in situ methylation analysis of chondrocytes obtained directly from human cartilage, strongly correlated with higher levels of gene expression. The methylation status of these sites had a significant impact on promoter activities in chondrocytes, as revealed in transfection experiments with site-directed CpG mutants in a CpG-free luciferase reporter. Methylation of the -110 and -299 CpG sites, which reside within a hypoxia-inducible factor (HIF) consensus motif in the respective MMP13 and IL1B promoters, produced the most marked suppression of their transcriptional activities. Methylation of the -110 bp CpG site in the MMP13 promoter inhibited its HIF-2α-driven transactivation and decreased HIF-2α binding to the MMP13 proximal promoter in chromatin immunoprecipitation assays. In contrast to HIF-2α, MMP13 transcriptional regulation by other positive (RUNX2, AP-1, ELF3) and negative (Sp1, GATA1, and USF1) factors was not affected by methylation status. However, unlike the MMP13 promoter, IL1B was not susceptible to HIF-2α transactivation, indicating that the -299 CpG site in the IL1B promoter must interact with other transcription factors to modulate IL1B transcriptional activity. Taken together, our data reveal that the methylation of different CpG sites in the proximal promoters of the human MMP13 and IL1B genes modulates their transcription by distinct mechanisms.

  16. 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.

  17. 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...

  18. PTSD and DNA Methylation in Select Immune Function Gene Promoter Regions: A Repeated Measures Case-Control Study of U.S. Military Service Members

    PubMed Central

    Rusiecki, Jennifer A.; Byrne, Celia; Galdzicki, Zygmunt; Srikantan, Vasantha; Chen, Ligong; Poulin, Matthew; Yan, Liying; Baccarelli, Andrea

    2013-01-01

    Background: The underlying molecular mechanisms of PTSD are largely unknown. Distinct expression signatures for PTSD have been found, in particular for immune activation transcripts. DNA methylation may be significant in the pathophysiology of PTSD, since the process is intrinsically linked to gene expression. We evaluated temporal changes in DNA methylation in select promoter regions of immune system-related genes in U.S. military service members with a PTSD diagnosis, pre- and post-diagnosis, and in controls. Methods: Cases (n = 75) had a post-deployment diagnosis of PTSD in their medical record. Controls (n = 75) were randomly selected service members with no PTSD diagnosis. DNA was extracted from pre- and post-deployment sera. DNA methylation (%5-mC) was quantified at specific CpG sites in promoter regions of insulin-like growth factor 2 (IGF2), long non-coding RNA transcript H19, interleukin-8 (IL8), IL16, and IL18 via pyrosequencing. We used multivariate analysis of variance and generalized linear models to calculate adjusted means (adjusted for age, gender, and race) to make temporal comparisons of %5-mC for cases (pre- to post-deployment) versus controls (pre- to post-deployment). Results: There were significant differences in the change of %5-mC pre- to post-deployment between cases and controls for H19 (cases: +0.57%, controls: −1.97%; p = 0.04) and IL18 (cases: +1.39%, controls: −3.83%; p = 0.01). For H19 the difference was driven by a significant reduction in %5-mC among controls; for IL18 the difference was driven by both a reduction in %5-mC among controls and an increase in %5-mC among cases. Stratified analyses revealed more pronounced differences in the adjusted means of pre-post H19 and IL18 methylation differences for cases versus controls among older service members, males, service members of white race, and those with shorter deployments (6–12 months). Conclusion: In the study of deployed personnel, those who did not

  19. Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters.

    PubMed

    Shen, Lanlan; Kondo, Yutaka; Guo, Yi; Zhang, Jiexin; Zhang, Li; Ahmed, Saira; Shu, Jingmin; Chen, Xinli; Waterland, Robert A; Issa, Jean-Pierre J

    2007-10-01

    The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.

  20. DNA Methylation in Promoter Region as Biomarkers in Prostate Cancer

    PubMed Central

    Yang, Mihi; Park, Jong Y.

    2013-01-01

    The prostate gland is the most common site of cancer and the second leading cause of cancer death in American men. Recent emerging molecular biological technologies help us to know that epigenetic alterations such as DNA methylation within the regulatory (promoter) regions of genes are associated with transcriptional silencing in cancer. Promoter hypermethylation of critical pathway genes could be potential biomarkers and therapeutic targets for prostate cancer. In this chapter, we updated current information on methylated genes associated with the development and progression of prostate cancer. Over 40 genes have been investigated for methylation in promoter region in prostate cancer. These methylated genes are involved in critical pathways, such as DNA repair, metabolism, and invasion/metastasis. The role of hypermethylated genes in regulation of critical pathways in prostate cancer is discussed. These findings may provide new information of the pathogenesis, the exciting potential to be predictive and to provide personalized treatment of prostate cancer. Indeed, some epigenetic alterations in prostate tumors are being translated into clinical practice for therapeutic use. PMID:22359288

  1. 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

  2. Analysis of adenomatous polyposis coli gene expression, APC locus-microsatellite instability and APC promoter methylation in the progression of melanocytic tumours.

    PubMed

    Korabiowska, Monika; Schlott, Thilo; Siems, Nils; Müller, Anegret; Cordon-Cardo, Carlos; Fischer, Gösta; Brinck, Ulrich

    2004-12-01

    Adenomatous polyposis coli gene (APC) defects have been demonstrated for the first time in familial adenomatous polyposis. Recent reports indicate that the APC gene is an intermediary between cell adhesion molecules and the cytoskeleton and that it may function as a gatekeeper of colonic epithelial proliferation. The objective of this study was to analyse APC's presence in lentigos, primary melanomas and melanoma metastases. By immunohistochemistry, APC was demonstrated in all lentigos, in 75 out of 88 primary melanomas and in 16 out of 28 melanoma lymphatic metastases. The percentage of immunolabelled tumour cells (APC index) in lentigos ranged between 5 and 69%, in primary melanomas between 0 and 98% and in melanoma metastases between 0 and 52%. Statistically significant differences between lentigos and primary melanomas and between lentigos and metastases in APC expression were found. In a multivariate analysis, APC showed an independent prognostic impact. Analysis of microsatellite instability in the APC locus was performed on 29 melanomas. Microsatellite instability was found in 5/29 melanomas and loss of heterozygosity in 1/29 melanomas. Promoter methylation of APC was found in 6/10 APC-negative primary melanomas and in 9/10 APC-negative melanoma lymphatic metastases investigated. We conclude about important role of APC alterations for melanoma progression.

  3. Methylation of coding region alone inhibits gene expression in plant protoplasts.

    PubMed Central

    Hohn, T; Corsten, S; Rieke, S; Müller, M; Rothnie, H

    1996-01-01

    Derivatives of the cauliflower mosaic virus 35S promoter lacking CG and CNG methylation targets were constructed and used to direct transcription of reporter gene constructs in transiently transformed protoplasts. Such methylation-target-free (MTF) promoters, although weaker than the 35S promoter, retain significant activity despite mutation of the as-1 element. The effect of methylation on gene expression in MTF- and 35S-promoter driven constructs was examined. Even when the promoter region was free of methylation targets, reporter gene expression was markedly reduced when cytosine residues in CG dinucleotides were methylated in vitro prior to transformation. Mosaic methylation experiments, in which only specific parts of the plasmids were methylated, revealed that methylation of the coding region alone has a negative effect on reporter gene expression. Methylation nearer the 5' end of the coding region was more inhibitory, consistent with inhibition of transcription elongation. Images Fig. 5 PMID:8710871

  4. 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

  5. Accumulated promoter methylation as a potential biomarker for esophageal cancer

    PubMed Central

    Shi, Peiyi; Wang, Jianping; Wang, Jianming

    2017-01-01

    We performed a two-stage molecular epidemiological study to explore DNA methylation profiles for potential biomarkers of esophageal squamous cell carcinoma (ESCC) in a Chinese population. Infinium Methylation 450K BeadChip was used to identify genes with differentially methylated CpG sites. Sixteen candidate genes were validated by sequencing 1160 CpG sites in their promoter regions using the Illumina MiSeq platform. When excluding sites with negative changes, 10 genes (BNIP3, BRCA1, CCND1, CDKN2A, HTATIP2, ITGAV, NFKB1, PIK3R1, PRDM16 and PTX3) showed significantly different methylation levels among cancer lesions, remote normal-appearing tissues, and healthy controls. PRDM16 had the highest diagnostic value with the AUC (95% CI) of 0.988 (0.965–1.000), followed by PIK3R1, with the AUC (95% CI) of 0.969 (0.928–1.000). In addition, the methylation status was higher in patients with advanced cancer stages. These results indicate that aberrant DNA methylation may be a potential biomarker for the diagnosis of ESCC. PMID:27893424

  6. Gene-Specific Promoter Methylation Status in Hormone-Receptor-Positive Breast Cancer Associates with Postmenopausal Body Size and Recreational Physical Activity

    PubMed Central

    McCullough, Lauren E.; Chen, Jia; White, Alexandra J.; Xu, Xinran; Cho, Yoon Hee; Bradshaw, Patrick T.; Eng, Sybil M.; Teitelbaum, Susan L.; Terry, Mary Beth; Garbowski, Gail; Neugut, Alfred I.; Hibshoosh, Hanina; Santella, Regina M.; Gammon, Marilie D.

    2015-01-01

    Introduction Breast cancer, the leading cancer diagnosis among American women, is positively associated with postmenopausal obesity and little or no recreational physical activity (RPA). However, the underlying mechanisms of these associations remain unresolved. Aberrant changes in DNA methylation may represent an early event in carcinogenesis, but few studies have investigated associations between obesity/RPA and gene methylation, particularly in postmenopausal breast tumors where these lifestyle factors are most relevant. Methods We used case-case unconditional logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CI) for the associations between body mass index (BMI=weight [kg]/height [m2]) in the year prior to diagnosis, or RPA (average hours/week), and methylation status (methylated vs. unmethylated) of 13 breast cancer-related genes in 532 postmenopausal breast tumor samples from the Long Island Breast Cancer Study Project. We also explored whether the association between BMI/RPA and estrogen/progesterone-receptor status (ER+PR+ vs. all others) was differential with respect to gene methylation status. Methylation-specific PCR and the MethyLight assay were used to assess gene methylation. Results BMI 25-29.9kg/m2, and perhaps BMI≥30kg/m2, was associated with methylated HIN1 in breast tumor tissue. Cases with BMI≥30kg/m2 were more likely to have ER+PR+ breast tumors in the presence of unmethylated ESR1 (OR=2.63, 95% CI 1.32-5.25) and women with high RPA were more likely to have ER+PR+ breast tumors with methylated GSTP1 (OR=2.33, 95% CI 0.79-6.84). Discussion While biologically plausible, our findings that BMI is associated with methylated HIN1 and BMI/RPA are associated with ER+PR+ breast tumors in the presence of unmethylated ESR1 and methylated GSTP1, respectively, warrant further investigation. Future studies would benefit from enrolling greater numbers of postmenopausal women and examining a larger panel of breast cancer

  7. Aberrant promoter methylation of SH3GL2 gene in vulvar squamous cell carcinoma correlates with clinicopathological characteristics and HPV infection status

    PubMed Central

    Li, Bo; He, Yinghui; Han, Xue; Zhang, Shitai; Xu, Yan; Zhou, Yang; Song, Zixuan; Ouyang, Ling

    2015-01-01

    Objective: This study attempted to examine the methylation status of SH3GL2 gene in different types of human vulvar lesions and its correlation with clinicopathological parameters. Methods: Immunohistochemical analysis was used to identify the expression status of SH3GL2 in vulvar squamous cell carcinoma (VSCC), vulvar intraepithelial neoplasia (VIN) and benign vulvar squamous epithelium tissues. Bisulfite genomic sequencing method was used to detect methylation status of the SH3GL2 gene. Clinicopathological correlation of the alterations was analysed by the chi-square tests. Results: Immunohistochemical analysis showed expression of SH3GL2 in VSCC was significantly downregulated than that in VIN and normal vulvar tissues. In accordance with higher frequency of methylation status in SH3GL2, statistical analysis showed methylation status of SH3GL2 was closely related to tumor TNM stage (P=0.003), but not related to age, tumor volume, tumor differentiation, lymph node metastasis and VIN grade. High-methylation status of SH3GL2 showed significant association with HPV infection status. Conclusions: Our results indicated that the methylation status of SH3GL2 gene was associated with the TNM staging and HPV infection status of VSCC, suggesting that it might play a synergistic role in the development of VSCC. PMID:26823912

  8. Promoter methylation of Wnt antagonist DKK1 gene and prognostic value in Korean patients with non-small cell lung cancers.

    PubMed

    Na, Yeonkyung; Lee, Su Man; Kim, Dong Sun; Park, Jae Yong

    2012-01-01

    Dickkopf-1 (DKK1) is known as a negative regulator of the Wnt signaling pathway, which plays a crucial role in carcinogenesis. However, aberrant expression and the role of DKK1 in human cancers remain controversial. To estimate the role of DKK1 and its prognostic potential in lung cancer, promoter methylation of DKK1 was evaluated in 139 primary non-small cell lung cancers (NSCLCs) by methylation-specific PCR and its association with clinical and prognostic parameters. DKK1 hypermethylation was detected in 48.9% of neoplastic lung tissues and was significantly more frequent in stage I than the more advanced stages II-IIIA (P=0.04). Additionally, patients with DKK1 methylation had a better overall survival than those with no methylation under univariate analysis. When stratified by clinicopathologic features, DKK1 methylation was significantly associated with a favorable survival in a subset of patients. The current findings suggested that DKK1 promoter methylation may be a tumor-associated event in the early stage of NSCLC and could also be useful prognostic indicator for NSCLC. Further work may clarify the molecular basis of DKK1 action in progression of NSCLC.

  9. Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells.

    PubMed

    Koutsimpelas, Dimitrios; Pongsapich, Warut; Heinrich, Ulf; Mann, Sylvia; Mann, Wolf J; Brieger, Jürgen

    2012-04-01

    Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs

  10. Transcriptional regulation of 15-lipoxygenase expression by promoter methylation.

    PubMed

    Liu, Cheng; Xu, Dawei; Sjöberg, Jan; Forsell, Pontus; Björkholm, Magnus; Claesson, Hans-Erik

    2004-07-01

    15-Lipoxygenase type 1 (15-LO), a lipid-peroxidating enzyme implicated in physiological membrane remodeling and the pathogenesis of atherosclerosis, inflammation, and carcinogenesis, is highly regulated and expressed in a tissue- and cell-type-specific fashion. It is known that interleukins (IL) 4 and 13 play important roles in transactivating the 15-LO gene. However, the fact that they only exert such effects on a few types of cells suggests additional mechanism(s) for the profile control of 15-LO expression. In the present study, we demonstrate that hyper- and hypomethylation of CpG islands in the 15-LO promoter region is intimately associated with the transcriptional repression and activation of the 15-LO gene, respectively. The 15-LO promoter was exclusively methylated in all examined cells incapable of expressing 15-LO (certain solid tumor and human lymphoma cell lines and human T lymphocytes) while unmethylated in 15-LO-competent cells (the human airway epithelial cell line A549 and human monocytes) where 15-LO expression is IL4-inducible. Inhibition of DNA methylation in L428 lymphoma cells restores IL4 inducibility to 15-LO expression. Consistent with this, the unmethylated 15-LO promoter reporter construct exhibited threefold higher activity in A549 cells compared to its methylated counterpart. Taken together, demethylation of the 15-LO promoter is a prerequisite for the gene transactivation, which contributes to tissue- and cell-type-specific regulation of 15-LO expression.

  11. RUNX3 Promoter Methylation Is Associated with Hepatocellular Carcinoma Risk: A Meta-Analysis.

    PubMed

    Zhang, Xueyan; He, Hairong; Zhang, Xu; Guo, Wenjie; Wang, Yueqi

    2015-04-01

    Runt-related transcription factor 3 (RUNX3) has been reported to be a tumor-suppressing gene in hepatocellular carcinoma. Association between hepatocellular carcinoma and RUNX3 promoter methylation has been investigated in studies with specific ethnic backgrounds and small sample sizes. In this study, a meta-analysis was adopted to combine the data from 11 studies of association between RUNX3 promoter methylation and hepatocellular carcinoma. Pooled odds ratio for RUNX3 promoter methylation status in hepatocellular carcinoma versus control liver tissue was 24.37 (95%CI: 12.14, 48.92), p < .00001, indicates a strong association between methylation of the RUNX3 promoter and hepatocellular carcinoma.

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

    PubMed

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

    2016-09-01

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

  13. Promoter analyses and transcriptional profiling of eggplant polyphenol oxidase 1 gene (SmePPO1) reveal differential response to exogenous methyl jasmonate and salicylic acid.

    PubMed

    Shetty, Santoshkumar M; Chandrashekar, Arun; Venkatesh, Yeldur P

    2012-05-01

    The transcriptional regulation of multigenic eggplant (Solanum melongena) polyphenol oxidase genes (SmePPO) is orchestrated by their corresponding promoters which mediate developmentally regulated expression in response to myriad biotic and abiotic factors. However, information on structural features of SmePPO promoters and modulation of their expression by plant defense signals are lacking. In the present study, SmePPOPROMOTERs were cloned by genome walking, and their transcription start sites (TSS) were determined by RLM-RACE. Extensive sequence analyses revealed the presence of evolutionarily conserved and over-represented putative cis-acting elements involved in light-regulated transcription, biosynthetic pathways (phenylpropanoid/flavonoid), hormone signaling (abscisic acid, gibberellic acid, jasmonate and salicylate), elicitor and stress responses (cold/dehydration responses), sugar metabolism and plant defense signaling (W-BOX/WRKY) that are common to SmePPOPROMOTER1 and 2. The TSS for SmePPO genes are located 9-15bp upstream of ATG with variable lengths of 5' untranslated regions. Transcriptional profiling of SmePPOs in eggplant seedlings has indicated differential response to methyl jasmonate (MeJA) or salicylic acid (SA) treatment. In planta, while MeJA elicited expression of all the six SmePPOs, SA was only able to induce the expression of SmePPO4-6. Interestingly, in dual treatment, SA considerably repressed the MeJA-induced expression of SmePPOs. Functional dissection of SmePPOPROMOTER1 by deletion analyses using Agrobacterium-mediated transient expression in tobacco leaves has shown that MeJA enhances the SmePPOPROMOTER1-β-glucuronidase (GUS) expression in vivo, while SA does not. Histochemical and quantitative GUS assays have also indicated the negative effect of SA on MeJA-induced expression of SmePPOPROMOTER1. By combining in silico analyses, transcriptional profiling and expression of SmePPOPROMOTER1-GUS fusions, the role of SA on the modulation

  14. 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

  15. Methylation status of a single CpG locus 3 bases upstream of TATA-box of receptor activator of nuclear factor-kappaB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis.

    PubMed

    Kitazawa, Riko; Kitazawa, Sohei

    2007-01-01

    Receptor activator of nuclear factor-kappaB ligand (RANKL) expression is tissue specific and limited to certain subsets of T-lymphocytes and stromal/osteoblastic cells. Even among osteoblasts, RANKL is expressed on about 20% of osteoblasts of the normal mouse. To clarify the mechanism of population-specific RANKL expression, we analyzed the effect of CpG methylation on its transcription, mRNA and protein expression as well as on osteoclastogenesis. Subpopulations of ST2 cells were used: P9, which expresses RANKL and supports osteoclastogenesis, and P16, which does not. By sodium bisulfite mapping, the rate of CpG methylation of the -65/+350 region, especially of CpG locus no. 1 three bases upstream of the TATA-box, was higher in P16 than in P9 ST2 cells. ChIP and gel shift assay showed that methylated CpG locus no. 1 was a target of MeCP2 binding that, in turn, blocked the binding of the TATA-box binding protein to the TATA-box. In vitro methylation by SssI of the promoter construct reduced its transcriptional activity at the steady state and its response to 1alpha,25(OH)2 vitamin D3. Conversely, treatment with DNA methylase inhibitor, 5-aza-2'-deoxycytidine, significantly restored RANKL expression and osteoclastogenesis in P16 cells. Except for primary cultured osteoblasts, CpG locus no. 1 was frequently methylated in various normal mouse tissues. We propose that the methylation status of the CpG locus three bases upstream of the TATA-box modulates the control of cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. The heterogeneity of stromal/ osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter.

  16. 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

  17. 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.

  18. 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.

  19. PTSD and DNA Methylation in Select Immune Function Gene Promoter Regions: A Repeated Measures Case-control Study of U.S. Military Service Members

    DTIC Science & Technology

    2013-06-24

    exclude possible confounding by other psychiatric illnesses , for which differential gene expression or methylation has been reported (Mill and Petronis...ICD-9 code 296), and manic phase bi-polar disorder (also ICD-9 code 296). The PTSD cases (n= 75) had existing pre- and post- deployment serum samples...Broad spec- trum of cytokine abnormalities in panic disorder and posttraumatic stress disorder. Depress . Anxiety 26, 447–455. doi:10.1002/da.20564

  20. Quantitative analysis of follistatin (FST) promoter methylation in peripheral blood of patients with polycystic ovary syndrome.

    PubMed

    Sang, Qing; Zhang, Shaozhen; Zou, Shien; Wang, Huan; Feng, Ruizhi; Li, Qiaoli; Jin, Li; He, Lin; Xing, Qinghe; Wang, Lei

    2013-02-01

    Epigenetic mechanisms may contribute to polycystic ovary syndrome (PCOS). To date, however, no studies have associated CpG methylation levels of any candidate gene with PCOS susceptibility. Follistatin (FST), an activin-binding protein, is expressed in numerous tissues and is shown to have linkage with PCOS. However, results from case-control association analyses between this gene and PCOS are inconsistent. Thus, this study investigated possible association of methylation levels in the promoter and 5'-untranscribed region (UTR) of the FST gene with PCOS incidence in peripheral blood leukocytes and endometrial tissue. Using mass array quantitative methylation analysis, first the 5'-UTR methylation in FST was analysed in 130 PCOS patients and 120 controls. The methylation level of the FST gene was further studied in endometrium from 24 controls and 24 PCOS patients. This study demonstrates that methylation levels of CpG sites in the FST promoter and 5'-UTR are not associated with PCOS. Nonetheless, this was the first study to quantitatively evaluate the methylation levels of a candidate gene in association with PCOS. Further studies should be performed to examine methylation in other candidate genes. Understanding the epigenetic mechanisms involved in PCOS may yield new insights into the pathophysiology of the disorder. Animal models demonstrate that epigenetic reprogramming may contribute to polycystic ovary syndrome (PCOS). To date, however, no studies have associated CpG methylation levels of any candidate gene with PCOS susceptibility. Follistatin (FST), an activin-binding protein, is expressed in numerous tissues and is a PCOS candidate gene. However, results from association analyses between this gene and PCOS are inconsistent. Thus, we investigated possible association of methylation levels in the promoter and 5'-UTR of the FST gene with PCOS incidence in peripheral blood leukocytes and endometrial tissue. Using mass array quantitative methylation analysis, we

  1. Methylation and expression analysis of 15 genes and three normally-methylated genes in 13 Ovarian cancer cell lines.

    PubMed

    Imura, Masayoshi; Yamashita, Satoshi; Cai, Li-Yi; Furuta, Jun-Ichi; Wakabayashi, Mika; Yasugi, Toshiharu; Ushijima, Toshikazu

    2006-09-28

    Aberrant methylation of CpG islands (CGIs) in promoter regions of tumor-suppressor genes causes their silencing, and aberrant demethylation of normally methylated CGIs in promoter regions causes aberrant expression of cancer-testis antigens. Here, we comprehensively analyzed aberrant methylation of 15 genes and demethylation of three normally methylated genes in 13 ovarian cancer cell lines. RASSF1A was most frequently methylated (complete methylation in 7 and partial methylation in 4 cell lines), followed by ESR1 (5 and 2, respectively), FLNC (4 and 4), HAND1 (4 and 2), LOX (3 and 2), HRASLS (3 and 2), MGMT (3 and 0), CDKN2A (3 and 0), THBD (2 and 1), hMLH1 (2 and 0), CDH1 (1 and 1) and GSTP1 (1 and 0). hTERC and TIMP3 were only partially methylated in 7 and 2 cell lines, respectively. BRCA1 was not methylated at all. Aberrant demethylation of MAGE-A3, -B2 and -A1 was detected in 8, 4 and 3 cell lines, respectively. Gene expression was consistently absent in cell lines without unmethylated DNA molecules. Aberrant methylation was frequently observed in MCAS, RMUG-L (mucinous cell carcinomas), RTSG (poorly-differentiated carcinoma) and TYK-nu (undifferentiated carcinoma) while infrequent in HTOA, JHOS-2, and OV-90 (serous cell carcinomas). Aberrant demethylation was frequently observed in OV-90, OVK-18, and ES-2 cell lines. It was shown that aberrant methylation and demethylation were frequently observed in ovarian cancer cell lines, and these data will provide a basis for further epigenetic analysis in ovarian cancers.

  2. Quantitative evaluation of DNMT3B promoter methylation in breast cancer patients using differential high resolution melting analysis.

    PubMed

    Naghitorabi, M; Mohammadi Asl, J; Mir Mohammad Sadeghi, H; Rabbani, M; Jafarian-Dehkordi, A; Javanmard, Haghjooye S

    2013-07-01

    DNA methylation plays an important role in carcinogenesis through epigenetic silencing of tumor suppressor genes. Aberrant methylation usually results from changes in the activity of DNA methyltransferases (DNMTs). Some studies show that the overexpression of the DNMTs may lead to aberrant methylation of tumor suppressor genes. Also the overexpression of DNMTs may be related to methylation status of their genes. Due to limited number of studies on DNMT3B promoter methylation, this study was performed to quantitatively measure the methylation level of DNMT3B gene in archival formalin fixed paraffin embedded (FFPE) tissues from breast cancer patients. Using differential high resolution melting analysis (D-HRMA) technology, the methylation level of DNMT3B gene promoter was quantified in 98 breast cancer FFPE tissues and also 10 fresh frozen normal tissue samples. Statistical analyses used for analyzing the correlation between the methylation and clinical variables. All the normal samples were found to be methylated at the DNMT3B promoter (the average methylation level 3.34%). Patients were identified as hypo-methylated (mean methylation level 0.8%), methylated (mean methylation level 2.48%) and hyper-methylated (mean methylation level 10.5%). Statistical analysis showed a significant correlation between the methylation status and the sample type, cancer type and tumor size. Also the methylation level was significantly associated with histologic grade. It is concluded that quantification of DNMT3B promoter methylation might be used as a reliable and sensitive diagnostic and prognostic tool in breast cancer. Also D-HRMA is demonstrated as a rapid and cost effective method for quantitative evaluation of promoter methylation.

  3. 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

  4. A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer

    PubMed Central

    Schwartzman, Jacob; Mongoue-Tchokote, Solange; Gibbs, Angela; Gao, Lina; Corless, Christopher L; Jin, Jennifer; Zarour, Luai; Higano, Celestia; True, Lawrence D; Vessella, Robert L; Wilmot, Beth; Bottomly, Daniel; McWeeney, Shannon K; Bova, G. Steven; Partin, Alan W; Mori, Motomi

    2011-01-01

    DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1,505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer. PMID:21946329

  5. Perinatal high methyl donor alters gene expression in IGF system in male offspring without altering DNA methylation

    PubMed Central

    Amarger, Valérie; Giudicelli, Fanny; Pagniez, Anthony; Parnet, Patricia

    2017-01-01

    Aim: To investigate the effect of a protein restriction and a supplementation with methyl donor nutrients during fetal and early postnatal life on the expression and epigenetic state of imprinted genes from the IGF system. Materials & methods: Pregnant female rats were fed a protein-restricted diet supplemented or not with methyl donor. Results: Gene expression of the Igf2, H19, Igf1, Igf2r and Plagl1 genes in the liver of male offspring at birth and weaning was strongly influenced by maternal diet. Whereas the methylation profiles of the Igf2, H19 and Igf2r genes were remarkably stable, DNA methylation of Plagl1 promoter was slightly modified. Conclusion: DNA methylation of most, but not all, imprinted gene regulatory regions was resistant to methyl group nutritional supply. PMID:28344827

  6. 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.

  7. Mitogen-activated protein kinase 6 mediates nuclear translocation of ORE3 to promote ORE9 gene expression in methyl jasmonate-induced leaf senescence.

    PubMed

    Zhang, Yushan; Liu, Jian; Chai, Jinyu; Xing, Da

    2016-01-01

    Methyl jasmonate (MeJA) is a potent promoter of plant senescence. ORESARA3 (ORE3)/ETHYLENE INSENSITIVE2 (EIN2), a protein similar to the members of the disease-related Nramp metal transporter family, is involved in cross-talk among several senescence processes related to abscisic acid, ethylene, MeJA, age and darkness. Nevertheless, the mechanism involved in the regulation of ORE3/EIN2 in exogenous MeJA-induced leaf senescence remains unclear. The C-terminal end of ORE3/EIN2 (CEND) was cleaved from ORE3/EIN2 located in the endoplasmic reticulum and then transferred to the nucleus during MeJA-induced senescence. Further analyses showed that mitogen-activated protein kinase 6 (MPK6) promoted CEND cleavage and nuclear translocation. Nuclear CEND accumulated ETHYLENE INSENSITIVE3 (EIN3), a transcription factor that accelerates MeJA-induced leaf senescence wherein ORESARA9 (ORE9) expression was suppressed in ein3, ore3, and mpk6 mutant plants. ChIP experiments revealed that EIN3 bound directly to the ORE9 promoter and this binding was enhanced in MeJA-induced leaf senescence. This study revealed the effect of the signalling pathway involving MPK6-ORE3-EIN3-ORE9 on regulating leaf senescence and provided insights into the mechanism of MeJA in promoting leaf senescence in Arabidopsis thaliana.

  8. DNA promoter methylation in breast tumors: no association with genetic polymorphisms in MTHFR and MTR.

    PubMed

    Tao, Meng Hua; Shields, Peter G; Nie, Jing; Marian, Catalin; Ambrosone, Christine B; McCann, Susan E; Platek, Mary; Krishnan, Shiva S; Xie, Bin; Edge, Stephen B; Winston, Janet; Vito, Dominica; Trevisan, Maurizio; Freudenheim, Jo L

    2009-03-01

    Aberrant promoter methylation is recognized as an important feature of breast carcinogenesis. We hypothesized that genetic variation of genes for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR), two critical enzymes in the one-carbon metabolism, may alter DNA methylation levels and thus influence DNA methylation in breast cancer. We evaluated case-control association of MTHFR C677T, A1298C, and MTR A2756G polymorphisms for cases strata-defined by promoter methylation status for each of three genes, E-cadherin, p16, and RAR-beta2 in breast cancer; in addition, we evaluated case-case comparisons of the likelihood of promoter methylation in relation to genotypes using a population-based case-control study conducted in Western New York State. Methylation was evaluated with real-time methylation-specific PCRs for 803 paraffin-embedded breast tumor tissues from women with primary, incident breast cancer. We applied unordered polytomous regression and unconditional logistic regression to derive adjusted odds ratios and 95% confidence intervals. We did not find any association of MTHFR and MTR polymorphisms with breast cancer risk stratified by methylation status nor between polymorphisms and likelihood of promoter methylation of any of the genes. There was no evidence of difference within strata defined by menopausal status, estrogen receptor status, folate intake, and lifetime alcohol consumption. Overall, we found no evidence that these common polymorphisms of the MTHFR and MTR genes are associated with promoter methylation of E-cadherin, p16, and RAR-beta2 genes in breast cancer.

  9. DNA promoter methylation in breast tumors: No association with genetic polymorphisms in MTHFR and MTR

    PubMed Central

    Tao, Meng Hua; Shields, Peter G.; Nie, Jing; Marian, Catalin; Ambrosone, Christine B.; McCann, Susan E.; Platek, Mary; Krishnan, Shiva S.; Xie, Bin; Edge, Stephen B.; Winston, Janet; Vito, Dominica; Trevisan, Maurizio; Freudenheim, Jo L.

    2013-01-01

    Aberrant promoter methylation is recognized as an important feature of breast carcinogenesis. We hypothesized that genetic variation of genes for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR), two critical enzymes in one-carbon metabolism, may alter DNA methylation levels, and thus influence DNA methylation in breast cancer. We evaluated case-control association of MTHFR C677T, A1298C, and MTR A2756G polymorphisms for cases strata defined by promoter methylation status for each of three genes, E- cadherin, p16, and RAR-β2 in breast cancer; in addition, we evaluated case-case comparisons of likelihood of promoter methylation in relation to genotypes using a population-based case-control study conducted in Western New York State. Methylation was evaluated with real time methylation-specific PCRs for 803 paraffin embedded breast tumor tissues from women with primary, incident breast cancer. We applied unordered polytomous regression and unconditional logistic regression to derive adjusted odds ratios (OR) and 95% confidence intervals (CI). We did not find any association of MTHFR and MTR polymorphisms with breast cancer risk stratified by methylation status nor between polymorphisms and likelihood of promoter methylation of any of the genes. There was no evidence of difference within strata defined by menopausal status, ER status, folate intake and lifetime alcohol consumption. Overall, we found no evidence that these common polymorphisms of the MTHFR and MTR genes are associated with promoter methylation of E- cadherin, p16, and RAR-β2 genes in breast cancer. PMID:19240236

  10. Delta DNMT3B variants regulate DNA methylation in a promoter-specific manner.

    PubMed

    Wang, Jie; Bhutani, Manisha; Pathak, Ashutosh K; Lang, Wenhua; Ren, Hening; Jelinek, Jaroslav; He, Rong; Shen, Lanlan; Issa, Jean-Pierre; Mao, Li

    2007-11-15

    DNA methyltransferase 3B (DNMT3B) is critical in de novo DNA methylation during development and tumorigenesis. We recently reported the identification of a DNMT3B subfamily, DeltaDNMT3B, which contains at least seven variants, resulting from alternative pre-mRNA splicing. DeltaDNMT3Bs are the predominant expression forms of DNMT3B in human lung cancer. A strong correlation was observed between the promoter methylation of RASSF1A gene but not p16 gene (both frequently inactivated by promoter methylation in lung cancer) and expression of DeltaDNMT3B4 in primary lung cancer, suggesting a role of DeltaDNMT3B in regulating promoter-specific methylation of common tumor suppressor genes in tumorigenesis. In this report, we provide first experimental evidence showing a direct involvement of DeltaDNMT3B4 in regulating RASSF1A promoter methylation in human lung cancer cells. Knockdown of DeltaDNMT3B4 expression by small interfering RNA resulted in a rapid demethylation of RASSF1A promoter and reexpression of RASSF1A mRNA but had no effect on p16 promoter in the lung cancer cells. Conversely, normal bronchial epithelial cells with stably transfected DeltaDNMT3B4 gained an increased DNA methylation in RASSF1A promoter but not p16 promoter. We conclude that promoter DNA methylation can be differentially regulated and DeltaDNMT3Bs are involved in regulation of such promoter-specific de novo DNA methylation.

  11. Exercise effects on methylation of ASC gene.

    PubMed

    Nakajima, K; Takeoka, M; Mori, M; Hashimoto, S; Sakurai, A; Nose, H; Higuchi, K; Itano, N; Shiohara, M; Oh, T; Taniguchi, S

    2010-09-01

    Chronic moderate exercise has been reported to reduce pro-inflammatory cytokines. To analyze the molecular mechanisms by which training exerts these effects, the epigenetic influences of age and exercise on the ASC gene, which is responsible for IL-1beta and IL-18 secretion, were investigated by ASC gene methylation. Further, the relationship between carcinogenesis and exercise, and methylation of the P15 tumor suppressive gene was also analyzed. High-intensity interval walking exercise, consisting of 3 min low-intensity walking at 40% of peak aerobic capacity followed by a 3 min high-intensity walking period above 70% of peak aerobic capacity, was continued for 6 months. Peripheral blood DNA extracts from young control (n=34), older control (n=153), and older exercise (n=230) groups were then analyzed by pyrosequencing for DNA methylation. Methylation of ASC decreased significantly with age (young control vs. older control, p<0.01), which is indicative of an age-dependent increase in ASC expression. Compared to the older control group, the degree of ASC methylation was higher in the older exercise group (older control vs. older exercise: p<0.01), and presumably lower ASC expression. Neither exercise nor age affected the methylation of the P15. In summary, chronic moderate exercise appears to attenuate the age-dependent decrease in ASC methylation, implying suppression of excess pro-inflammatory cytokines through reduction of ASC expression.

  12. Regulation of Hox orthologues in the oyster Crassostrea gigas evidences a functional role for promoter DNA methylation in an invertebrate.

    PubMed

    Saint-Carlier, Emma; Riviere, Guillaume

    2015-06-04

    DNA methylation within promoter regions (PRDM) controls vertebrate early gene transcription and thereby development, but is neglected outside this group. However, epigenetic features in the oyster Crassostrea gigas suggest functional significance of PDRM in invertebrates. To investigate this, reporter constructs containing in vitro methylated oyster Hox gene promoters were transfected into oyster embryos. The influence of in vivo methylation was studied using bisulfite sequencing and DNA methyltransferase inhibition during development. Our results demonstrate that methylation controls the transcriptional activity of the promoters investigated, unraveling a functional role for PRDM in a lophotrochozoan, an important finding regarding the evolution of epigenetic regulation.

  13. Differential Methylation of the Arsenic (III) Methyltransferase Promoter According to Arsenic Exposure

    PubMed Central

    Gribble, Matthew O.; Tang, Wan-yee; Shang, Yan; Pollak, Jonathan; Umans, Jason G.; Francesconi, Kevin A.; Goessler, Walter; Silbergeld, Ellen K.; Guallar, Eliseo; Cole, Shelley A.; Fallin, M. Daniele; Navas-Acien, Ana

    2013-01-01

    Inorganic arsenic is methylated in the body by arsenic (III) methyltransferase. Arsenic methylation is thought to play a role in arsenic-related epigenetic phenomena including aberrant DNA and histone methylation. However, it is unclear whether the promoter of the AS3MT gene, which codes for arsenic (III) methyltransferase, is differentially methylated as a function of arsenic exposure. In this study we evaluated AS3MT promoter methylation according to exposure, assessed by urinary arsenic excretion in a stratified random sample of 48 participants from the Strong Heart Study who had urine arsenic measured at baseline and DNA available from 1989–1991 and 1998–1999. For this study, all data are from the 1989–1991 visit. We measured AS3MT promoter methylation at its 48 CpG loci by bisulphite sequencing. We compared mean % methylation at each CpG locus by arsenic exposure group using linear regression adjusted for study centre, age and sex. A hypomethylated region in the AS3MT promoter was associated with higher arsenic exposure. In vitro, arsenic induced AS3MT promoter hypomethylation and it increased AS3MT expression in human peripheral blood mononuclear cells. These findings may suggest that arsenic exposure influences the epigenetic regulation of a major arsenic metabolism gene. PMID:24154821

  14. Promoter Methylation Analysis Reveals that KCNA5 Ion Channel Silencing Supports Ewing Sarcoma Cell Proliferation

    PubMed Central

    Ryland, Katherine E; Hawkins, Allegra G.; Weisenberger, Daniel J.; Punj, Vasu; Borinstein, Scott C.; Laird, Peter W.; Martens, Jeffrey R.; Lawlor, Elizabeth R.

    2015-01-01

    Polycomb proteins are essential regulators of gene expression in stem cells and development. They function to reversibly repress gene transcription via post-translational modification of histones and chromatin compaction. In many human cancers, genes that are repressed by polycomb in stem cells are subject to more stable silencing via DNA methylation of promoter CpG islands. Ewing sarcoma is an aggressive bone and soft tissue tumor that is characterized by over-expression of polycomb proteins. This study investigates the DNA methylation status of polycomb target gene promoters in Ewing sarcoma tumors and cell lines and observes that the promoters of differentiation genes are frequent targets of CpG-island DNA methylation. In addition, the promoters of ion channel genes are highly differentially methylated in Ewing sarcoma compared to non-malignant adult tissues. Ion channels regulate a variety of biological processes, including proliferation, and dysfunction of these channels contributes to tumor pathogenesis. In particular, reduced expression of the voltage-gated Kv1.5 channel has been implicated in tumor progression. These data show that DNA methylation of the KCNA5 promoter contributes to stable epigenetic silencing of Kv1.5 channel. This epigenetic repression is reversed by exposure to the DNA methylation inhibitor decitabine, which inhibits Ewing sarcoma cell proliferation through mechanisms that include restoration of Kv1.5 channel function. Implications This study demonstrates that promoters of ion channels are aberrantly methylated in Ewing sarcoma and that epigenetic silencing of KCNA5 contributes to tumor cell proliferation, thus providing further evidence of the importance of ion channel dyregulation to tumorigenesis. PMID:26573141

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. Methylization analysis of the FMR1 gene in carrier females

    SciTech Connect

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

    1994-09-01

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

  18. Promoter DNA Methylation Pattern Identifies Prognostic Subgroups in Childhood T-Cell Acute Lymphoblastic Leukemia

    PubMed Central

    Borssén, Magnus; Palmqvist, Lars; Karrman, Kristina; Abrahamsson, Jonas; Behrendtz, Mikael; Heldrup, Jesper; Forestier, Erik; Roos, Göran; Degerman, Sofie

    2013-01-01

    Background Treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has improved, but there is a considerable fraction of patients experiencing a poor outcome. There is a need for better prognostic markers and aberrant DNA methylation is a candidate in other malignancies, but its potential prognostic significance in T-ALL is hitherto undecided. Design and Methods Genome wide promoter DNA methylation analysis was performed in pediatric T-ALL samples (n = 43) using arrays covering >27000 CpG sites. Clinical outcome was evaluated in relation to methylation status and compared with a contemporary T-ALL group not tested for methylation (n = 32). Results Based on CpG island methylator phenotype (CIMP), T-ALL samples were subgrouped as CIMP+ (high methylation) and CIMP− (low methylation). CIMP− T-ALL patients had significantly worse overall and event free survival (p = 0.02 and p = 0.001, respectively) compared to CIMP+ cases. CIMP status was an independent factor for survival in multivariate analysis including age, gender and white blood cell count. Analysis of differently methylated genes in the CIMP subgroups showed an overrepresentation of transcription factors, ligands and polycomb target genes. Conclusions We identified global promoter methylation profiling as being of relevance for subgrouping and prognostication of pediatric T-ALL. PMID:23762353

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

    PubMed

    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

    2016-02-15

    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.

  20. Expression analysis and promoter methylation under osmotic and salinity stress of TaGAPC1 in wheat (Triticum aestivum L).

    PubMed

    Fei, Ying; Xue, Yuanxia; Du, Peixiu; Yang, Shushen; Deng, Xiping

    2017-03-01

    Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) catalyzes a key reaction in glycolysis and encoded by a multi-gene family which showed instability expression under abiotic stress. DNA methylation is an epigenetic modification that plays an important role in gene regulation in response to abiotic stress. The comprehension of DNA methylation at promoter region of TaGAPC1 can provide insights into the transcription regulation mechanisms of plant genes under abiotic stress. In this study, we cloned TaGAPC1 genes and its promoters from two wheat genomes, then investigated the expression patterns of TaGAPC1 under osmotic and salinity stress, and analyzed the promoter sequences. Moreover, the methylation patterns of promoters under stress were confirmed. Expression analysis indicated that TaGAPC1 was induced inordinately by stresses in two wheat genotypes with contrasting drought tolerance. Several stress-related cis-acting elements (MBS, DRE, GT1 and LTR et al.) were located in its promoters. Furthermore, the osmotic and salinity stress induced the demethylation of CG and CHG nucleotide in the promoter region of Changwu134. The methylation level of CHG and CHH in promoter of Zhengyin1 was always increased under stresses, and the CG contexts remained unchanged. The cytosine loci of stress-related cis-acting elements also showed different methylation changes in this process. These results provide insights into the relationship between promoter methylation and gene expression, promoting the function investigation of GAPC.

  1. Detection of MGMT promoter methylation in glioblastoma using pyrosequencing.

    PubMed

    Xie, Hao; Tubbs, Raymond; Yang, Bin

    2015-01-01

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

  2. Detection of MGMT promoter methylation in glioblastoma using pyrosequencing.

    PubMed

    Xie, Hao; Tubbs, Raymond; Yang, Bin

    2015-01-01

    Recent clinical trials on patients with glioblastoma revealed that O(6)-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient's response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation.

  3. Deoxyribonucleic acid (DNA) methyltransferase contributes to p16 promoter CpG island methylation in lung adenocarcinoma with smoking.

    PubMed

    Sun, Rongju; Liu, Jiahong; Wang, Bo; Ma, Lingyun; Quan, Xiaojiao; Chu, Zhixiang; Li, Tanshi

    2015-01-01

    In this study, the relationship between CpG island methylation and smoking and DNA methyltransferase in the occurrence and development of lung adenocarcinoma was explored by detecting p16 promoter methylation status. Protein and mRNA levels of p16 were detected by immunohistochemistry and in situ hybridization assays. p16 gene promoter and exon 1 CpG island locus Hap II sites methylation status was analyzed with the methylation-specific PCR. Only 4 of 40 p16-positive cases were detected to methylate on CpG islands with 10% methylating rate whereas 18 of p16-negative cases were methylated up to 36.73% of methylating rate. The methylating rates of both p16-positive and p16-negative groups were significantly different. 17 of 50 cases with smoking from total 89 lung adenocarcinoma cases were detected to methylate on CpG islands while only 5 of the remaining 39 non-smokers to methylate. The difference of the methylating rates in both smokers and non-smokers was significant to suggest the closely association of CpG island methylation of p16 with smoking. Furthermore, p16 promoter CpG islands were detected to methylate in 15 of 35 cases with higher DNA methyltransferase activity whereas only 7 detected to methylate in the remaining 54 cases with lower DNA methyltransferase activity. p16 promoter CpG island methylation likely made p16 expressing silence thus contributed to the tumorigenesis of lung adenocarcinoma. Smoking is likely to promote p16 CpG island methylation or by its effect of the activity and metabolism of DNA methyltransferase 1 (DNMT) on CpG island methylation status.

  4. TNF-alpha promoter methylation as a predictive biomarker for weight-loss response.

    PubMed

    Campión, Javier; Milagro, Fermin I; Goyenechea, Estibaliz; Martínez, J Alfredo

    2009-06-01

    Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine which is commonly elevated in obese subjects and whose promoter is susceptible to be regulated by cytosine methylation. The aim of this research was to analyze whether epigenetic regulation of human TNF-alpha promoter by cytosine methylation could be involved in the predisposition to lose body weight after following a balanced hypocaloric diet. Twenty-four patients (12 women/12 men) with excessive body weight-for-height (BMI: 30.5+/-0.32 kg/m2; age: 34+/-4 years old) followed an 8-week energy-restricted diet. Blood mononuclear cell DNA, isolated before the nutritional intervention, was treated with bisulfite and a region of TNF-alpha gene promoter (from -360 to +50 bp) was sequenced. Obese men with successful weight loss (>or=5% of initial body weight) showed lower levels of total TNF-alpha promoter methylation (r=0.74; P=0.021), especially in the positions -170 bp (r=0.75, P=0.005) and -120 bp (r=0.70, P=0.011). Baseline TNF-alpha circulating levels were positively associated with total promoter methylation (r=0.84, P=0.005) and methylation at position -245 bp (r=0.75, P=0.020). TNF-alpha promoter methylation could be a good inflammation marker predicting the hypocaloric diet-induced weight-loss, and constitutes a first step toward personalized nutrition based on epigenetic criteria.

  5. GPER Promoter Methylation Controls GPER Expression in Breast Cancer Patients.

    PubMed

    Weissenborn, Christine; Ignatov, Tanja; Nass, Norbert; Kalinski, Thomas; Dan Costa, Serban; Zenclussen, Ana Claudia; Ignatov, Atanas

    2017-02-07

    Recently, we found that G-protein-coupled estrogen receptor (GPER) protein expression decreased during breast carcinogenesis, and that GPER promoter is methylated. Here we analyzed GPER promoter methylation in 260 primary breast cancer specimens by methylation-specific polymerized chain reaction. The results demonstrated that GPER protein down-regulation significantly correlated with GPER promoter hypermethylation (p < .001). Comparison of 108 tumors and matched normal breast tissues indicated a significant GPER down-regulation in cancer tissues correlating with GPER promoter hypermethylation (p < .001). The latter was an unfavorable factor for overall survival of patients with triple-negative breast cancer (p = .025). Thus GPER promoter hypermethylation might be used as a prognostic factor.

  6. Meta-analysis of the association between APC promoter methylation and colorectal cancer

    PubMed Central

    Ding, Zhenyu; Jiang, Tong; Piao, Ying; Han, Tao; Han, Yaling; Xie, Xiaodong

    2015-01-01

    Previous studies investigating the association between adenomatous polyposis coli (APC) gene promoter methylation and colorectal cancer (CRC) have yielded conflicting results. The aim of this study was to comprehensively evaluate the potential application of the detection of APC promoter methylation to the prevention and treatment of CRC. PubMed, Embase, and MEDLINE (results updated to October 2014) were searched for relevant studies. The effect size was defined as the weighted odds ratio (OR), which was calculated using either the fixed-effects or random-effects model. Prespecified subgroup and sensitivity analyses were conducted to evaluate potential heterogeneity among the included studies. Nineteen studies comprising 2,426 participants were selected for our meta-analysis. The pooled results of nine studies comprising a total of 740 subjects indicated that APC promoter methylation was significantly associated with CRC risk (pooled OR 5.53; 95% confidence interval [CI] 3.50–8.76; P<0.01). Eleven studies with a total of 1,219 patients evaluated the association between APC promoter methylation and the presence of CRC metastasis, and the pooled OR was 0.80 (95% CI 0.44–1.46; P=0.47). A meta-analysis conducted with four studies with a total of 467 patients found no significant correlation between APC promoter methylation and the presence of colorectal adenoma (pooled OR 1.85; 95% CI 0.67–5.10; P=0.23). No significant correlation between APC promoter methylation and patients’ Dukes’ stage, TNM stage, differentiation grade, age, or sex was identified. In conclusion, APC promoter methylation was found to be significantly associated with a higher risk of developing CRC. The findings indicate that APC promoter methylation may be a potential biomarker for the carcinogenesis of CRC. PMID:25632237

  7. Meta-analysis of the association between APC promoter methylation and colorectal cancer.

    PubMed

    Ding, Zhenyu; Jiang, Tong; Piao, Ying; Han, Tao; Han, Yaling; Xie, Xiaodong

    2015-01-01

    Previous studies investigating the association between adenomatous polyposis coli (APC) gene promoter methylation and colorectal cancer (CRC) have yielded conflicting results. The aim of this study was to comprehensively evaluate the potential application of the detection of APC promoter methylation to the prevention and treatment of CRC. PubMed, Embase, and MEDLINE (results updated to October 2014) were searched for relevant studies. The effect size was defined as the weighted odds ratio (OR), which was calculated using either the fixed-effects or random-effects model. Prespecified subgroup and sensitivity analyses were conducted to evaluate potential heterogeneity among the included studies. Nineteen studies comprising 2,426 participants were selected for our meta-analysis. The pooled results of nine studies comprising a total of 740 subjects indicated that APC promoter methylation was significantly associated with CRC risk (pooled OR 5.53; 95% confidence interval [CI] 3.50-8.76; P<0.01). Eleven studies with a total of 1,219 patients evaluated the association between APC promoter methylation and the presence of CRC metastasis, and the pooled OR was 0.80 (95% CI 0.44-1.46; P=0.47). A meta-analysis conducted with four studies with a total of 467 patients found no significant correlation between APC promoter methylation and the presence of colorectal adenoma (pooled OR 1.85; 95% CI 0.67-5.10; P=0.23). No significant correlation between APC promoter methylation and patients' Dukes' stage, TNM stage, differentiation grade, age, or sex was identified. In conclusion, APC promoter methylation was found to be significantly associated with a higher risk of developing CRC. The findings indicate that APC promoter methylation may be a potential biomarker for the carcinogenesis of CRC.

  8. Increased promoter methylation in exfoliated breast epithelial cells in women with a previous breast biopsy.

    PubMed

    Browne, Eva P; Punska, Elizabeth C; Lenington, Sarah; Otis, Christopher N; Anderton, Douglas L; Arcaro, Kathleen F

    2011-12-01

    Accurately identifying women at increased risk of developing breast cancer will provide greater opportunity for early detection and prevention. DNA promoter methylation is a promising biomarker for assessing breast cancer risk. Breast milk contains large numbers of exfoliated epithelial cells that are ideal for methylation analyses. Exfoliated epithelial cells were isolated from the milk obtained from each breast of 134 women with a history of a non-proliferative benign breast biopsy (Biopsy Group). Promoter methylation of three tumor suppressor genes, RASSF1, SFRP1 and GSTP1, was assessed by pyrosequencing of bisulfite-modified DNA. Methylation scores from the milk of the 134 women in the Biopsy Group were compared to scores from 102 women for whom a breast biopsy was not a recruitment requirement (Reference Group). Mean methylation scores for RASSF1 and GSTP1 were significantly higher in the Biopsy than in the Reference Group. For all three genes the percentage of outlier scores was greater in the Biopsy than in the Reference Group but reached statistical significance only for GSTP1. A comparison between the biopsied and non-biopsied breasts of the Biopsy Group revealed higher mean methylation and a greater number of outlier scores in the biopsied breast for both SFRP1 and RASSF1, but not for GSTP1. This is the first evidence of CpG island methylation in tumor suppressor genes of women who may be at increased risk of developing breast cancer based on having had a prior breast biopsy.

  9. DNA methylation profiling of the fibrinogen gene landscape in human cells and during mouse and zebrafish development.

    PubMed

    Vorjohann, Silja; Pitetti, Jean-Luc; Nef, Serge; Gonelle-Gispert, Carmen; Buhler, Leo; Fish, Richard J; Neerman-Arbez, Marguerite

    2013-01-01

    The fibrinogen genes FGA, FGB and FGG show coordinated expression in hepatocytes. Understanding the underlying transcriptional regulation may elucidate how their tissue-specific expression is maintained and explain the high variability in fibrinogen blood levels. DNA methylation of CpG-poor gene promoters is dynamic with low methylation correlating with tissue-specific gene expression but its direct effect on gene regulation as well as implications of non-promoter CpG methylation are not clear. Here we compared methylation of CpG sites throughout the fibrinogen gene cluster in human cells and mouse and zebrafish tissues. We observed low DNA methylation of the CpG-poor fibrinogen promoters and of additional regulatory elements (the liver enhancers CNC12 and PFE2) in fibrinogen-expressing samples. In a gene reporter assay, CpG-methylation in the FGA promoter reduced promoter activity, suggesting a repressive function for DNA methylation in the fibrinogen locus. In mouse and zebrafish livers we measured reductions in DNA methylation around fibrinogen genes during development that were preceded by increased fibrinogen expression and tri-methylation of Histone3 lysine4 (H3K4me3) in fibrinogen promoters. Our data support a model where changes in hepatic transcription factor expression and histone modification provide the switch for increased fibrinogen gene expression in the developing liver which is followed by reduction of CpG methylation.

  10. PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches.

    PubMed

    Heitzer, Ellen; Bambach, Isabella; Dandachi, Nadia; Horn, Michael; Wolf, Peter

    2010-10-01

    Basal cell carcinoma (BCC) is the most common form of skin cancer. Mutations of the PTCH hallmark gene are detected in about 50-60% of BCCs, which raises the question whether other mechanisms such as promoter methylation can inactivate PTCH. Therefore, we performed methylation analysis of the PTCH promoter in a total of 56 BCCs. The sensitivity of three different methods, including direct bisulphite sequencing PCR, MethyLight and high-resolution melting (HRM), was applied and compared. We found that HRM analysis of DNA from fresh tissue [rather than formalin-fixed and paraffin-embedded tissue (FFPE)] was the most sensitive method to detect methylation. Low-level methylation of the PTCH promoter was detected in five out of 16 analysed BCCs (31%) on DNA from fresh tissue but only in two (13%) samples on short-time stored FFPE DNA from the very same tumors. In contrast, we were unable to detect methylation by HRM on long-time stored DNA in any of the remaining 40 BCC samples. Our data suggest that (i) HRM on DNA extracted from fresh tissue is the most sensitive method to detect methylation and (ii) methylation of the PTCH promoter may only play a minor role in BCC carcinogenesis.

  11. 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.

  12. DAPK Promoter Methylation and Bladder Cancer Risk: A Systematic Review and Meta-Analysis

    PubMed Central

    Zhang, Zhensheng; Zeng, Shuxiong; Liu, Anwei; Tang, Shijie; Ren, Qian; Sun, Yinghao; Xu, Chuanliang

    2016-01-01

    Background Methylation of tumor suppressor gene promoter leads to transcription inactivation and is involved in tumorigenesis. Several studies demonstrate a potential association between the Death-Associated Protein Kinase (DAPK) gene promoter methylation and bladder cancer risk, tumor stage and histological grade. Due to inconsistent results of these studies, we performed this meta-analysis to ascertain the association. Methods Studies were retrieved from the PubMed, Embase, Web of Science and the Cochrane Library databases. Study selection and data extraction were executed by two reviewers independently. Meta-analysis was performed using Stata 13.0 and Review Manager 5.3 software. Results A total of 21 articles involving 15 case control and 8 case series studies were included in this meta-analysis. DAPK promoter methylation was associated with bladder cancer risk (OR: 5.81; 95%CI = 3.83–8.82, P<0.00001). The frequency of DAPK promoter methylation was equal in bladder cancer tissue and paired adjacent normal tissue (OR: 0.87; 95%CI = 0.31–2.48, P = 0.794). Furthermore, DAPK promoter methylation was associated with higher histological grade (OR: 1.52; 95%CI = 1.10–2.09, P = 0.011) but not associated with tumor stage (OR: 1.12; 95%CI = 0.67–1.87, P = 0.668). Conclusions The result suggests that DAPK promoter methylation is significantly increased in bladder cancer patients compared to normal controls. DAPK promoter methylation could serve as a biomarker for bladder cancer detection and management. PMID:27907054

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

    PubMed

    Zhang, Li; Long, Xinghua

    2015-12-08

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

  14. Role of CDH1 promoter polymorphism and DNA methylation in bladder carcinogenesis: a meta-analysis.

    PubMed

    Wang, Yi; Kong, Chui-Ze; Zhang, Zhe; Yang, Chun-Ming; Li, Jun

    2014-04-01

    Increasing scientific evidences suggest that CDH1 gene promoter polymorphism and DNA methylation may contribute to the development and progression of bladder cancer, but many existing studies have yielded inconclusive results. This meta-analysis aims to assess the role of CDH1 gene promoter polymorphism and methylation in bladder carcinogenesis. An extensive literature search for relevant studies was conducted in PubMed, Embase, Web of Science, Cochrane Library, and CBM databases from their inception through April 1, 2013. This meta-analysis was performed using the STATA 12.0 software. The crude odds ratio with 95% confidence interval was calculated. Fifteen studies were included in this meta-analysis with a total of 824 bladder cancer patients and 818 healthy controls being assessed. Our meta-analysis revealed that the A variant of CDH1 -160C/A polymorphism was associated with an increased risk of bladder cancer. Further analysis by pathological subtype indicated that patients with invasive carcinoma had a higher frequency of CDH1 -160A variant than those with superficial carcinoma. We analyzed the methylation frequency of CDH1 gene in 608 bladder cancer samples and 338 normal bladder samples. Our data strongly suggest that the CDH1 promoter methylation frequencies in bladder cancer tissues were greater than those in normal control tissues. In conclusion, our meta-analysis indicates that promoter polymorphism and methylation of CDH1 gene may be involved in the development and progression of bladder cancer. CDH1 gene promoter polymorphism and methylation might be promising biomarkers for the diagnosis and prognosis of bladder cancer.

  15. Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

    PubMed Central

    Shah, Nameeta; Lin, Biaoyang; Sibenaller, Zita; Ryken, Timothy; Lee, Hwahyung; Yoon, Jae-Geun; Rostad, Steven; Foltz, Greg

    2011-01-01

    O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089–13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301–7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting. PMID

  16. 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.

  17. Outcome in unresectable glioblastoma: MGMT promoter methylation makes the difference.

    PubMed

    Thon, Niklas; Thorsteinsdottir, Jun; Eigenbrod, Sabina; Schüller, Ulrich; Lutz, Jürgen; Kreth, Simone; Belka, Claus; Tonn, Jörg-Christian; Niyazi, Maximilian; Kreth, Friedrich Wilhelm

    2017-02-01

    In 2011, we reported a predominant prognostic/predictive role of MGMT promoter methylation status on progression-free survival (PFS) in unresectable glioblastoma patients undergoing upfront radiotherapy plus concomitant and maintenance temozolomide (RTX/TMZ → TMZ). We, here, present the final results of this prospective study focussing on the prognostic/predictive value of MGMT promoter methylation status for death risk stratification. Overall, 56 adult patients with unresectable, biopsy proven glioblastoma were prospectively assigned to upfront RTX/TMZ → TMZ treatment between March 2006 and August 2008. Last follow-up was performed in June 2016. MGMT promoter methylation was determined using methylation-specific PCR (MSP) and sodium bisulfite sequencing. Analyses were done by intention to treat. Prognostic factors were obtained from proportional hazard models. At the time of the final analysis 55 patients showed progressive disease and 53 patients had died. MGMT promoter was methylated (unmethylated) in 30 (26) patients. Methylation of the MGMT promoter was the strongest favorable predictor for overall survival (OS, median: 20.3 vs. 7.3 months, p < 0.001, HR 0.30, 95% CI 0.16-0.55), and PFS (median: 15.0 vs. 6.1 months, p < 0.001, HR 0.31, 95% CI 0.17-0.57) and was also associated with higher frequencies of treatment response and prolonged post-recurrence survival (PRS, median: 4.5 vs. 1.4 months, p < 0.002, HR 0.39, 95% CI 0.21-0.71). Knowledge of MGMT promoter methylation status is essential for patients' counseling, prognostic evaluation, and for the design of future trials dealing with unresectable glioblastomas.

  18. Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize

    PubMed Central

    Li, Xinxin; Miclaus, Mihai; Messing, Joachim

    2016-01-01

    An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences. PMID:26741504

  19. Gene structure, expression, and DNA methylation characteristics of sea cucumber cyclin B gene during aestivation.

    PubMed

    Zhu, Aijun; Chen, Muyan; Zhang, Xiumei; Storey, Kenneth B

    2016-12-05

    The sea cucumber, Apostichopus japonicus, is a good model for studying environmentally-induced aestivation by a marine invertebrate. One of the central requirements of aestivation is the repression of energy-expensive cellular processes such as cell cycle progression. The present study identified the gene structure of the cell cycle regulator, cyclin B, and detected the expression levels of this gene over three stages of the annual aestivation-arousal cycle. Furthermore, the DNA methylation characteristics of cyclin B were analyzed in non-aestivation and deep-aestivation stages of sea cucumbers. We found that the cyclin B promoter contains a CpG island, three CCAAT-boxes and three cell cycle gene homology regions (CHRs). Application of qRT-PCR analysis showed significant downregulation of cyclin B transcript levels during deep-aestivation in comparison with non-aestivation in both intestine and longitudinal muscle, and these returned to basal levels after arousal from aestivation. Methylation analysis of the cyclin B core promoter revealed that its methylation level showed significant differences between non-aestivation and deep-aestivation stages (p<0.05) and interestingly, a positive correlation between Cyclin B transcripts expression and methylation levels of the core promoter was also observed. Our findings suggest that cell cycle progression may be reversibly arrested during aestivation as indicated by the changes in cyclin B expression levels and we propose that DNA methylation is one of the regulatory mechanisms involved in cyclin B transcriptional variation.

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

    PubMed Central

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

    2016-01-01

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

  1. TP53 Promoter Methylation in Primary Glioblastoma: Relationship with TP53 mRNA and Protein Expression and Mutation Status

    PubMed Central

    Szybka, Malgorzata; Malachowska, Beata; Fendler, Wojciech; Potemski, Piotr; Piaskowski, Sylwester; Jaskolski, Dariusz; Papierz, Wielislaw; Skowronski, Wieslaw; Och, Waldemar; Kordek, Radzislaw

    2014-01-01

    Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway. PMID:24506545

  2. 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.

  3. Quantitative analysis of promoter methylation in exfoliated epithelial cells isolated from breast milk of healthy women.

    PubMed

    Wong, Chung M; Anderton, Douglas L; Smith-Schneider, Sallie; Wing, Megan A; Greven, Melissa C; Arcaro, Kathleen F

    2010-10-01

    Promoter methylation analysis of genes frequently silenced in breast cancer is a promising indicator of breast cancer risk, as these methylation events are thought to occur long before presentation of disease. The numerous exfoliated epithelial cells present in breast milk may provide the breast epithelial DNA needed for detailed methylation analysis and assessment of breast cancer risk. Fresh breast milk samples and health, lifestyle, and reproductive history questionnaires were collected from 111 women. Pyrosequencing analysis was conducted on DNA isolated from the exfoliated epithelial cells immunomagnetically separated from the total cell population in the breast milk of 102 women. A total of 65 CpG sites were examined in six tumor suppressor genes: PYCARD (also known as ASC or TMS1), CDH1, GSTP1, RBP1 (also known as CRBP1), SFRP1, and RASSF1. A sufficient quantity of DNA was obtained for meaningful analysis of promoter methylation; women donated an average of 86 ml of milk with a mean yield of 32,700 epithelial cells per ml. Methylation scores were in general low as expected of benign tissue, but analysis of outlier methylation scores revealed a significant relationship between breast cancer risk, as indicated by previous biopsy, and methylation score for several CpG sites in CDH1, GSTP1, SFRP1, and RBP1. Methylation of RASSF1 was positively correlated with women's age irrespective of her reproductive history. Promoter methylation patterns in DNA from breast milk epithelial cells can likely be used to assess breast cancer risk. Additional studies of women at high breast cancer risk are warranted.

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

    PubMed

    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.

  7. 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

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

    PubMed

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

    2014-01-01

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

  9. Promoter histone H3 lysine 9 di-methylation is associated with DNA methylation and aberrant expression of p16 in gastric cancer cells.

    PubMed

    Meng, Chun-Feng; Zhu, Xin-Jiang; Peng, Guo; Dai, Dong-Qiu

    2009-11-01

    In the course of gastric cancer development, gene silencing by DNA hypermethylation is an important mechanism. While DNA methylation often co-exists with histone modifications to regulate gene expression, the function of histone modifications in gene silencing in gastric cancer has not been evaluated in detail. p16, a well-known tumor suppressor gene, is frequently silenced in DNA hypermethylation manner in gastric cancer. Accordingly, we chose p16 to clarify whether there is a correlation among histone H3 lysine 9 (H3-K9) di-methylation, H3-K9 acetylation, DNA methylation and p16 expression in human gastric cancer. Three gastric cancer cells, MKN-45, SGC-7901 and BGC-823, were treated with 5-aza-2'-deoxycytidine (5-Aza-dC) and/or trichostatin A (TSA). We investigated p16 promoter DNA methylation status, p16 mRNA levels, regional and global levels of di-methyl-H3-K9 and acetyl-H3-K9 in four groups: i) 5-Aza-dC, ii) TSA, iii) the combination of 5-Aza-dC and TSA and iv) control group with no treatments. p16 silencing is characterized by DNA hypermethylation, H3-K9 hypoacetylation and H3-K9 hypermethylation at the promoter region. Treatment with TSA, increased H3-K9 acetylation at the hypermethylated promoter, but did not affect H3-K9 di-methylation or p16 expression. By contrast, treatment with 5-Aza-dC, reduced H3-K9 di-methylation, increased H3-K9 acetylation at the hypermethylated promoter and reactivated the expression of p16. Combined treatment restored the expression of p16 synergistically. In addition, 5-Aza-dC and the combined treatment did not result in global alteration of H3-K9 di-methylation. These results suggest that H3-K9 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence p16 in gastric cancer. The decreased H3-K9 di-methylation correlates with DNA demethylation and reactivation of p16. H3-K9 di-methylation as well as DNA methylation related to p16 silencing is limited to the promoter region. In addition to its effect

  10. APC gene methylation is inversely correlated with features of the CpG island methylator phenotype in colorectal cancer.

    PubMed

    Iacopetta, Barry; Grieu, Fabienne; Li, Wei; Ruszkiewicz, Andrew; Caruso, Maria; Moore, James; Watanabe, Goh; Kawakami, Kazuyuki

    2006-11-15

    The notion of a CpG island methylator phenotype (CIMP) was proposed to describe a subset of colorectal cancers (CRC) displaying frequent and concordant methylation of CpG islands located within gene promoter regions. Some workers have failed to observe associations between CIMP and specific clinicopathological features of CRC, possibly because of the choice of genes used to define this phenotype. The aim of the current study was to determine whether the aberrant methylation of 6 genes implicated in CRC development was associated with the same phenotypic features of this tumour type. The MethyLight assay was used to provide quantitative estimates of MLH1, P16, TIMP3, P14, DAPK and APC methylation levels in 199 unselected colorectal tumours. The methylation of MLH1, P16, TIMP3 and P14 was highly concordant (p < 0.0001 for each pair) but that of DAPK and APC was not. An inverse association was observed between the methylation of APC and TIMP3 (p = 0.004). Methylation of the MLH1, P16, TIMP3 and P14 genes was associated with tumour infiltrating lymphocytes (p < 0.05), microsatellite instability (p < 0.001), BRAF mutation (p < 0.0001) and elevated concentrations of the methyl group carriers tetrahydrofolate (THF) and 5,10-methylene THF (p < 0.05). In contrast, APC methylation was associated with wildtype BRAF (p = 0.003) and with lower concentrations of methyl group carriers (p < 0.05). These findings highlight the importance of gene selection in studies that aim to characterize the biological features and clinical behaviour of CIMP+ tumours.

  11. 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.

  12. Pre-B cell to macrophage transdifferentiation without significant promoter DNA methylation changes.

    PubMed

    Rodríguez-Ubreva, Javier; Ciudad, Laura; Gómez-Cabrero, David; Parra, Maribel; Bussmann, Lars H; di Tullio, Alessandro; Kallin, Eric M; Tegnér, Jesper; Graf, Thomas; Ballestar, Esteban

    2012-03-01

    Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBPα-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.

  13. 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

  14. SHISA3 Promoter Methylation Is a Potential Diagnostic and Prognostic Biomarker for Laryngeal Squamous Cell Carcinoma

    PubMed Central

    Zhou, Chongchang; Li, Jinyun; Ye, Dong; Deng, Hongxia; Cao, Bin; Hao, Wenjuan; Lin, Lexi

    2017-01-01

    The purpose of this study was to evaluate the contribution of SHISA3 promoter methylation to laryngeal squamous cell carcinoma (LSCC). SHISA3 promoter methylation status and expression were determined using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (qRT-PCR) in 93 paired LSCC and adjacent normal tissues, respectively. Furthermore, the regulatory function of the SHISA3 promoter fragment was analyzed using a luciferase reporter assay. The results reveal that there is a significant increase in SHISA3 methylation in LSCC tissues compared with corresponding nontumor tissues (P = 4.58E − 12). The qRT-PCR results show a significant association between SHISA3 methylation and expression in LSCC (P = 1.67E − 03). In addition, the area under the receiver operating characteristic curve was 0.91. Consequently, a log-rank test and multivariate Cox analysis suggest that SHISA3 promoter hypermethylation is a predictor of poor overall survival for LSCC (log-rank P = 0.024; HR = 2.71; 95% CI = 1.024–7.177; P = 0.047). The results indicate that SHISA3 promoter hypermethylation might increase the risk of LSCC through regulation of gene expression and is a potential diagnostic and prognostic biomarker for LSCC. PMID:28299336

  15. High fat diet-induced obesity modifies the methylation pattern of leptin promoter in rats.

    PubMed

    Milagro, F I; Campión, J; García-Díaz, D F; Goyenechea, E; Paternain, L; Martínez, J A

    2009-03-01

    Leptin is an adipokine involved in body weight and food intake regulation whose promoter region presents CpG islands that could be subject to dynamic methylation. This methylation process could be affected by environmental (e.g. diet) or endogenous (e.g., adipocyte differentiation, inflammation, hypoxia) factors, and could influence adipocyte leptin gene expression. The aim of this article was to study whether a high-energy diet may affect leptin gene promoter methylation in rats. A group of eleven male Wistar rats were assigned into two dietary groups, one fed on a control diet for 11 weeks and the other on a high-fat cafeteria diet. Rats fed a high-energy diet become overweight and hyperleptinemic as compared to the controls. DNA isolated from retroperitoneal adipocytes was treated with bisulfite and a distal portion of leptin promoter (from -694 to -372 bp) including 13 CpG sites was amplified by PCR and sequenced. The studied promoter portion was slightly more methylated in the cafeteria-fed animals, which was statistically significant (p < 0.05) for one of the CpG sites (located at the position -443). In obese rats, such methylation was associated to lower circulating leptin levels, suggesting that this position could be important in the regulation of leptin gene expression, probably by being a target sequence of different transcription factors. Our findings reveal, for the first time, that leptin methylation pattern can be influenced by diet-induced obesity, and suggest that epigenetic mechanisms could be involved in obesity by regulating the expression of important epiobesigenic genes.

  16. Quantitative assessment of the diagnostic role of FHIT promoter methylation in non-small cell lung cancer

    PubMed Central

    Tan, Yulong; Lu, Zhouyi; Wang, An; Tan, Lixing; Chen, Sidi; Guo, Shicheng; Wang, Jiucun; Chen, Xiaofeng

    2017-01-01

    Aberrant methylation of CpG islands acquired in promoter regions plays an important role in carcinogenesis. Accumulated evidence demonstrates FHIT gene promoter hyper-methylation is involved in non-small cell lung cancer (NSCLC). To test the diagnostic ability of FHIT methylation status on NSCLC, thirteen studies, including 2,119 samples were included in our meta-analysis. Simultaneously, four independent DNA methylation datasets from TCGA and GEO database were analyzed for validation. The pooled odds ratio of FHIT promoter methylation in cancer samples was 3.43 (95% CI: 1.85 to 6.36) compared with that in controls. In subgroup analysis, significant difference of FHIT gene promoter methylation status in NSCLC and controls was found in Asians but not in Caucasian population. In validation stage, 950 Caucasian samples, including 126 paired samples from TCGA, 568 cancer tissues and 256 normal controls from GEO database were analyzed, and all 8 CpG sites near the promoter region of FHIT gene were not significantly differentially methylated. Thus the diagnostic role of FHIT gene in the lung cancer may be relatively limited in the Caucasian population but useful in the Asians. PMID:28036263

  17. CXCL12 methylation-mediated epigenetic regulation of gene expression in papillary thyroid carcinoma.

    PubMed

    Zhang, Sijia; Wang, Yihan; Chen, Meijun; Sun, Lulu; Han, Jun; Elena, V Kazakova; Qiao, Hong

    2017-03-08

    Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer, and its incidence rate is rapidly growing. It is necessary to understand the pathogenesis of PTC to develop effective diagnosis methods. Promoter methylation has been recognized to contribute to the alterations in gene expression observed in tumorigenesis. Our RNA-seq data identified 1191 differentially expressed mRNAs and 147 differentially expressed lncRNAs in PTC. Next, promoter methylation of these genes was detected by reduced representation bisulfite sequencing (RRBS) technology and comprehensively analyzed to identify differential methylation. In total, 14 genes (13 mRNAs and 1 lncRNA), in which methylation was intimately involved in regulating gene expression, were proposed as novel diagnostic biomarkers. To gain insights into the relationships among these 14 genes, a core co-function network was constructed based on co-expression, co-function and co-methylation data. Notably, CXCL12 was identified as an essential gene in the network that was closely connected with the other genes. These data suggested that CXCL12 down-regulation in PTC may be caused by promoter hypermethylation. Our study was the first to perform an RRBS analysis for PTC and suggested that CXCL12 may contribute to PTC development by methylation-mediated epigenetic regulation of gene expression.

  18. CXCL12 methylation-mediated epigenetic regulation of gene expression in papillary thyroid carcinoma

    PubMed Central

    Zhang, Sijia; Wang, Yihan; Chen, Meijun; Sun, Lulu; Han, Jun; Elena, V. Kazakova; Qiao, Hong

    2017-01-01

    Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer, and its incidence rate is rapidly growing. It is necessary to understand the pathogenesis of PTC to develop effective diagnosis methods. Promoter methylation has been recognized to contribute to the alterations in gene expression observed in tumorigenesis. Our RNA-seq data identified 1191 differentially expressed mRNAs and 147 differentially expressed lncRNAs in PTC. Next, promoter methylation of these genes was detected by reduced representation bisulfite sequencing (RRBS) technology and comprehensively analyzed to identify differential methylation. In total, 14 genes (13 mRNAs and 1 lncRNA), in which methylation was intimately involved in regulating gene expression, were proposed as novel diagnostic biomarkers. To gain insights into the relationships among these 14 genes, a core co-function network was constructed based on co-expression, co-function and co-methylation data. Notably, CXCL12 was identified as an essential gene in the network that was closely connected with the other genes. These data suggested that CXCL12 down-regulation in PTC may be caused by promoter hypermethylation. Our study was the first to perform an RRBS analysis for PTC and suggested that CXCL12 may contribute to PTC development by methylation-mediated epigenetic regulation of gene expression. PMID:28272462

  19. Aging related methylation influences the gene expression of key control genes in colorectal cancer and adenoma

    PubMed Central

    Galamb, Orsolya; Kalmár, Alexandra; Barták, Barbara Kinga; Patai, Árpád V; Leiszter, Katalin; Péterfia, Bálint; Wichmann, Barnabás; Valcz, Gábor; Veres, Gábor; Tulassay, Zsolt; Molnár, Béla

    2016-01-01

    AIM To analyze colorectal carcinogenesis and age-related DNA methylation alterations of gene sequences associated with epigenetic clock CpG sites. METHODS In silico DNA methylation analysis of 353 epigenetic clock CpG sites published by Steve Horvath was performed using methylation array data for a set of 123 colonic tissue samples [64 colorectal cancer (CRC), 42 adenoma, 17 normal; GEO accession number: GSE48684]. Among the differentially methylated age-related genes, secreted frizzled related protein 1 (SFRP1) promoter methylation was further investigated in colonic tissue from 8 healthy adults, 19 normal children, 20 adenoma and 8 CRC patients using bisulfite-specific PCR followed by methylation-specific high resolution melting (MS-HRM) analysis. mRNA expression of age-related “epigenetic clock” genes was studied using Affymetrix HGU133 Plus2.0 whole transcriptome data of 153 colonic biopsy samples (49 healthy adult, 49 adenoma, 49 CRC, 6 healthy children) (GEO accession numbers: GSE37364, GSE10714, GSE4183, GSE37267). Whole promoter methylation analysis of genes showing inverse DNA methylation-gene expression data was performed on 30 colonic samples using methyl capture sequencing. RESULTS Fifty-seven age-related CpG sites including hypermethylated PPP1R16B, SFRP1, SYNE1 and hypomethylated MGP, PIPOX were differentially methylated between CRC and normal tissues (P < 0.05, Δβ ≥ 10%). In the adenoma vs normal comparison, 70 CpG sites differed significantly, including hypermethylated DKK3, SDC2, SFRP1, SYNE1 and hypomethylated CEMIP, SPATA18 (P < 0.05, Δβ ≥ 10%). In MS-HRM analysis, the SFRP1 promoter region was significantly hypermethylated in CRC (55.0% ± 8.4 %) and adenoma tissue samples (49.9% ± 18.1%) compared to normal adult (5.2% ± 2.7%) and young (2.2% ± 0.7%) colonic tissue (P < 0.0001). DNA methylation of SFRP1 promoter was slightly, but significantly increased in healthy adults compared to normal young samples (P < 0.02). This correlated

  20. Methylation-Sensitive Melt Curve Analysis of the Reprimo Gene Methylation in Gastric Cancer

    PubMed Central

    Lai, Junzhong; Luo, Qianping; Ke, Huican; Chen, Qi

    2016-01-01

    Reprimo (RPRM) is a p53-induced tumor suppressor gene. Its aberrant DNA methylation is correlated with carcinogenesis and may be used as a surrogate marker for the early detection of gastric cancer. However, the detail information regarding its DNA methylation has not been revealed. Here, we investigated the RPRM gene methylation in gastric cancer tumor and plasma samples by methylation-sensitive melt curve analysis (MS-MCA) and bisulfite sequencing in depth. We developed a semi-quantitative method based on MS-MCA for detecting DNA methylation and unraveled the RPRM gene methylation pattern in gastric cancer. This study provides a solid foundation for the future application of detecting RPRM gene methylation in human plasma or serum samples to help diagnose gastric cancer or for prognosis evaluation. PMID:27992600

  1. 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.

  2. 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

  3. Human hedgehog interacting protein expression and promoter methylation in medulloblastoma cell lines and primary tumor samples

    PubMed Central

    Shahi, Mehdi H.; Afzal, Mohammad; Sinha, Subrata; Eberhart, Charles G.; Rey, Juan A.; Fan, Xing

    2015-01-01

    Medulloblastoma is the most common pediatric brain tumor and its development is affected by genetic and epigenetic factors. In this study we found there is low or no expression of the hedgehog interacting protein (HHIP), a negative regulator of the sonic hedgehog pathway, in most medulloblastoma cell lines and primary samples explored. We proceeded to promoter methylation assays of this gene by MCA-Meth, and found that HHIP was hypermethylated in all medulloblastoma cell lines, but only in 2 out of 14 (14%) primary tumor samples. Methylation correlated with low or unexpressed HHIP in cell lines but not in primary tumor samples. These results suggest the possibility of epigenetic regulation of HHIP in medulloblastoma, similarly to gastric, hepatic and pancreatic cancer. However, HHIP seems to be not only under regulation of promoter methylation, but under other factors involved in the control of its low levels of expression in medulloblastoma. PMID:20853133

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

    PubMed Central

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

    2007-01-01

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

  5. Associations Between Early Life Stress and Gene Methylation in Children

    PubMed Central

    Romens, Sarah E; McDonald, Jennifer; Svaren, John; Pollak, Seth D

    2015-01-01

    Children exposed to extreme stress are at heightened risk for developing mental and physical disorders. However, little is known about mechanisms underlying these associations in humans. An emerging insight is that children's social environments change gene expression, which contributes to biological vulnerabilities for behavioral problems. Epigenetic changes in the glucocorticoid receptor gene, a critical component of stress regulation, were examined in whole blood from 56 children aged 11–14 years. Children exposed to physical maltreatment had greater methylation within exon 1F in the NR3C1 promoter region of the gene compared to nonmaltreated children, including the putative NGFI-A (nerve growth factor) binding site. These results highlight molecular mechanisms linking childhood stress with biological changes that may lead to mental and physical disorders. PMID:25056599

  6. Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression

    PubMed Central

    Rivier-Cordey, Anne-Sophie; Caetano, Carlos; Fish, Richard J.; Kruithof, Egbert K. O.

    2016-01-01

    Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter. PMID:27973546

  7. Methylation of the BRCA1 promoter in peripheral blood DNA is associated with triple-negative and medullary breast cancer.

    PubMed

    Gupta, Satish; Jaworska-Bieniek, Katarzyna; Narod, Steven A; Lubinski, Jan; Wojdacz, Tomasz K; Jakubowska, Anna

    2014-12-01

    It has been proposed that methylation signatures in blood-derived DNA may correlate with cancer risk. In this study, we evaluated whether methylation of the promoter region of the BRCA1 gene detectable in DNA from peripheral blood cells is a risk factor for breast cancer, in particular for tumors with pathologic features characteristic for cancers with BRCA1 gene mutations. We conducted a case-control study of 66 breast cancer cases and 36 unaffected controls. Cases were triple-negative or of medullary histology, or both; 30 carried a constitutional BRCA1 mutation and 36 did not carry a mutation. Blood for DNA methylation analysis was taken within three months of diagnosis. Methylation of the promoter of the BRCA1 gene was measured in cases and controls using methylation-sensitive high-resolution melting (MS-HRM). A sample with any detectable level of methylation was considered to be positive. Methylation of the BRCA1 promoter was detected in 15 of 66 cases and in 2 of 36 controls (OR 5.0, p = 0.03). Methylation was present in 15 of 36 women with breast cancer and without germline BRCA1 mutation, but in none of 30 women with breast cancer and a germline mutation (p < 0.01). The association between methylation and breast cancer was restricted to women with no constitutional BRCA1 mutation (OR 12.1, p = 0.0006). Methylation of the promoter of the BRCA1 gene detectable in peripheral blood DNA may be a marker of increased susceptibility to triple-negative or medullary breast cancer.

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

    PubMed

    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

    2014-10-21

    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

  9. Elevated OPRD1 promoter methylation in Alzheimer’s disease patients

    PubMed Central

    Chen, Zhongming; Zhou, Dongsheng; Xu, Xuting; Cui, Wei; Hong, Qingxiao; Jiang, Liting; Li, Jinfeng; Zhou, Xiaohui; Li, Ying; Guo, Zhiping; Zha, Qin; Niu, Yanfang; Weng, Qiuyan; Duan, Shiwei; Wang, Qinwen

    2017-01-01

    Aberrant DNA methylation has been observed in the patients with Alzheimer’s disease (AD), a common neurodegenerative disorder in the elderly. OPRD1 encodes the delta opioid receptor, a member of the opioid family of G-protein-coupled receptors. In the current study, we compare the DNA methylation levels of OPRD1 promoter CpG sites (CpG1, CpG2, and CpG3) between 51 AD cases and 63 controls using the bisulfite pyrosequencing technology. Our results show that significantly higher CpG3 methylation is found in AD cases than controls. Significant associations are found between several biochemical parameters (including HDL-C and ALP) and CpG3 methylation. Subsequent luciferase reporter gene assay shows that DNA fragment containing the three OPRD1 promoter CpGs is able to regulate gene expression. In summary, our results suggest that OPRD1 promoter hypermethylation is associated with the risk of AD. PMID:28253273

  10. DNA Methylation Analysis of BRD1 Promoter Regions and the Schizophrenia rs138880 Risk Allele

    PubMed Central

    Dyrvig, Mads; Qvist, Per; Lichota, Jacek; Larsen, Knud; Nyegaard, Mette; Børglum, Anders D.

    2017-01-01

    The bromodomain containing 1 gene, BRD1 is essential for embryogenesis and CNS development. It encodes a protein that participates in histone modifying complexes and thereby regulates the expression of a large number of genes. Genetic variants in the BRD1 locus show association with schizophrenia and bipolar disorder and risk alleles in the promoter region correlate with reduced BRD1 expression. Insights into the transcriptional regulation of BRD1 and the pathogenic mechanisms associated with BRD1 risk variants, however, remain sparse. By studying transcripts in human HeLa and SH-SY5Y cells we provide evidence for differences in relative expression of BRD1 transcripts with three alternative 5’ UTRs (exon 1C, 1B, and 1A). We further show that expression of these transcript variants covaries negatively with DNA methylation proportions in their upstream promoter regions suggesting that promoter usage might be regulated by DNA methylation. In line with findings that the risk allele of the rs138880 SNP in the BRD1 promoter region correlates with reduced BRD1 expression, we find that it is also associated with moderate regional BRD1 promoter hypermethylation in both adipose tissue and blood. Importantly, we demonstrate by inspecting available DNA methylation and expression data that these regions undergo changes in methylation during fetal brain development and that differences in their methylation proportions in fetal compared to postnatal frontal cortex correlate significantly with BRD1 expression. These findings suggest that BRD1 may be dysregulated in both the developing and mature brain of risk allele carriers. Finally, we demonstrate that commonly used mood stabilizers Lithium, Valproate, and Carbamazepine affect the expression of BRD1 in SH-SY5Y cells. Altogether this study indicates a link between genetic risk and epigenetic dysregulation of BRD1 which raises interesting perspectives for targeting the mechanisms pharmacologically. PMID:28095495

  11. DNA promoter and histone H3 methylation downregulate NGX6 in gastric cancer cells.

    PubMed

    Liu, Jian; Zhu, Xinjiang; Xu, Xiaoyang; Dai, Dongqiu

    2014-01-01

    Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a novel candidate tumor metastasis suppressor gene. Our study was to determine whether DNA hypermethylation and histone modification at the NGX6 gene promoter play important roles in silencing NGX6 expression in gastric cancer. NGX6 expression was downregulated in all gastric cancer cells and 76.19 % tissues. In three GC cell lines, hypermethylated NGX6 loci were characterized by histone H3-K9 hypoacetylation and hypermethylation. Trichostatin A treatment could moderately increase H3-K9 acetylation at the silenced loci; however, it had no effect on DNA and H3-K9 methylation and minimal effects on NGX6 expression. In contrast, 5'aza-2'-deoxycytidine treatment could rapidly decrease DNA and H3-K9 methylation at the silenced loci, leading to the reexpression of NGX6. Combined treatment with 5'aza-2'-deoxycytidine and trichostatin A had synergistic effects on the reexpression of NGX6 at the hypermethylation loci. Our current study shows that NGX6 expression is downregulated in GC cancer cells and tissues due to NGX6 promoter methylation and H3-K9 methylation, but not H3-K9 acetylation. Our findings indicate that the downregulation of NGX6 expression contributes to the development and progression of gastric cancer. More studies are needed to determine the precise mechanism of NGX6 in the progression of gastric cancer.

  12. The Correlation of MGMT Promoter Methylation and Clinicopathological Features in Gastric Cancer: A Systematic Review and Meta-Analysis.

    PubMed

    Ding, Yong; Yang, Qihua; Wang, Bojun; Ye, Guoliang; Tong, Xiaoqiong

    2016-01-01

    The silencing of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) by promoter methylation commonly occurs in human cancers. The relationship between MGMT promoter methylation and gastric cancer (GC) remains inconsistent. This study aimed to evaluate the potential value of MGMT promoter methylation in GC patients. Electronic databases were searched to identify eligible studies. The pooled odds ratio (OR) and the corresponding 95% confidence interval (95% CI) were used to evaluate the effects of MGMT methylation on GC risk and clinicopathological characteristics. In total, 31 eligible studies including 2988 GC patients and 2189 nonmalignant controls were involved in meta-analysis. In the pooled analysis, MGMT promoter methylation was significantly associated with GC risk (OR = 3.34, P < 0.001) and substantial heterogeneity (P < 0.001). Meta-regression and subgroup analyses based on the testing method, sample material and ethnicity failed to explain the sources of heterogeneity. Interestingly, MGMT methylation showed a trend associated with gender, and methylation is lower in males compared with females (OR = 0.76, 95% CI = 0.56-1.03). We did not find a significant association in relation to tumor types, clinical stage, age status or H. pylori status in cancer (all P > 0.1). MGMT promoter methylation may be correlated with the prognosis of GCs in disease free survival (DFS) or overall survival (OS) for univariate analysis. MGMT promoter methylation may play a crucial role in the carcinogenesis and prognosis of GC. MGMT methylation was not correlated with tumor types, clinical stage, age status, H. pylori status. However, the result of the association of MGMT methylation and gender should be considered with caution.

  13. The Correlation of MGMT Promoter Methylation and Clinicopathological Features in Gastric Cancer: A Systematic Review and Meta-Analysis

    PubMed Central

    Ding, Yong; Yang, Qihua; Wang, Bojun; Ye, Guoliang; Tong, Xiaoqiong

    2016-01-01

    The silencing of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) by promoter methylation commonly occurs in human cancers. The relationship between MGMT promoter methylation and gastric cancer (GC) remains inconsistent. This study aimed to evaluate the potential value of MGMT promoter methylation in GC patients. Electronic databases were searched to identify eligible studies. The pooled odds ratio (OR) and the corresponding 95% confidence interval (95% CI) were used to evaluate the effects of MGMT methylation on GC risk and clinicopathological characteristics. In total, 31 eligible studies including 2988 GC patients and 2189 nonmalignant controls were involved in meta-analysis. In the pooled analysis, MGMT promoter methylation was significantly associated with GC risk (OR = 3.34, P < 0.001) and substantial heterogeneity (P < 0.001). Meta-regression and subgroup analyses based on the testing method, sample material and ethnicity failed to explain the sources of heterogeneity. Interestingly, MGMT methylation showed a trend associated with gender, and methylation is lower in males compared with females (OR = 0.76, 95% CI = 0.56–1.03). We did not find a significant association in relation to tumor types, clinical stage, age status or H. pylori status in cancer (all P > 0.1). MGMT promoter methylation may be correlated with the prognosis of GCs in disease free survival (DFS) or overall survival (OS) for univariate analysis. MGMT promoter methylation may play a crucial role in the carcinogenesis and prognosis of GC. MGMT methylation was not correlated with tumor types, clinical stage, age status, H. pylori status. However, the result of the association of MGMT methylation and gender should be considered with caution. PMID:27824946

  14. 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.

  15. BRCA1 promoter methylation is a marker of better response to anthracycline-based therapy in sporadic TNBC.

    PubMed

    Ignatov, T; Poehlmann, A; Ignatov, A; Schinlauer, A; Costa, S D; Roessner, A; Kalinski, T; Bischoff, J

    2013-09-01

    The aim of the current study was to investigate the role of BRCA1 gene aberrations in sporadic triple-negative breast cancer (TNBC) and its impact on anthracycline-based therapy. BRCA1 promoter methylation was analyzed in 70 TNBC and compared with the clinical and pathologic characteristics. As a control group, we used 70 patients with non-TNBC. BRCA1 promoter methylation was observed in 65.2 % of patients and was similar in both groups. BRCA1 promoter methylation was associated with decreased intensity of BRCA1 protein expression (P = 0.002) and significant increase of median disease-free survival (DFS) of TNBC patients receiving adjuvant anthracycline-based chemotherapy (P = 0.001). Multivariate analysis revealed that BRCA1 promoter methylation remains a favorable factor in regard to DFS (HR 0.224; 95 % CI 0.092-0.546, P = 0.001) in TNBC after adjustment for other prognostic factors. In contrast, in non-TNBC, BRCA1 promoter methylation was not associated with any clinical and pathologic parameters. BRCA1 promoter methylation is a common mechanism of BRCA1 gene aberration in sporadic breast cancer and is predictive for better response to anthracycline-based therapies.

  16. DNA Methylation Profiling Reveals Correlation of Differential Methylation Patterns with Gene Expression in Human Epilepsy.

    PubMed

    Wang, Liang; Fu, Xinwei; Peng, Xi; Xiao, Zheng; Li, Zhonggui; Chen, Guojun; Wang, Xuefeng

    2016-05-01

    DNA methylation plays important roles in regulating gene expression and has been reported to be related with epilepsy. This study aimed to define differential DNA methylation patterns in drug-refractory epilepsy patients and to investigate the role of DNA methylation in human epilepsy. We performed DNA methylation profiling in brain tissues from epileptic and control patients via methylated-cytosine DNA immunoprecipitation microarray chip. Differentially methylated loci were validated by bisulfite sequencing PCR, and the messenger RNA (mRNA) levels of candidate genes were evaluated by reverse transcriptase PCR. We found 224 genes that showed differential DNA methylation between epileptic patients and controls. Among the seven candidate genes, three genes (TUBB2B, ATPGD1, and HTR6) showed relative transcriptional regulation by DNA methylation. TUBB2B and ATPGD1 exhibited hypermethylation and decreased mRNA levels, whereas HTR6 displayed hypomethylation and increased mRNA levels in the epileptic samples. Our findings suggest that certain genes become differentially regulated by DNA methylation in human epilepsy.

  17. DNA methylation profile of genes involved in inflammation and autoimmunity in inflammatory bowel disease.

    PubMed

    Karatzas, Pantelis S; Mantzaris, Gerassimos J; Safioleas, Michael; Gazouli, Maria

    2014-12-01

    The contribution of epigenetic alterations to disease pathogenesis is emerging as a research priority. In this study, we aimed to seek DNA methylation changes in peripheral blood and tissue biopsies from patients with inflammatory bowel disease. The promoter methylation status of genes involved in inflammation and autoimmunity was profiled using the Human Inflammatory Response and Autoimmunity EpiTect Methyl II Signature PCR Array profiles. Methylation was considered to be hypermethylated if >20% according to the instructions of the manufacturer. The microarrays were validated with Quantitative Real-time PCR. Regarding Crohn disease (CD) no gene appeared hypermethylated compared to healthy controls. In ulcerative colitis (UC) 5 genes (CXCL14, CXCL5, GATA3, IL17C, and IL4R) were hypermethylated compared to healthy controls. Some of the examined genes show different methylation patterns between CD and UC. Concerning tissue samples we found that all hypermethylated genes appear the same methylation pattern and confirmed a moderate-strong correlation between methylation levels in colon biopsies and peripheral blood (Pearson coefficients r=0.089-0.779, and r=0.023-0.353, respectively). The epigenetic changes observed in this study indicate that CD and UC exhibit specific DNA methylation signatures with potential clinical applications in IBD non-invasive diagnosis and prognosis.

  18. 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

  19. DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation.

    PubMed

    Peralta-Arrieta, Irlanda; Hernández-Sotelo, Daniel; Castro-Coronel, Yaneth; Leyva-Vázquez, Marco Antonio; Illades-Aguiar, Berenice

    2017-01-01

    Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo. The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer.

  20. DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation

    PubMed Central

    Peralta-Arrieta, Irlanda; Hernández-Sotelo, Daniel; Castro-Coronel, Yaneth; Leyva-Vázquez, Marco Antonio; Illades-Aguiar, Berenice

    2017-01-01

    Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo. The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer. PMID:28123849

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

    PubMed

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

    2016-01-01

    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.

  2. 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

  3. 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.

  4. 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.

  5. DAPK1 Promoter Methylation and Cervical Cancer Risk: A Systematic Review and a Meta-Analysis

    PubMed Central

    Agodi, Antonella; Barchitta, Martina; Quattrocchi, Annalisa; Maugeri, Andrea; Vinciguerra, Manlio

    2015-01-01

    Objective The Death-Associated Protein Kinase 1 (DAPK1) gene has been frequently investigated in cervical cancer (CC). The aim of the present study was to carry out a systematic review and a meta-analysis in order to evaluate DAPK1 promoter methylation as an epigenetic marker for CC risk. Methods A systematic literature search was carried out. The Cochrane software package Review Manager 5.2 was used. The fixed-effects or random-effects models, according to heterogeneity across studies, were used to calculate odds ratios (ORs) and 95% Confidence Intervals (CIs). Furthermore, subgroup analyses were conducted by histological type, assays used to evaluate DAPK1 promoter methylation, and control sample source. Results A total of 20 papers, published between 2001 and 2014, on 1929 samples, were included in the meta-analysis. DAPK1 promoter methylation was associated with an increased CC risk based on the random effects model (OR: 21.20; 95%CI = 11.14–40.35). Omitting the most heterogeneous study, the between study heterogeneity decreased and the association increased (OR: 24.13; 95% CI = 15.83–36.78). The association was also confirmed in all the subgroups analyses. Conclusions A significant strong association between DAPK1 promoter methylation and CC was shown and confirmed independently by histological tumor type, method used to evaluate methylation and source of control samples. Methylation markers may have value in early detection of CC precursor lesions, provide added reassurances of safety for women who are candidates for less frequent screens, and predict outcomes of women infected with human papilloma virus. PMID:26267895

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

    PubMed Central

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

    2014-01-01

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

  7. A genome-wide study of DNA methylation patterns and gene expression levels in multiple human and chimpanzee tissues.

    PubMed

    Pai, Athma A; Bell, Jordana T; Marioni, John C; Pritchard, Jonathan K; Gilad, Yoav

    2011-02-01

    The modification of DNA by methylation is an important epigenetic mechanism that affects the spatial and temporal regulation of gene expression. Methylation patterns have been described in many contexts within and across a range of species. However, the extent to which changes in methylation might underlie inter-species differences in gene regulation, in particular between humans and other primates, has not yet been studied. To this end, we studied DNA methylation patterns in livers, hearts, and kidneys from multiple humans and chimpanzees, using tissue samples for which genome-wide gene expression data were also available. Using the multi-species gene expression and methylation data for 7,723 genes, we were able to study the role of promoter DNA methylation in the evolution of gene regulation across tissues and species. We found that inter-tissue methylation patterns are often conserved between humans and chimpanzees. However, we also found a large number of gene expression differences between species that might be explained, at least in part, by corresponding differences in methylation levels. In particular, we estimate that, in the tissues we studied, inter-species differences in promoter methylation might underlie as much as 12%-18% of differences in gene expression levels between humans and chimpanzees.

  8. Endothelial Cell–Specific Expression of Roundabout 4 Is Regulated by Differential DNA Methylation of the Proximal Promoter

    PubMed Central

    Okada, Yoshiaki; Funahashi, Nobuaki; Tanaka, Toru; Nishiyama, Yuji; Yuan, Lei; Shirakura, Keisuke; Turjman, Alexis S.; Kano, Yoshihiro; Naruse, Hiroki; Suzuki, Ayano; Sakai, Miki; Zhixia, Jiang; Kitajima, Kenji; Ishimoto, Kenji; Hino, Nobumasa; Kondoh, Masuo; Mukai, Yohei; Nakagawa, Shinsaku; García-Cardeña, Guillermo; Aird, William C.; Doi, Takefumi

    2017-01-01

    Objective The molecular basis of endothelial cell (EC)–specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5′-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression. Methods and Results Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the −42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type–specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell–derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation. Conclusions The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression. PMID:24855053

  9. Long-term survival in glioblastoma: methyl guanine methyl transferase (MGMT) promoter methylation as independent favourable prognostic factor

    PubMed Central

    Smrdel, Uros; Zwitter, Matjaz; Bostjancic, Emanuela; Zupan, Andrej; Kovac, Viljem; Glavac, Damjan; Bokal, Drago; Jerebic, Janja

    2016-01-01

    Abstract Background In spite of significant improvement after multi-modality treatment, prognosis of most patients with glioblastoma remains poor. Standard clinical prognostic factors (age, gender, extent of surgery and performance status) do not clearly predict long-term survival. The aim of this case-control study was to evaluate immuno-histochemical and genetic characteristics of the tumour as additional prognostic factors in glioblastoma. Patients and methods Long-term survivor group were 40 patients with glioblastoma with survival longer than 30 months. Control group were 40 patients with shorter survival and matched to the long-term survivor group according to the clinical prognostic factors. All patients underwent multimodality treatment with surgery, postoperative conformal radiotherapy and temozolomide during and after radiotherapy. Biopsy samples were tested for the methylation of MGMT promoter (with methylation specific polymerase chain reaction), IDH1 (with immunohistochemistry), IDH2, CDKN2A and CDKN2B (with multiplex ligation-dependent probe amplification), and 1p and 19q mutations (with fluorescent in situ hybridization). Results Methylation of MGMT promoter was found in 95% and in 36% in the long-term survivor and control groups, respectively (p < 0.001). IDH1 R132H mutated patients had a non-significant lower risk of dying from glioblastoma (p = 0.437), in comparison to patients without this mutation. Other mutations were rare, with no significant difference between the two groups. Conclusions Molecular and genetic testing offers additional prognostic and predictive information for patients with glioblastoma. The most important finding of our analysis is that in the absence of MGMT promoter methylation, longterm survival is very rare. For patients without this mutation, alternative treatments should be explored. PMID:27904447

  10. 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.

  11. Linking ATM Promoter Methylation to Cell Cycle Protein Expression in Brain Tumor Patients: Cellular Molecular Triangle Correlation in ATM Territory.

    PubMed

    Mehdipour, P; Karami, F; Javan, Firouzeh; Mehrazin, M

    2015-08-01

    Ataxia telangiectasia mutated (ATM) is a key gene in DNA double-strand break (DSB), and therefore, most of its disabling genetic alterations play an important initiative role in many types of cancer. However, the exact role of ATM gene and its epigenetic alterations, especially promoter methylation in different grades of brain tumors, remains elusive. The current study was conducted to query possible correlations among methylation statue of ATM gene, ATM/ retinoblastoma (RB) protein expression, D1853N ATM polymorphism, telomere length (TL), and clinicopathological characteristics of various types of brain tumors. Isolated DNA from 30 fresh tissues was extracted from different types of brain tumors and two brain tissues from deceased normal healthy individuals. DNAs were treated with bisulfate sodium using DNA modification kit (Qiagen). Methylation-specific polymerase chain reaction (MSP-PCR) was implicated to determine the methylation status of treated DNA templates confirmed by promoter sequencing. Besides, the ATM and RB protein levels were determined by immunofluorescence (IF) assay using monoclonal mouse antihuman against ATM, P53, and RB proteins. To achieve an interactive correlation, the methylation data were statistically analyzed by considering TL and D1853N ATM polymorphism. More than 73% of the brain tumors were methylated in ATM gene promoter. There was strong correlation between ATM promoter methylation and its protein expression (p < 0.001). As a triangle, meaningful correlation was also found between methylated ATM promoter and ATM protein expression with D1853N ATM polymorphism (p = 0.01). ATM protein expression was not in line with RB protein expression while it was found to be significantly correlated with ATM promoter methylation (p = 0.01). There was significant correlation between TL neither with ATM promoter methylation nor with ATM protein expression nor with D1853N polymorphism. However, TL has shown strong correlation with patient's age and

  12. Virus-induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation.

    PubMed

    Zhao, Mingmin; San León, David; Delgadillo, Ma Otilia; García, Juan Antonio; Simón-Mateo, Carmen

    2014-08-01

    We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so-called virus-induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5' end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3' end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3' end, and an overall increase in CG methylation in the 5' end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.

  13. 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.

  14. 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.

  15. Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting duct.

    PubMed

    Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

    2013-10-01

    Aldosterone increases tubular Na(+) absorption largely by increasing α-epithelial Na(+) channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.

  16. Prognostic Relevance of Tumor Purity and TERT Promoter Mutations on MGMT Promoter Methylation in Glioblastoma.

    PubMed

    Schulze Heuling, Eva; Knab, Felix; Radke, Josefine; Eskilsson, Eskil; Martinez-Ledesma, Emmanuel; Koch, Arend; Czabanka, Marcus; Dieterich, Christoph; Verhaak, Roel G; Harms, Christoph; Euskirchen, Philipp

    2017-02-01

    Promoter methylation status of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, is a critical biomarker in glioblastoma multiforme (GBM) as treatment decisions and clinical trial inclusion rely on its accurate assessment. However, interpretation of results is complicated by poor inter-assay reproducibility as well as weak a correlation between methylation status and expression levels of MGMT. The present study systematically investigates the influence of tumor purity on tissue subjected to MGMT analysis. A quantitative, allele-specific real-time PCR (qAS-PCR) assay was developed to determine genotype and mutant allele frequency of telomerase promoter (pTERT) mutations as a direct measure of tumor purity. We studied tumor purity, pTERT mutation by Sanger sequencing, MGMT methylation by pyrosequencing, IDH1 mutation status, and clinical parameters in a cohort of high-grade gliomas (n=97). The qAS-PCR reliably predicted pTERT genotype and tumor purity compared with independent methods. Tumor purity positively and significantly correlated with the extent of methylation in MGMT methylated GBMs. Extent of MGMT methylation differed significantly with respect to pTERT mutation hotspot (C228T vs. C250T). Interestingly, frontal lobe tumors showed greater tumor purity than those in other locations. Above all, tumor purity was identified as an independent prognostic factor in GBM. In conclusion, we determined mutual associations of tumor purity with MGMT methylation and pTERT mutations and found that the extent of MGMT methylation reflects tumor purity. In turn, tumor purity is prognostic in IDH1 wildtype GBM.

  17. MIEN1 is tightly regulated by SINE Alu methylation in its promoter

    PubMed Central

    Van Treuren, Timothy; Klinkebiel, David L.; Vishwanatha, Jamboor K.

    2016-01-01

    Migration and invasion enhancer 1 (MIEN1) is a novel gene involved in prostate cancer progression by enhancing prostate cancer cell migration and invasion. DNA methylation, an important epigenetic regulation, is one of the most widely altered mechanisms in prostate cancer. This phenomenon frames the basis to study the DNA methylation patterns in the promoter region of MIEN1. Bisulfite pyrosequencing demonstrates the MIEN1 promoter contains a short interspersed nuclear Alu element (SINE Alu) repeat sequence. Validation of methylation inhibition on MIEN1 was performed using nucleoside analogs and non-nucleoside inhibitors and resulted in an increase in both MIEN1 RNA and protein in normal cells. MIEN1 mRNA and protein increases upon inhibition of individual DNA methyltransferases using RNA interference technologies. Furthermore, dual luciferase reporter assays, in silico analysis, and chromatin immunoprecipitation assays identified a sequence upstream of the transcription start site that has a site for binding of the USF transcription factors. These results suggest the MIEN1 promoter has a SINE Alu region that is hypermethylated in normal cells leading to repression of the gene. In cancer, the hypomethylation of a part of this repeat, in addition to the binding of USF, results in MIEN1 expression. PMID:27589566

  18. 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

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

    PubMed Central

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

    2014-01-01

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

  20. 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.

  1. The expression of RUNDC3B is associated with promoter methylation in lymphoid malignancies.

    PubMed

    Burmeister, Dane W; Smith, Emily H; Cristel, Robert T; McKay, Stephanie D; Shi, Huidong; Arthur, Gerald L; Davis, Justin Wade; Taylor, Kristen H

    2017-03-01

    DNA methylation is an epigenetic modification that plays an important role in the regulation of gene expression. The function of RUNDC3B has yet to be determined, although its dysregulated expression has been associated with malignant potential of both breast and lung carcinoma. To elucidate the potential of using DNA methylation in RUNDC3B as a biomarker in lymphoid malignancies, the methylation status of six regions spanning the CpG island in the promoter region of RUNDC3B was determined in cancer cell lines. Lymphoid malignancies were found to have more prominent methylation and did not express RUNDC3B compared with myeloid malignancies and solid tumours, supporting the potential use of DNA methylation in this region as a biomarker for lymphoid malignancies. RUNDC3B contains a RUN domain in its N-terminal region that mediates interaction with Rap2, an important component of the mitogen-activated protein kinase (MAPK) cascade, which regulates cellular proliferation and differentiation. The protein sequence of RUNDC3B also contains characteristic binding sites for MAPK intermediates. Therefore, it is possible that RUNDC3B serves as a mediator between Rap2 and the MAPK signalling cascade. Three genes with MAPK-inducible expression were downregulated in a methylated leukaemia cell line (HSPA5, Jun and Fos). Jun and Fos combine to form the activating protein 1 transcription factor, and loss of this factor is associated with the dysregulation of genes involved in differentiation and proliferation. We hypothesize that the loss of RUNDC3B secondary to aberrant hypermethylation of the early growth response 3 transcription factor binding site results in dysregulated MAPK signalling and carcinogenesis in lymphoid malignancies. © 2015 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.

  2. The expression of RUNDC3B is associated with promoter methylation in lymphoid malignancies

    PubMed Central

    Burmeister, Dane W.; Smith, Emily H.; Cristel, Robert T.; McKay, Stephanie D.; Shi, Huidong; Arthur, Gerald L.; Davis, Justin Wade

    2015-01-01

    Abstract DNA methylation is an epigenetic modification that plays an important role in the regulation of gene expression. The function of RUNDC3B has yet to be determined, although its dysregulated expression has been associated with malignant potential of both breast and lung carcinoma. To elucidate the potential of using DNA methylation in RUNDC3B as a biomarker in lymphoid malignancies, the methylation status of six regions spanning the CpG island in the promoter region of RUNDC3B was determined in cancer cell lines. Lymphoid malignancies were found to have more prominent methylation and did not express RUNDC3B compared with myeloid malignancies and solid tumours, supporting the potential use of DNA methylation in this region as a biomarker for lymphoid malignancies. RUNDC3B contains a RUN domain in its N‐terminal region that mediates interaction with Rap2, an important component of the mitogen‐activated protein kinase (MAPK) cascade, which regulates cellular proliferation and differentiation. The protein sequence of RUNDC3B also contains characteristic binding sites for MAPK intermediates. Therefore, it is possible that RUNDC3B serves as a mediator between Rap2 and the MAPK signalling cascade. Three genes with MAPK‐inducible expression were downregulated in a methylated leukaemia cell line (HSPA5, Jun and Fos). Jun and Fos combine to form the activating protein 1 transcription factor, and loss of this factor is associated with the dysregulation of genes involved in differentiation and proliferation. We hypothesize that the loss of RUNDC3B secondary to aberrant hypermethylation of the early growth response 3 transcription factor binding site results in dysregulated MAPK signalling and carcinogenesis in lymphoid malignancies. © 2015 The Authors. Hematological Oncology published by John Wiley & Sons Ltd PMID:26011749

  3. Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats.

    PubMed

    López-Álvarez, Guadalupe S; Wojdacz, Tomasz K; García-Cuellar, Claudia M; Monroy-Ramírez, Hugo C; Rodríguez-Segura, Miguel A; Pacheco-Rivera, Ruth A; Valencia-Antúnez, Carlos A; Cervantes-Anaya, Nancy; Soto-Reyes, Ernesto; Vásquez-Garzón, Verónica R; Sánchez-Pérez, Yesennia; Villa-Treviño, Saúl

    2017-01-15

    The association between the downregulation of genes and DNA methylation in their CpG islands has been extensively studied as a mechanism that favors carcinogenesis. The objective of this study was to analyze the methylation of a set of genes selected based on their microarray expression profiles during the process of hepatocarcinogenesis. Rats were euthanized at: 24 h, 7, 11, 16 and 30 days and 5, 9, 12 and 18 months post-treatment. We evaluated the methylation status in the CpG islands of four deregulated genes (Casp3, Cldn1, Pex11a and Nox4) using methylation-sensitive high-resolution melting technology for the samples obtained from different stages of hepatocarcinogenesis. We did not observe methylation in Casp3, Cldn1 or Pex11a. However, Nox4 exhibited altered methylation patterns, reaching a maximum of 10%, even during the early stages of hepatocarcinogenesis. We observed downregulation of mRNA and protein of Nox4 (97.5% and 40%, respectively) after the first carcinogenic stimulus relative to the untreated samples. Our results suggest that Nox4 downregulation is associated with DNA methylation of the CpG island in its promoter. We propose that methylation is a mechanism that can silence the expression of Nox4, which could contribute to the acquisition of neoplastic characteristics during hepatocarcinogenesis in rats.

  4. Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats

    PubMed Central

    López-Álvarez, Guadalupe S.; Wojdacz, Tomasz K.; García-Cuellar, Claudia M.; Monroy-Ramírez, Hugo C.; Rodríguez-Segura, Miguel A.; Pacheco-Rivera, Ruth A.; Valencia-Antúnez, Carlos A.; Cervantes-Anaya, Nancy; Soto-Reyes, Ernesto; Vásquez-Garzón, Verónica R.; Sánchez-Pérez, Yesennia; Villa-Treviño, Saúl

    2017-01-01

    ABSTRACT The association between the downregulation of genes and DNA methylation in their CpG islands has been extensively studied as a mechanism that favors carcinogenesis. The objective of this study was to analyze the methylation of a set of genes selected based on their microarray expression profiles during the process of hepatocarcinogenesis. Rats were euthanized at: 24 h, 7, 11, 16 and 30 days and 5, 9, 12 and 18 months post-treatment. We evaluated the methylation status in the CpG islands of four deregulated genes (Casp3, Cldn1, Pex11a and Nox4) using methylation-sensitive high-resolution melting technology for the samples obtained from different stages of hepatocarcinogenesis. We did not observe methylation in Casp3, Cldn1 or Pex11a. However, Nox4 exhibited altered methylation patterns, reaching a maximum of 10%, even during the early stages of hepatocarcinogenesis. We observed downregulation of mRNA and protein of Nox4 (97.5% and 40%, respectively) after the first carcinogenic stimulus relative to the untreated samples. Our results suggest that Nox4 downregulation is associated with DNA methylation of the CpG island in its promoter. We propose that methylation is a mechanism that can silence the expression of Nox4, which could contribute to the acquisition of neoplastic characteristics during hepatocarcinogenesis in rats. PMID:27895046

  5. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells.

    PubMed

    Ball, Madeleine P; Li, Jin Billy; Gao, Yuan; Lee, Je-Hyuk; LeProust, Emily M; Park, In-Hyun; Xie, Bin; Daley, George Q; Church, George M

    2009-04-01

    Studies of epigenetic modifications would benefit from improved methods for high-throughput methylation profiling. We introduce two complementary approaches that use next-generation sequencing technology to detect cytosine methylation. In the first method, we designed approximately 10,000 bisulfite padlock probes to profile approximately 7,000 CpG locations distributed over the ENCODE pilot project regions and applied them to human B-lymphocytes, fibroblasts and induced pluripotent stem cells. This unbiased choice of targets takes advantage of existing expression and chromatin immunoprecipitation data and enabled us to observe a pattern of low promoter methylation and high gene-body methylation in highly expressed genes. The second method, methyl-sensitive cut counting, generated nontargeted genome-scale data for approximately 1.4 million HpaII sites in the DNA of B-lymphocytes and confirmed that gene-body methylation in highly expressed genes is a consistent phenomenon throughout the human genome. Our observations highlight the usefulness of techniques that are not inherently or intentionally biased towards particular subsets like CpG islands or promoter regions.

  6. Response to psychotherapy in borderline personality disorder and methylation status of the BDNF gene

    PubMed Central

    Perroud, N; Salzmann, A; Prada, P; Nicastro, R; Hoeppli, M-E; Furrer, S; Ardu, S; Krejci, I; Karege, F; Malafosse, A

    2013-01-01

    Downregulation of brain-derived neurotrophic factor (BDNF) gene expression with corresponding increased methylation at specific promoters has been associated with stressful experiences in early life and may explain later adulthood psychopathology. We measured the percentage of methylation at BDNF CpG exons I and IV as well as plasma BDNF protein levels in 115 subjects with borderline personality disorder (BPD) and 52 controls. BPD subjects then underwent a 4-week course of intensive dialectical behavior therapy (I-DBT). BDNF methylation status and protein levels were re-assessed at the end of treatment. BPD subjects had significantly higher methylation status in both CpG regions than controls. In addition, the higher the number of childhood trauma, the higher was the methylation status. In BPD subjects, BDNF methylation significantly increased after I-DBT. Nonresponders accounted for the majority of this increase, whereas responders showed a decrease in methylation status over time. Accordingly, the changes in methylation status over time were significantly associated with changes in depression scores, hopelessness scores and impulsivity. No association was found between protein levels and BDNF methylation status. We here found a relationship between child maltreatment and higher DNA methylation of BDNF. These results moreover support the idea that these epigenetic marks may be changed through psychotherapeutic approaches and that these changes underline changes in cognitive functions. PMID:23422958

  7. Methylation of BRCA1 promoter region is associated with unfavorable prognosis in women with early-stage breast cancer.

    PubMed

    Hsu, Nicholas C; Huang, Ya-Fang; Yokoyama, Kazunari K; Chu, Pei-Yi; Chen, Fang-Ming; Hou, Ming-Feng

    2013-01-01

    BRCA1-associated breast cancers are associated with particular features such as early onset, poor histological differentiation, and hormone receptor negativity. Previous studies conducted in Taiwanese population showed that the mutation of BRCA1 gene does not play a significant role in the occurrence of breast cancer. The present study explored methylation of BRCA1 promoter and its relationship to clinical features and outcome in Taiwanese breast cancer patients. Tumor specimens from a cohort of 139 early-stage breast cancer patients were obtained during surgery before adjuvant treatment for DNA extraction. Methylation of BRCA1 promoter region was determined by methylation-specific PCR and the results were related to clinical features and outcome of patients using statistical analysis. Methylation of the BRCA1 promoter was detected in 78 (56%) of the 139 tumors. Chi-square analysis indicated that BRCA1 promoter methylation correlated significantly with triple-negative (ER-/PR-/HER2-) status of breast cancer patients (p = 0.041). The Kaplan-Meier method showed that BRCA1 promoter methylation was significantly associated with poor overall survival (p = 0.026) and disease-free survival (p = 0.001). Multivariate analysis which incorporated variables of patients' age, tumor size, grade, and lymph node metastasis revealed that BRCA1 promoter methylation was associated with overall survival (p = 0.027; hazard ratio, 16.38) and disease-free survival (p = 0.003; hazard ratio, 12.19) [corrected].Our findings underscore the clinical relevance of the methylation of BRCA1 promoter in Taiwanese patients with early-stage breast cancer.

  8. 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.

  9. 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

  10. Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression

    PubMed Central

    Ponnaluri, V. K. Chaithanya; Ehrlich, Kenneth C.; Zhang, Guoqiang; Lacey, Michelle; Johnston, Douglas; Pradhan, Sriharsa; Ehrlich, Melanie

    2017-01-01

    ABSTRACT Differentially methylated or hydroxymethylated regions (DMRs) in mammalian DNA are often associated with tissue-specific gene expression but the functional relationships are still being unraveled. To elucidate these relationships, we studied 16 human genes containing myogenic DMRs by analyzing profiles of their epigenetics and transcription and quantitatively assaying 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) at specific sites in these genes in skeletal muscle (SkM), myoblasts, heart, brain, and diverse other samples. Although most human promoters have little or no methylation regardless of expression, more than half of the genes that we chose to study—owing to their myogenic DMRs—overlapped tissue-specific alternative or cryptic promoters displaying corresponding tissue-specific differences in histone modifications. The 5mC levels in myoblast DMRs were significantly associated with 5hmC levels in SkM at the same site. Hypermethylated myogenic DMRs within CDH15, a muscle- and cerebellum-specific cell adhesion gene, and PITX3, a homeobox gene, were used for transfection in reporter gene constructs. These intragenic DMRs had bidirectional tissue-specific promoter activity that was silenced by in vivo-like methylation. The CDH15 DMR, which was previously associated with an imprinted maternal germline DMR in mice, had especially strong promoter activity in myogenic host cells. These findings are consistent with the controversial hypothesis that intragenic DNA methylation can facilitate transcription and is not just a passive consequence of it. Our results support varied roles for tissue-specific 5mC- or 5hmC-enrichment in suppressing inappropriate gene expression from cryptic or alternative promoters and in increasing the plasticity of gene expression required for development and rapid responses to tissue stress or damage. PMID:27911668

  11. Gene-Specific Methylation Analysis in Thymomas of Patients with Myasthenia Gravis

    PubMed Central

    Lopomo, Angela; Ricciardi, Roberta; Maestri, Michelangelo; De Rosa, Anna; Melfi, Franca; Lucchi, Marco; Mussi, Alfredo; Coppedè, Fabio; Migliore, Lucia

    2016-01-01

    Thymomas are uncommon neoplasms that arise from epithelial cells of the thymus and are often associated with myasthenia gravis (MG), an autoimmune disease characterized by autoantibodies directed to different targets at the neuromuscular junction. Little is known, however, concerning epigenetic changes occurring in thymomas from MG individuals. To further address this issue, we analyzed DNA methylation levels of genes involved in one-carbon metabolism (MTHFR) and DNA methylation (DNMT1, DNMT3A, and DNMT3B) in blood, tumor tissue, and healthy thymic epithelial cells from MG patients that underwent a surgical resection of a thymic neoplasm. For the analyses we applied the methylation-sensitive high-resolution melting technique. Both MTHFR and DNMT3A promoters showed significantly higher methylation in tumor tissue with respect to blood, and MTHFR also showed significantly higher methylation levels in tumor tissue respect to healthy adjacent thymic epithelial cells. Both DNMT1 and DNMT3B promoter regions were mostly hypomethylated in all the investigated tissues. The present study suggests that MTHFR methylation is increased in thymomas obtained from MG patients; furthermore, some degrees of methylation of the DNMT3A gene were observed in thymic tissue with respect to blood. PMID:27999265

  12. Interactions between CYP11B2 Promoter Methylation and Smoking Increase Risk of Essential Hypertension

    PubMed Central

    Mao, Shuqi; Fan, Rui; Zhong, Fade; Zhu, Fubao; Hao, Lingmei

    2016-01-01

    Aldosterone synthase (CYP11B2) is closely linked to essential hypertension (EH). However, it remains unclear whether the methylation of the CYP11B2 promoter is involved in the development of EH in humans. Our study is aimed at evaluating the contribution of CYP11B2 promoter methylation to the risk of EH. Methylation levels were measured using pyrosequencing technology in 192 participants in a hospital-based case-control study. Logistic regression and multiple linear regression analyses were utilized to adjust for confounding factors and the GMDR method was applied to investigate high-order gene-environment interactions. Although no significant result was observed linking the four analyzed CpG sites to EH, GMDR detected significant interactions among CpG1, CpG3, CpG4, and smoking correlated with an increased risk of EH (OR = 4.62, adjusted P = 0.011). In addition, CpG2 (adjusted P = 0.013) and CpG3 (adjusted P = 0.039) methylation was significantly lower in healthy males than in healthy females. Likewise, after adjusting for confounding factors, CpG2 methylation (adjusted P = 0.007) still showed significant gender-specific differences among the participants of the study. CpG1 (P = 0.009) site was significantly positively correlated with age, and CpG3 (P = 0.007) and CpG4 (P = 0.006) were both inversely linked to smoking. Our findings suggest that gene-environment interactions are associated with the pathogenesis and progression of EH. PMID:28078278

  13. Chronic exposure to trichloroethylene increases DNA methylation of the Ifng promoter in CD4(+) T cells.

    PubMed

    Gilbert, Kathleen M; Blossom, Sarah J; Erickson, Stephen W; Broadfoot, Brannon; West, Kirk; Bai, Shasha; Li, Jingyun; Cooney, Craig A

    2016-10-17

    CD4(+) T cells in female MRL+/+ mice exposed to solvent and water pollutant trichloroethylene (TCE) skew toward effector/memory CD4(+) T cells, and demonstrate seemingly non-monotonic alterations in IFN-γ production. In the current study we examined the mechanism for this immunotoxicity using effector/memory and naïve CD4(+) T cells isolated every 6 weeks during a 40 week exposure to TCE (0.5mg/ml in drinking water). A time-dependent effect of TCE exposure on both Ifng gene expression and IFN-γ protein production was observed in effector/memory CD4(+) T cells, with an increase after 22 weeks of exposure and a decrease after 40 weeks of exposure. No such effect of TCE was observed in naïve CD4(+) T cells. A cumulative increase in DNA methylation in the CpG sites of the promoter of the Ifng gene was observed in effector/memory, but not naïve, CD4(+) T cells over time. Also unique to the Ifng promoter was an increase in methylation variance in effector/memory compared to naïve CD4(+) T cells. Taken together, the CpG sites of the Ifng promoter in effector/memory CD4(+) T cells were especially sensitive to the effects of TCE exposure, which may help explain the regulatory effect of the chemical on this gene.

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

    PubMed

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

    2016-08-02

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

  15. Investigation of methylation and protein expression of the Runx3 gene in colon carcinogenesis.

    PubMed

    He, Shao-Ya; Jiang, Ren-Fa; Jiang, Jie; Xiang, Yang-Sheng; Wang, Ling

    2015-09-01

    In the present study, the methylation and protein expression of the runt-related transcription factor 3 (Runx3) gene was detected in sporadic colorectal cancer, colonic adenoma and normal colon tissue to evaluate their clinical significance in colorectal carcinogenesis. A total of 34 colonic cancer specimens, 34 colonic adenoma specimens and 34 normal colonic tissue specimens were used in the study. The CpG island methylation status of the Runx3 gene was detected by methylation-specific polymerase chain reaction and the protein expression of Runx3 was detected by immunohistochemistry. The results showed that the rates of methylation of the Runx3 gene in colonic cancer and colonic adenomas were significantly higher than that in the normal colonic tissue (23.5, 20.6 vs. 0.0%; P<0.05). There was no significant difference in the percentage of methylation of the Runx3 gene between colonic adenoma and colonic cancer (P>0.05). The positive percentage of Runx3 protein expression was significantly lower in colonic cancer compared with colonic adenoma and normal tissue (17.7 vs. 61.8, 76.5%; P<0.05). Methylation of the promoter CpG islands of the Runx3 gene is an important genetic event of colon carcinogenesis and may be associated with an altered protein level of Runx3.

  16. N-hexane inhalation during pregnancy alters DNA promoter methylation in the ovarian granulosa cells of rat offspring.

    PubMed

    Li, Hong; Liu, Jin; Sun, Yan; Wang, Wenxiang; Weng, Shaozheng; Xiao, Shihua; Huang, Huiling; Zhang, Wenchang

    2014-08-01

    The N-hexane-induced impact on the reproductive system of the offspring of animals exposed to n-hexane has caused great concern. Pregnant Wistar rats inhaled 500, 2 500 or 12 500 ppm n-hexane during gestational days 1-20. Clinical characteristics and developmental indices were observed. Ovarian granulosa cells were extracted from F1 rats, the number of follicles was determined in ovarian slices and promoter methylation was assessed using MeDIP-Chip. Several methods were used to analyze the scanned genes, including the Gene Ontology Consortium tools, the DAVID Functional Annotation Clustering Tool, hierarchical clustering and KEGG pathway analysis. The results indicated that the live pups/litter ratio was significantly lowest in the 12 500 ppm group. A significant decrease in secondary follicles and an increase in atresic follicles were observed in the 12 500 ppm group. The number of shared demethylated genes was higher than that of the methylated genes, and the differentially methylated genes were enriched in cell death and apoptosis, cell growth and hormone regulation. The methylation profiles of the offspring from the 500 ppm and control groups were different from those of the 2500 and 12 500 ppm groups. Furthermore, the methylation status of genes in the PI3K-Akt and NF-kappa B signaling pathways was changed after n-hexane exposure. The Cyp11a1, Cyp17a1, Hsd3b1, Cyp1a1 and Srd5a1 promoters were hypermethylated in the n-hexane-exposed groups. These results indicate that the developmental toxicity of n-hexane in F1 ovaries is accompanied by the altered methylation of promoters of genes associated with apoptotic processes and steroid hormone biosynthesis.

  17. The mom gene of bacteriophage Mu: the mechanism of methylation-dependent expression.

    PubMed Central

    Seiler, A; Blöcker, H; Frank, R; Kahmann, R

    1986-01-01

    Transcription of the DNA modification gene (mom) of bacteriophage Mu requires methylation of three GATC sites upstream of the mom promoter by the Escherichia coli deoxyadenosine methylation function (Dam). The three sites map within a 40-bp segment termed region I. Small deletions, inversions, duplications and specific point mutations have been introduced in region I. Their effect on mom expression has been studied in dam+ and dam strains. Dam-dependent expression of the mom gene requires a specific arrangement of the three GATC sites and the presence of the methylated base in at least two of the three sites. We show that mom specific modification is regulated by a host protein. The Mom function is expressed in dam strains if they are defective in one component of the methylation-instructed mismatch correction system, mutH. We suggest that the product of mutH functions as a transcriptional repressor by binding to region I. PMID:3536483

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

    PubMed

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

    2013-11-01

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

  19. DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes.

    PubMed

    Couvert, P; Poirier, K; Carrié, A; Chalas, C; Jouannet, P; Beldjord, C; Bienvenu, T; Chelly, J; Kerjean, A

    2003-02-01

    The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.

  20. IDH1/2 Mutation and MGMT Promoter Methylation - the Relevant Survival Predictors in Czech Patients with Brain Gliomas.

    PubMed

    Kramář, F; Minárik, M; Benešová, L; Halková, T; Netuka, D; Bradáč, O; Beneš, V

    2016-01-01

    Gliomas are a heterogeneous group of tumours varying in prognosis, treatment approach, and overall survival. Recently, novel markers have been identified which are linked to patient prognosis and therapeutic response. Especially the mutation of the enzyme isocitrate dehydrogenase 1 or 2 (IDH1/2) gene and the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status seem to be the most important predictors of survival. From 2012 to 2015, 94 Czech patients with primary brain tumours were enrolled into the study. The IDH1/2 mutation was detected by denaturing capillary electrophores.The methylation status of the MGMT gene and other 46 genes was revealed by MS-MLPA. In all 94 patients, the clinical data were correlated with molecular markers by Kaplan-Meier analyses and Cox regression model. The MGMT promoter methylation status was established and compared to clinical data. In our study eight different probes were used to elucidate the MGMT methylation status; hypermethylation was proclaimed if four and more probes were positive. This 3 : 5 ratio was tested and confirmed by Kaplan-Meier and Cox analyses. The study confirmed the importance of the IDH1/2 mutation and hypermethylation of the MGMT gene promoter being present in tumour tissue. Both markers are independent positive survival predictors; in the Cox model the IDH hazard ratio was 0.10 and in the case of MGMT methylation it reached 0.32. The methylation analysis of the panel of additional 46 genes did not reveal any other significant epigenetic markers; none of the candidate genes have been confirmed in the Cox regression analyses as an independent prognostic factor.

  1. Association between RASSF1A promoter methylation and renal cell cancer susceptibility: a meta-analysis.

    PubMed

    Huang, Y Q; Guan, H; Liu, C H; Liu, D C; Xu, B; Jiang, L; Lin, Z X; Chen, M

    2016-04-25

    Epigenetic inactivation of Ras-associated domain family 1A (RASSF1A) by hyper-methylation of its promoter region has been identified in various cancers. However, the role of RASSF1A in renal cancer has neither been thoroughly investigated nor reviewed. In this study, we reviewed and performed a meta-analysis of 13 published studies reporting correlations between methylation frequency of the RASSF1A promoter region and renal cancer risk. The odds ratios (ORs) of eligible studies and their corresponding 95% confidence intervals (95%CIs) were used to correlate RASSF1A promoter methylation with renal cell cancer risk and clinical or pathological variables, respectively. RASSF1A promoter methylation was significantly associated with the risk of renal cell cancer (OR = 19.35, 95%CI = 9.57-39.13). RASSF1A promoter methylation was significantly associated with pathological tumor grade (OR = 3.32, 95%CI = 1.55-7.12), and a possible positive correlation between RASSF1A promoter methylation status and tumor stage was noted (OR = 1.89, 95%CI = 1.00-3.56, P = 0.051). Overall, this meta-analysis demonstrated that RASSF1A promoter methylation is significantly associated with increased risk of renal cell cancer. RASSF1A promoter methylation frequency was positively correlated with pathological tumor grade, but not the clinical stage. This study showed that RASSF1A promoter methylation could be utilized to predict renal cell cancer prognosis.

  2. Berberine reduces methylation of the MTTP promoter and alleviates fatty liver induced by a high-fat diet in rats[S

    PubMed Central

    Chang, XinXia; Yan, HongMei; Fei, Jing; Jiang, MingHong; Zhu, HongGuang; Lu, DaRu; Gao, Xin

    2010-01-01

    High-calorie food leads to nonalcoholic fatty liver disease (NAFLD) through dysregulation of genes involved in lipid metabolism, but the precise mechanism remains unclear. DNA methylation represents one of the mechanisms that contributes to dysregulation of gene expression via interaction with environmental factors. Berberine can alleviate fatty liver in db/db and ob/ob mice. Here, we investigated whether DNA methylation is involved in the pathogenesis of NAFLD induced by a high-fat diet (HFD) and whether berberine improves NAFLD through influencing the methylation status of promoters of key genes. HFD markedly decreased the mRNA levels encoding CPT-1α, MTTP, and LDLR in the liver. In parallel, DNA methylation levels in the MTTP promoter of rats with NAFLD were elevated in the liver. Interestingly, berberine reversed the downregulated expression of these genes and selectively inhibited HFD-induced increase in the methylation of MTTP. Consistently, berberine increased hepatic triglyceride (TG) export and ameliorated HFD-induced fatty liver. Furthermore, a close negative correlation was observed between the MTTP expression and its DNA methylation (at sites −113 and −20). These data indicate that DNA methylation of the MTTP promoter likely contributes to its downregulation during HFD-induced NAFLD and, further, that berberine can partially counteract the HFD-elicited dysregulation of MTTP by reversing the methylation state of its promoter, leading to reduced hepatic fat content. PMID:20567026

  3. Methylation of the Claudin 1 Promoter Is Associated with Loss of Expression in Estrogen Receptor Positive Breast Cancer

    PubMed Central

    Di Cello, Francescopaolo; Cope, Leslie; Li, Huili; Jeschke, Jana; Wang, Wei; Baylin, Stephen B.; Zahnow, Cynthia A.

    2013-01-01

    Downregulation of the tight junction protein claudin 1 is a frequent event in breast cancer and is associated with recurrence, metastasis, and reduced survival, suggesting a tumor suppressor role for this protein. Tumor suppressor genes are often epigenetically silenced in cancer. Downregulation of claudin 1 via DNA promoter methylation may thus be an important determinant in breast cancer development and progression. To investigate if silencing of claudin 1 has an epigenetic etiology in breast cancer we compared gene expression and methylation data from 217 breast cancer samples and 40 matched normal samples available through the Cancer Genome Atlas (TCGA). Moreover, we analyzed claudin 1 expression and methylation in 26 breast cancer cell lines. We found that methylation of the claudin 1 promoter CpG island is relatively frequent in estrogen receptor positive (ER+) breast cancer and is associated with low claudin 1 expression. In contrast, the claudin 1 promoter was not methylated in most of the ER-breast cancers samples and some of these tumors overexpress claudin 1. In addition, we observed that the demethylating agents, azacitidine and decitabine can upregulate claudin 1 expression in breast cancer cell lines that have a methylated claudin 1 promoter. Taken together, our results indicate that DNA promoter methylation is causally associated with downregulation of claudin 1 in a subgroup of breast cancer that includes mostly ER+ tumors, and suggest that epigenetic therapy to restore claudin 1 expression might represent a viable therapeutic strategy in this subtype of breast cancer. PMID:23844228

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

    SciTech Connect

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

    2006-02-03

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

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

    PubMed

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

    2015-11-03

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

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

    PubMed Central

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

    2015-01-01

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

  7. A study on the correlation between MTHFR promoter methylation and diabetic nephropathy

    PubMed Central

    Yang, Xiao-Hui; Cao, Ren-Fang; Yu, Yang; Sui, Miao; Zhang, Tao; Xu, Jing-Yi; Wang, Xiao-Mei

    2016-01-01

    Objective: In order to observe the relationship between MTHFR promoter and DN, the determinations on MTHFR promoter methylation level and expression of HCY from DN patients have been carried out. Methods: According to the Diabetes diagnosis and classification standard from WHO in 1999, 85 patients with DM diagnosed by Endocrinology and 30 healthy participants from our medical examination center were chosen as control specimen to study in this paper. All this specimen were divided into A, B, C and D four groups , which are corresponding simple diabetes mellitus group (SDM), early diabetic nephropathy group (EDN), clinical diabetic nephropathy group (CDN) and normal control group. And then, all common materials and clinical experiments data have been collected respectively. (1) Extracted the peripheral blood DNA of each group and determinate the methylation status of MTHFR gene promoter by PCR (MSP). (2) Determinated the serum HCY protein expression of each group. Results: (1) The MTHFR promoter methylation of SDM and diabetic nephropathy group are wear off comparied with normal control group. And MTHFR promoter was in demethylation state in normal control group, a slightly weak in SDN, a obviously weak in early diabetic nephropathy group; the MTHFR promoter was in methylation state in clinical diabetic nephropathy group. (2) The HCY protein of simple diabetes mellitus group, early diabetic nephropathy group and clinical diabetic nephropathy group are Pitch with normal control group. HCY protein level of each group are as 7.41±1.61 umol/L, 10.34±2.89 umol/L, 10.95±5.89 umol/L and 13.03±6.14 umol/L corresponding normal control group, simple diabetes mellitus group, early diabetic nephropathy group and clinical diabetic nephropathy group. And there is no statistical significance about the differences among four groups. Conclusion: The demethylation state of MTHFR promoter was obviously weaker in clinical diabetic nephropathy group than in SDM. The level of serum

  8. Differential methylation of genes and repeats in land plants.

    PubMed

    Rabinowicz, Pablo D; Citek, Robert; Budiman, Muhammad A; Nunberg, Andrew; Bedell, Joseph A; Lakey, Nathan; O'Shaughnessy, Andrew L; Nascimento, Lidia U; McCombie, W Richard; Martienssen, Robert A

    2005-10-01

    The hypomethylated fraction of plant genomes is usually enriched in genes and can be selectively cloned using methylation filtration (MF). Therefore, MF has been used as a gene enrichment technology in sorghum and maize, where gene enrichment was proportional to genome size. Here we apply MF to a broad variety of plant species spanning a wide range of genome sizes. Differential methylation of genic and non-genic sequences was observed in all species tested, from non-vascular to vascular plants, but in some cases, such as wheat and pine, a lower than expected level of enrichment was observed. Remarkably, hexaploid wheat and pine show a dramatically large number of gene-like sequences relative to other plants. In hexaploid wheat, this apparent excess of genes may reflect an abundance of methylated pseudogenes, which may thus be more prevalent in recent polyploids.

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

    PubMed

    Matthaios, Dimitrios; Balgkouranidou, Ioanna; Karayiannakis, Anastasios; Bolanaki, Helen; Xenidis, Nikolaos; Amarantidis, Kyriakos; Chelis, Leonidas; Romanidis, Konstantinos; Chatzaki, Aikaterini; Lianidou, Evi; Trypsianis, Grigorios; Kakolyris, Stylianos

    2016-07-01

    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

  10. Benzo[a]pyrene decreases global and gene specific DNA methylation during zebrafish development

    PubMed Central

    Fang, Xiefan; Thornton, Cammi; Scheffler, Brian E.; Willett, Kristine L.

    2013-01-01

    DNA methylation is important for gene regulation and is vulnerable to early-life exposure to environmental contaminants. We found that direct waterborne benzo[a]pyrene (BaP) exposure at 24 μg/L from 2.5 to 96 hours post fertilization (hpf) to zebrafish embryos significantly decreased global cytosine methylation by 44.8% and promoter methylation in vasa by 17%. Consequently, vasa expression was significantly increased by 33%. In contrast, BaP exposure at environmentally relevant concentrations did not change CpG island methylation or gene expression in cancer genes such as ras-association domain family member 1 (rassf1), telomerase reverse transcriptase (tert), c-jun, and c-myca. Similarly, BaP did not change gene expression of DNA methyltransferase 1 (dnmt1) and glycine N-methyltransferase (gnmt). While total DNMT activity was not affected, GNMT enzyme activity was moderately increased. In summary, BaP is an epigenetic modifier for global and gene specific DNA methylation status in zebrafish larvae. PMID:23542452

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. Selective DNA methylation of BDNF promoter in bipolar disorder: differences among patients with BDI and BDII.

    PubMed

    D'Addario, Claudio; Dell'Osso, Bernardo; Palazzo, Maria Carlotta; Benatti, Beatrice; Lietti, Licia; Cattaneo, Elisabetta; Galimberti, Daniela; Fenoglio, Chiara; Cortini, Francesca; Scarpini, Elio; Arosio, Beatrice; Di Francesco, Andrea; Di Benedetto, Manuela; Romualdi, Patrizia; Candeletti, Sanzio; Mari, Daniela; Bergamaschini, Luigi; Bresolin, Nereo; Maccarrone, Mauro; Altamura, A Carlo

    2012-06-01

    The etiology of bipolar disorder (BD) is still poorly understood, involving genetic and epigenetic mechanisms as well as environmental contributions. This study aimed to investigate the degree of DNA methylation at the promoter region of the brain-derived neurotrophic factor (BDNF) gene, as one of the candidate genes associated with major psychoses, in peripheral blood mononuclear cells isolated from 94 patients with BD (BD I=49, BD II=45) and 52 healthy controls. A significant BDNF gene expression downregulation was observed in BD II 0.53±0.11%; P<0.05), but not in BD I (1.13±0.19%) patients compared with controls (CONT: 1±0.2%). Consistently, an hypermethylation of the BDNF promoter region was specifically found in BD II patients (CONT: 24.0±2.1%; BDI: 20.4±1.7%; BDII: 33.3±3.5%, P<0.05). Of note, higher levels of DNA methylation were observed in BD subjects on pharmacological treatment with mood stabilizers plus antidepressants (34.6±4.2%, predominantly BD II) compared with those exclusively on mood-stabilizing agents (21.7±1.8%; P<0.01, predominantly BD I). Moreover, among the different pharmacological therapies, lithium (20.1±3.8%, P<0.05) and valproate (23.6±2.9%, P<0.05) were associated with a significant reduction of DNA methylation compared with other drugs (35.6±4.6%). Present findings suggest selective changes in DNA methylation of BDNF promoter in subjects with BD type II and highlight the importance of epigenetic factors in mediating the onset and/or susceptibility to BD, providing new insight into the mechanisms of gene expression. Moreover, they shed light on possible mechanisms of action of mood-stabilizing compounds vs antidepressants in the treatment of BD, pointing out that BDNF regulation might be a key target for their effects.

  17. 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

  18. 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

  19. Aberrant Promoter Methylation of Caveolin-1 Is Associated with Favorable Response to Taxane-Platinum Combination Chemotherapy in Advanced NSCLC

    PubMed Central

    Brodie, Seth A.; Lombardo, Courtney; Li, Ge; Kowalski, Jeanne; Gandhi, Khanjan; You, Shaojin; Khuri, Fadlo R.; Marcus, Adam; Vertino, Paula M.; Brandes, Johann C.

    2014-01-01

    Purpose Aberrant promoter DNA methylation can serve as a predictive biomarker for improved clinical responses to certain chemotherapeutics. One of the major advantages of methylation biomarkers is the ease of detection and clinical application. In order to identify methylation biomarkers predictive of a response to a taxane-platinum based chemotherapy regimen in advanced NSCLC we performed an unbiased methylation analysis of 1,536 CpG dinucleotides in cancer-associated gene loci and correlated results with clinical outcomes. Methods We studied a cohort of 49 patients (median age 62 years) with advanced NSCLC treated at the Atlanta VAMC between 1999 and 2010. Methylation analysis was done on the Illumina GoldenGate Cancer panel 1 methylation microarray platform. Methylation data were correlated with clinical response and adjusted for false discovery rates. Results Cav1 methylation emerged as a powerful predictor for achieving disease stabilization following platinum taxane based chemotherapy (p = 1.21E-05, FDR significance  = 0.018176). In Cox regression analysis after multivariate adjustment for age, performance status, gender, histology and the use of bevacizumab, CAV1 methylation was significantly associated with improved overall survival (HR 0.18 (95%CI: 0.03–0.94)). Silencing of CAV1 expression in lung cancer cell lines(A549, EKVX)by shRNA led to alterations in taxane retention. Conclusions CAV1 methylation is a predictor of disease stabilization and improved overall survival following chemotherapy with a taxane-platinum combination regimen in advanced NSCLC. CAV1 methylation may predict improved outcomes for other chemotherapeutic agents which are subject to cellular clearance mediated by caveolae. PMID:25222296

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

    PubMed Central

    Gonzalez, Diego; Collier, Justine

    2013-01-01

    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. PMID:23480529

  1. Gene silencing by DNA methylation and dual inheritance in Chinese hamster ovary cells.

    PubMed

    Paulin, R P; Ho, T; Balzer, H J; Holliday, R

    1998-06-01

    Chinese hamster ovary (CHO) cells strain D422, which has one copy of the adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine. Colonies were isolated and shown to be reactivated to APRT+ by 5-aza-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of independent origin and subjected to bisulphite treatment. This deaminates cytosine to uracil in single-stranded DNA but does not deaminate 5-methyl cytosine. PCR, cloning and sequencing revealed the methylation pattern of CpG doublets in the promoter region of the APRT- gene, whereas the active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT+ gene, could also be silenced by the same procedure but at a lower frequency. The availability of the 5-methyl dCTP-induced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulphonate, makes it possible to follow concomitantly the inheritance of active, mutant or silenced gene copies. This analysis demonstrates "dual inheritance" at the APRT locus in CHO cells.

  2. Programming gene expression with combinatorial promoters

    PubMed Central

    Cox, Robert Sidney; Surette, Michael G; Elowitz, Michael B

    2007-01-01

    Promoters control the expression of genes in response to one or more transcription factors (TFs). The architecture of a promoter is the arrangement and type of binding sites within it. To understand natural genetic circuits and to design promoters for synthetic biology, it is essential to understand the relationship between promoter function and architecture. We constructed a combinatorial library of random promoter architectures. We characterized 288 promoters in Escherichia coli, each containing up to three inputs from four different TFs. The library design allowed for multiple −10 and −35 boxes, and we observed varied promoter strength over five decades. To further analyze the functional repertoire, we defined a representation of promoter function in terms of regulatory range, logic type, and symmetry. Using these results, we identified heuristic rules for programming gene expression with combinatorial promoters. PMID:18004278

  3. Epididymal Region-Specific miRNA Expression and DNA Methylation and Their Roles in Controlling Gene Expression in Rats

    PubMed Central

    Hu, Shuanggang; Zhang, Jinsong; Xie, Shengsong; Ma, Wubin; Ni, Minjie; Tang, Chunhua; Zhou, Lu; Zhou, Yuchuan; Liu, Mofang; Li, Yixue; Zhang, Yonglian

    2015-01-01

    Region-specific gene expression is an intriguing feature of the mammalian epididymis. This unique property is essential for sperm maturation and storage, and it also implicates stringent and multi-level regulations of gene expression. Over the past decade, the androgen-driven activation of epididymal gene transcription has been extensively studied. However, it still remains largely unexplored whether and how other regulatory mechanisms, such as miRNAs and DNA methylation, are involved in controlling regional gene expression in the epididymis. Using microarray-based approaches, we studied the regional miRNA expression and DNA methylation profiles in 4 distinct epididymal regions (initial segment, caput, corpus and cauda) of rats. We found that the miR-200 family members were more expressed in caput, compared with cauda. By GSEA analysis, the differential expression of miR-200 family between caput and cauda was shown to be negatively correlated with their predicted target genes, among which 4 bona fide targets were verified by luciferase reporter assay. Predicted target genes of miR-200 family have enriched functions in anti-apoptosis, cell transportation and development, implying the regional diversity in epididymal functions. On the other hand, we revealed epididymal DNA methylation of 2002 CpG islands and 2771 gene promoters (-3.88-0.97kb), among which 1350 (67.43%) CpG islands and 2095 (75.60%) promoters contained region-specific DNA methylation. We observed significant and distinct functional enrichment in genes with specifically methylated promoters in each epididymal regions, but these DNA methylations did not show significant correlation with repressed gene transcription in the mature epididymis. Conclusively, we investigated the regional miRNA expression and DNA methylation in the rat epididymis and revealed a potential role of miR-200 family in gene expression regulation between caput and cauda. This may contribute to the distinct physiological function in

  4. 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.

  5. Aberrant methylation of MUC1 and MUC4 promoters are potential prognostic biomarkers for pancreatic ductal adenocarcinomas

    PubMed Central

    Yokoyama, Seiya; Higashi, Michiyo; Kitamoto, Sho; Oeldorf, Monika; Knippschild, Uwe; Kornmann, Marko; Maemura, Kosei; Kurahara, Hiroshi; Wiest, Edwin; Hamada, Tomofumi; Kitazono, Ikumi; Goto, Yuko; Tasaki, Takashi; Hiraki, Tsubasa; Hatanaka, Kazuhito; Mataki, Yuko; Taguchi, Hiroki; Hashimoto, Shinichi; Batra, Surinder K.; Tanimoto, Akihide; Yonezawa, Suguru; Hollingsworth, Michael A.

    2016-01-01

    Pancreatic cancer is still a disease of high mortality despite availability of diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic neoplasms. MUC1 and MUC4 are high molecular weight transmembrane mucins. These are overexpressed in many carcinomas, and high expression of these molecules is a risk factor associated with poor prognosis. We evaluated the methylation status of MUC1 and MUC4 promoter regions in pancreatic tissue samples from 169 patients with various pancreatic lesions by the methylation specific electrophoresis (MSE) method. These results were compared with expression of MUC1 and MUC4, several DNA methylation/demethylation factors (e.g. ten-eleven translocation or TET, and activation-induced cytidine deaminase or AID) and CAIX (carbonic anhydrase IX, as a hypoxia biomarker). These results were also analyzed with clinicopathological features including time of overall survival of PDAC patients. We show that the DNA methylation status of the promoters of MUC1 and MUC4 in pancreatic tissue correlates with the expression of MUC1 and MUC4 mRNA. In addition, the expression of several DNA methylation/demethylation factors show a significant correlation with MUC1 and MUC4 methylation status. Furthermore, CAIX expression significantly correlates with the expression of MUC1 and MUC4. Interestingly, our results indicate that low methylation of MUC1 and/or MUC4 promoters correlates with decreased overall survival. This is the first report to show a relationship between MUC1 and/or MUC4 methylation status and prognosis. Analysis of epigenetic changes in mucin genes may be of diagnostic utility and one of the prognostic predictors for patients with PDAC. PMID:27283771

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

    PubMed

    Hong, Qingxiao; Chen, Xiaoying; Ye, Huadan; Zhou, Annan; Gao, Yuting; Jiang, Danjie; Wu, Xiaodong; Tian, Bingru; Chen, Youfen; Wang, Ming; Xie, Jiping; Xia, Yongming; Duan, Shiwei

    2016-04-01

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

  7. 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

  8. Sexual epigenetics: gender-specific methylation of a gene in the sex determining region of Populus balsamifera

    PubMed Central

    Bräutigam, Katharina; Soolanayakanahally, Raju; Champigny, Marc; Mansfield, Shawn; Douglas, Carl; Campbell, Malcolm M.; Cronk, Quentin

    2017-01-01

    Methylation has frequently been implicated in gender determination in plants. The recent discovery of the sex determining region (SDR) of balsam poplar, Populus balsamifera, pinpointed 13 genes with differentiated X and Y copies. We tested these genes for differential methylation using whole methylome sequencing of xylem tissue of multiple individuals grown under field conditions in two common gardens. The only SDR gene to show a marked pattern of gender-specific methylation is PbRR9, a member of the two component response regulator (type-A) gene family, involved in cytokinin signalling. It is an ortholog of Arabidopsis genes ARR16 and ARR17. The strongest patterns of differential methylation (mostly male-biased) are found in the putative promoter and the first intron. The 4th intron is strongly methylated in both sexes and the 5th intron is unmethylated in both sexes. Using a statistical learning algorithm we find that it is possible accurately to assign trees to gender using genome-wide methylation patterns alone. The strongest predictor is the region coincident with PbRR9, showing that this gene stands out against all genes in the genome in having the strongest sex-specific methylation pattern. We propose the hypothesis that PbRR9 has a direct, epigenetically mediated, role in poplar sex determination. PMID:28345647

  9. Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer–promoter interactions

    PubMed Central

    Forrester, William C.; Fernández, Luis A.; Grosschedl, Rudolf

    1999-01-01

    The immunoglobulin intragenic μ enhancer region acts as a locus control region that mediates transcriptional activation over large distances in germ line transformation assays. In transgenic mice, but not in transfected tissue culture cells, the activation of a variable region (VH) promoter by the μ enhancer is dependent on flanking nuclear matrix attachment regions (MARs). Here, we examine the effects of DNA methylation, which occurs in early mouse development, on the function of the μ enhancer and the MARs. We find that methylation of rearranged μ genes in vitro, before transfection, represses the ability of the μ enhancer to activate the VH promoter over the distance of 1.2 kb. However, methylation does not affect enhancer-mediated promoter activation over a distance of 150 bp. In methylated DNA templates, the μ enhancer alone induces only local chromatin remodeling, whereas in combination with MARs, the μ enhancer generates an extended domain of histone acetylation. These observations provide evidence that DNA methylation impairs the distance independence of enhancer function and thereby imposes a requirement for additional regulatory elements, such as MARs, which facilitate long-range chromatin remodeling. PMID:10580007

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing

    PubMed Central

    Zheng, Xianwu; Zhu, Jianhua; Kapoor, Avnish; Zhu, Jian-Kang

    2007-01-01

    Argonautes (AGOs) are conserved proteins that contain an RNA-binding PAZ domain and an RNase H-like PIWI domain. In Arabidopsis, except for AGO1, AGO4 and AGO7, the roles of seven other AGOs in gene silencing are not known. We found that a mutation in AGO6 partially suppresses transcriptional gene silencing in the DNA demethylase mutant ros1-1. In ago6-1ros1-1 plants, RD29A promoter short interfering RNAs (siRNAs) are less abundant, and cytosine methylation at both transgenic and endogenous RD29A promoters is reduced, compared to that in ros1-1. Interestingly, the ago4-1 mutation has a stronger suppression of the transcriptional silencing phenotype of ros1-1 mutant. Analysis of cytosine methylation at the endogenous MEA-ISR, AtREP2 and SIMPLEHAT2 loci revealed that the CpNpG and asymmetric methylation levels are lower in either of the ago6-1 and ago4-1 single mutants than those in the wild type, and the levels are the lowest in the ago6-1ago4-1 double mutant. These results suggest that AGO6 is important for the accumulation of specific heterochromatin-related siRNAs, and for DNA methylation and transcriptional gene silencing, this function is partly redundant with AGO4. PMID:17332757

  12. DNA methylation determines nucleosome occupancy in the 5'-CpG islands of tumor suppressor genes.

    PubMed

    Portela, A; Liz, J; Nogales, V; Setién, F; Villanueva, A; Esteller, M

    2013-11-21

    Promoter CpG island hypermethylation of tumor suppressor genes is an epigenetic hallmark of human cancer commonly associated with nucleosome occupancy and the transcriptional silencing of the neighboring gene. Nucleosomes can determine the underlying DNA methylation status. Herein, we show that the opposite is also true: DNA methylation can determine nucleosome positioning. Using a cancer model and digital nucleosome positioning techniques, we demonstrate that the induction of DNA hypomethylation events by genetic (DNMT1/DNMT3B deficient cells) or drug (a DNA demethylating agent) approaches is associated with the eviction of nucleosomes from previously hypermethylated CpG islands of tumor suppressor genes. Most importantly, the establishment of a stable cell line that restores DNMT1/DNMT3B deficiency shows that nucleosomes reoccupy their positions in de novo methylated CpG islands. Finally, we extend these results to the genomic level, combining a DNA methylation microarray and the nucleosome positioning technique. Using this global approach, we observe the dependency of nucleosome occupancy upon the DNA methylation status. Thus, our results suggest that there is a close association between hypermethylated CpG islands and the presence of nucleosomes, such that each of these epigenetic mechanisms can determine the recruitment of the other.

  13. 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.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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

  16. Integrated analysis of genome-wide DNA methylation and gene expression profiles identifies potential novel biomarkers of rectal cancer

    PubMed Central

    Zhang, Jinning; Zhou, Yuhui; Dang, Shuwei; Chen, Hongsheng; Wu, Qiong; Liu, Ming

    2016-01-01

    DNA methylation was regarded as the promising biomarker for rectal cancer diagnosis. However, the optimal methylation biomarkers with ideal diagnostic performance for rectal cancer are still limited. To identify new molecular markers for rectal cancer, we mapped DNA methylation and transcriptomic profiles in the six rectal cancer and paired normal samples. Further analysis revealed the hypermethylated probes in cancer prone to be located in gene promoter. Meanwhile, transcriptome analysis presented 773 low-expressed and 1,161 over-expressed genes in rectal cancer. Correction analysis identified a panel of 36 genes with an inverse correlation between methylation and gene expression levels, including 10 known colorectal cancer related genes. From the other 26 novel marker genes, GFRA1 and GSTM2 were selected for further analysis on the basis of their biological functions. Further experiment analysis confirmed their methylation and expression status in a larger number (44) of rectal cancer samples, and ROC curves showed higher AUC than SEPT9, which has been used as a biomarker in rectal cancer. Our data suggests that aberrant DNA methylation of contiguous CpG sites in methylation array may be potential diagnostic markers of rectal cancer. PMID:27566576

  17. Hypermethylation of Wnt antagonist gene promoters and activation of Wnt pathway in myelodysplastic marrow cells.

    PubMed

    Masala, Erico; Valencia, Ana; Buchi, Francesca; Nosi, Daniele; Spinelli, Elena; Gozzini, Antonella; Sassolini, Francesca; Sanna, Alessandro; Zecchi, Sandra; Bosi, Alberto; Santini, Valeria

    2012-10-01

    We observed aberrant gene methylation of Wnt antagonists: sFRP1, sFRP2, sFRP4, sFRP5 and DKK1 in marrow cells of 55 MDS cases. Methylation of Wnt antagonist genes was associated with activation of the Wnt signaling pathway, consistent with the up-regulation of the Wnt downstream genes TCF1 and LEF1. Azacitidine exposure induced demethylation of Wnt-antagonist gene promoters and reduction of the non-phosphorylated β-catenin (NPBC) which is prevalent during Wnt pathway inactivation. Presence of ≥5% of bone marrow blasts was associated with methylation of sFRP1 and DKK1 and with methylation of more than two of the five Wnt antagonist genes.

  18. MLH1 promoter methylation, diet, and lifestyle factors in mismatch repair deficient colorectal cancer patients from EPIC-Norfolk.

    PubMed

    Gay, Laura J; Arends, Mark J; Mitrou, Panagiota N; Bowman, Richard; Ibrahim, Ashraf E; Happerfield, Lisa; Luben, Robert; McTaggart, Alison; Ball, Richard Y; Rodwell, Sheila A

    2011-01-01

    There is conflicting evidence for the role diet and lifestyle play in the development of mismatch repair (MMR)-deficient colorectal cancers (CRC). In this study, associations between MMR deficiency, clinicopathological characteristics, and dietary and lifestyle factors in sporadic CRC were investigated. Tumor samples from 185 individuals in the EPIC-Norfolk study were analyzed for MLH1 gene promoter methylation and microsatellite instability (MSI). Dietary and lifestyle data were collected prospectively using 7-day food diaries (7dd) and questionnaires. MMR-deficient tumor cases (MLH1 promoter methylation positive, MSI-H) were more likely to be female, older at diagnosis, early Dukes' stage (A/B), and proximal in location (MSI-H P = 0.03, 0.03, 0.02, and 0.001, respectively). Tumors with positive MLH1 promoter methylation (>20%) were associated with poor differentiation (P = 0.03). Low physical activity was associated with cases without MSI (P = 0.05). MMR deficiency was not significantly associated with cigarette smoking or alcohol, folate, fruit, vegetable, or meat consumption. We conclude that MMR-deficient tumors represent a distinct subset of sporadic CRC that are proximal in location, early Dukes' stage, and poorly differentiated, in cases that are female and older at diagnosis. There is no overall role for diet and lifestyle in MMR status in CRC, consistent with age-related susceptibility to MLH1 promoter methylation.

  19. Unstable expression of transgene is associated with the methylation of CAG promoter in the offspring from the same litter of homozygous transgenic mice.

    PubMed

    Zhou, Yang; Zhang, Teng; Zhang, Qin-Kai; Jiang, Ying; Xu, Deng-Gao; Zhang, Min; Shen, Wei; Pan, Qing-Jie

    2014-08-01

    Transgenic animals have been established for studying gene function, improving animals' production traits, and providing organ models for the exploration of human diseases. However, the stability of inheritance and transgene expression in transgenic animals has gained extensive attention. The unstable expression of transgene through DNA methyltransferase (DNMT) targeting to the methylation of transgenic DNA such as CAG promoter and Egfp coding region in homozygous transgenic animals is still unknown. In the present study, the offspring from the same litter of homozygous transgenic mice carrying ubiquitously expressed enhanced green fluorescence protein driven by CMV early enhancer/chicken β-actin (CAG) promoter was observed to have unstable expression of transgene Egfp, quantitative PCR, western blot and bisulfite sequencing were conducted to quantify the expressional characteristics and methylation levels in various tissues. The correlation between transgene expression and methylation was analyzed. We have found that transgene expression is dependent on the methylation of CAG promoter, but not Egfp coding region. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the offspring from the same litter of homozygous transgenic mice.

  20. 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.

  1. Aberrant DNA methylation of cancer-related genes in giant breast fibroadenoma: a case report

    PubMed Central

    2011-01-01

    Introduction Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes) and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas. Case presentation A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44. Conclusion In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion. PMID:22011321

  2. MGMT promoter methylation in serum and cerebrospinal fluid as a tumor-specific biomarker of glioma.

    PubMed

    Wang, Zheng; Jiang, Wei; Wang, Yahong; Guo, Yang; Cong, Zheng; DU, Fangfang; Song, Bin

    2015-07-01

    O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation is a conventional technique to predict the prognosis or individualized treatment of glioma in tumor tissue following surgery or biopsy. However, the technique cannot be applied in those glioma patients with concomitant neurological dysfunctions or advanced age. The present study aimed to find a new minimally invasive and efficient alternative method for the detection of MGMT promoter methylation. The expression of MGMT promoter methylation was assessed in peripheral blood and cerebrospinal fluid (CSF), and compared to the corresponding tumor tissue from glioma patients. The 89 patients in the study [32 World Health Organization (WHO) grade II, 19 WHO grade III and 38 WHO grade IV) were pathologically-diagnosed glioma and received radiation therapy following sample collection. The resected glioma tumor tissue (89), corresponding serum (89) and CSF (78) samples were collected for the detection of MGMT promoter methylation using methylation-specific polymerase chain reaction. The sensitivity and specificity of detecting MGMT promoter methylation in CSF and serum were compared. Among the tumor tissue samples, 51/89 (57.3%) showed MGMT promoter methylation. The specificity of the detection in the CSF and serum samples reached 100%. The sensitivity of MGMT promoter methylation detection in CSF and serum were 26/40 (65.0%) and 19/51 (37.3%), respectively (P<0.05). In the WHO II, III and IV subgroups, the sensitivities of MGMT promoter methylation detection using CSF were 8/12 (66.7%), 11/18 (61.1%) and 7/10 (70.0%), respectively, which were significantly higher than the sensitivities using serum (7/21, 33.3%; 7/19, 36.8%; and 5/11, 45.5%, respectively P<0.05). Among patients with residual postoperative tumors, the sensitivities of detecting MGMT promoter methylation using CSF and serum were 18/25 (72.0%) and 10/24 (41.7%), respectively, both of which were significantly higher than the corresponding

  3. 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

  4. Altered promoter nucleosome positioning is an early event in gene silencing.

    PubMed

    Hesson, Luke B; Sloane, Mathew A; Wong, Jason Wh; Nunez, Andrea C; Srivastava, Sameer; Ng, Benedict; Hawkins, Nicholas J; Bourke, Michael J; Ward, Robyn L

    2014-10-01

    Gene silencing in cancer frequently involves hypermethylation and dense nucleosome occupancy across promoter regions. How a promoter transitions to this silent state is unclear. Using colorectal adenomas, we investigated nucleosome positioning, DNA methylation, and gene expression in the early stages of gene silencing. Genome-wide gene expression correlated with highly positioned nucleosomes upstream and downstream of a nucleosome-depleted transcription start site (TSS). Hypermethylated promoters displayed increased nucleosome occupancy, specifically at the TSS. We investigated 2 genes, CDH1 and CDKN2B, which were silenced in adenomas but lacked promoter hypermethylation. Instead, silencing correlated with loss of nucleosomes from the -2 position upstream of the TSS relative to normal mucosa. In contrast, permanent CDH1 silencing in carcinoma cells was characterized by promoter hypermethylation and dense nucleosome occupancy. Our findings suggest that silenced genes transition through an intermediary stage involving altered promoter nucleosome positioning, before permanent silencing by hypermethylation and dense nucleosome occupancy.

  5. Role of p14ARF and p15INK4B promoter methylation in patients with lung cancer: a systematic meta-analysis

    PubMed Central

    Yang, Xinmei; Yang, Lei; Dai, Wanrong; Ye, Bo

    2016-01-01

    Background The cyclin-dependent kinase inhibitors p14ARF and p15INK4B are tumor suppressor genes that have been reported to be silenced through promoter methylation in many human cancers. However, the strength of association between p14ARF or p15INK4B promoter methylation and lung cancer remains unclear. Thus, we first determined whether p14ARF and p15INK4B promoter methylation played a key role in the carcinogenesis of lung cancer. Methods Eligible studies were selected from the online electronic databases. The pooled odds ratios or hazard ratios and 95% confidence intervals were calculated and summarized. Results Finally, 12 studies with 625 lung cancer samples and 488 nontumor samples were included under the fixed-effects model. The pooled odds ratio showed that p14ARF promoter methylation was observed to be significantly higher in non-small-cell lung cancer (NSCLC) than in nontumor samples (P<0.001). No significant correlation was found between p15INK4B promoter methylation and lung cancer (P=0.27). Subgroup analysis of ethnicity revealed that p14ARF promoter methylation was significantly related to the risk of NSCLC in Asian and Caucasian populations. Subgroup analysis of sample type demonstrated that p14ARF promoter methylation was correlated with the risk of NSCLC in tissue samples (P<0.001), but not in bronchoalveolar lavage fluid and blood samples. P14ARF promoter methylation from one study was not significantly correlated with overall survival of patients with NSCLC. Promoter methylation of p14ARF and p15INK4B was not correlated with clinicopathological characteristics, such as gender status, smoking status, tumor differentiation, and tumor stage (P>0.05). Conclusion Our findings suggested that p14ARF promoter methylation may play an important role in the carcinogenesis of lung cancer, but not p15INK4B promoter methylation. Promoter methylation of p14ARF and p15INK4B was not associated with clinicopathological parameters. However, more extensive large

  6. X chromosome gene methylation in peripheral lymphocytes from monozygotic twins discordant for scleroderma

    PubMed Central

    Selmi, C; Feghali-Bostwick, C A; Lleo, A; Lombardi, S A; De Santis, M; Cavaciocchi, F; Zammataro, L; Mitchell, M M; LaSalle, J M; Medsger Jr, T; Gershwin, M E

    2012-01-01

    Scleroderma (SSc) is a rare connective tissue disease characterized by fibrosis, microvasculopathy and autoimmune features. The role of genetics is limited in SSc, as suggested by similar concordance rates in monozygotic and dizygotic twin pairs, while environmental factors may act through epigenetic changes, as demonstrated for specific genes. Further, sex chromosome changes have been reported in SSc and may explain the female preponderance. In the present study we compared the methylation profile of all X chromosome genes in peripheral blood mononuclear cells from monozygotic twins discordant (n = 7) and concordant (n = 1) for SSc. Methylated DNA immunoprecipitations from each discordant twin pair were hybridized to a custom-designed array included 998 sites encompassing promoters of all X chromosome genes and randomly chosen autosomal genes. Biostatistical tools identified sites with an elevated probability to be consistently hypermethylated (n = 18) or hypomethylated (n = 25) in affected twins. Identified genes include transcription factors (ARX, HSFX1, ZBED1, ZNF41) and surface antigens (IL1RAPL2, PGRMC1), and pathway analysis suggests their involvement in cell proliferation (PGK1, SMS, UTP14A, SSR4), apoptosis (MTM1), inflammation (ARAF) and oxidative stress (ENOX2). In conclusion, we propose that X chromosome genes with different methylation profiles in monozygotic twin pairs may constitute candidates for SSc susceptibility. PMID:22861365

  7. Changes in SCD gene DNA methylation after bariatric surgery in morbidly obese patients are associated with free fatty acids

    PubMed Central

    Morcillo, Sonsoles; Martín-Núñez, Gracia Mª; García-Serrano, Sara; Gutierrez-Repiso, Carolina; Rodriguez-Pacheco, Francisca; Valdes, Sergio; Gonzalo, Montserrat; Rojo-Martinez, Gemma; Moreno-Ruiz, Francisco J.; Rodriguez-Cañete, Alberto; Tinahones, Francisco; García-Fuentes, Eduardo

    2017-01-01

    Stearoyl CoA Desaturase-1 (SCD) is considered as playing an important role in the explanation of obesity. The aim of this study was to evaluate whether the DNA methylation SCD gene promoter is associated with the metabolic improvement in morbidly obese patients after bariatric surgery. The study included 120 subjects with morbid obesity who underwent a laparoscopic Roux-en Y gastric by-pass (RYGB) and a control group of 30 obese subjects with a similar body mass index (BMI) to that found in morbidly obese subjects six months after RYGB. Fasting blood samples were obtained before and at six months after RYGB. DNA methylation was measured by pyrosequencing technology. DNA methylation levels of the SCD gene promoter were lower in morbidly obese subjects before bariatric surgery but increased after RYGB to levels similar to those found in the control group. Changes of DNA methylation SCD gene were associated with the changes of free fatty acids levels (r = −0.442, p = 0.006) and HOMA-IR (r = −0.249, p = 0.035) after surgery. RYGB produces an increase in the low SCD methylation promoter levels found in morbidly obese subjects. This change of SCD methylation levels is associated with changes in FFA and HOMA-IR. PMID:28393901

  8. Methamphetamine and HIV-Tat Alter Murine Cardiac DNA Methylation and Gene Expression

    PubMed Central

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

    2015-01-01

    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

  9. 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.

  10. Racial differences in genome-wide methylation profiling and gene expression in breast tissues from healthy women

    PubMed Central

    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

  11. Alterations of the retinoblastoma and p16 pathway correlate with promoter methylation in malignant fibrous histiocytomas.

    PubMed

    Brinck, Ulrich; Schlott, Thilo; Störber, Steffi; Stachura, Jerzy; Bortkiewicz, Pawel; Nagel, Wolf-Dieter; Hasse, Frank Michael; Cordon-Cardo, Carlos; Fischer, Gösta; Korabiowska, Monika

    2006-01-01

    Recent reports indicate that the alterations in the p16 and pRb pathways can influence tumour progression and poor prognosis in several tumours. The objective of this study was to analyse p16 and pRb expression in161 patients with malignant fibrous histiocytomas (MFH). By immunohistochemistry, p16 and pRb were demonstrated in 25% and 56% of MFH, respectively. Cox regression analysis demonstrated an independent prognostic influence of both genes. Generally, the loss of p16 and pRb expression correlated with poorer prognosis. Promoter methylation of p16 was found in 16/42 of p16 negative MFH and of pRb in 2/42 of pRb-negative MFH. It can be concluded that p16 and pRb alterations play an important role in the progression of soft tissue sarcomas.

  12. The progeny of Arabidopsis thaliana plants exposed to salt exhibit changes in DNA methylation, histone modifications and gene expression.

    PubMed

    Bilichak, Andriy; Ilnystkyy, Yaroslav; Hollunder, Jens; Kovalchuk, Igor

    2012-01-01

    Plants are able to acclimate to new growth conditions on a relatively short time-scale. Recently, we showed that the progeny of plants exposed to various abiotic stresses exhibited changes in genome stability, methylation patterns and stress tolerance. Here, we performed a more detailed analysis of methylation patterns in the progeny of Arabidopsis thaliana (Arabidopsis) plants exposed to 25 and 75 mM sodium chloride. We found that the majority of gene promoters exhibiting changes in methylation were hypermethylated, and this group was overrepresented by regulators of the chromatin structure. The analysis of DNA methylation at gene bodies showed that hypermethylation in the progeny of stressed plants was primarily due to changes in the 5' and 3' ends as well as in exons rather than introns. All but one hypermethylated gene tested had lower gene expression. The analysis of histone modifications in the promoters and coding sequences showed that hypermethylation and lower gene expression correlated with the enrichment of H3K9me2 and depletion of H3K9ac histones. Thus, our work demonstrated a high degree of correlation between changes in DNA methylation, histone modifications and gene expression in the progeny of salt-stressed plants.

  13. Bivalent domains enforce transcriptional memory of DNA methylated genes in cancer cells.

    PubMed

    Rodriguez, Jairo; Muñoz, Mar; Vives, Laura; Frangou, Costas G; Groudine, Mark; Peinado, Miguel A

    2008-12-16

    Silencing of multiple cancer-related genes is associated with de novo methylation of linked CpG islands. Additionally, bivalent histone modification profiles characterized by the juxtaposition of active and inactive histone marks have been observed in genes that become hypermethylated in cancer. It is unknown how these ambiguous epigenetic states are maintained and how they interrelate with adjacent genomic regions with different epigenetic landscapes. Here, we present the analysis of a set of neighboring genes, including many frequently silenced in colon cancer cells, in a chromosomal region at 5q35.2 spanning 1.25 Mb. Promoter DNA methylation occurs only at genes maintained at a low transcriptional state and is characterized by the presence of bivalent histone marks, namely trimethylation of lysines 4 and 27 in histone 3. Chemically induced hyperacetylation and DNA demethylation lead to up-regulation of silenced genes in this locus yet do not resolve bivalent domains into a domain-wide active chromatin conformation. In contrast, active genes in the region become down-regulated after drug treatment, accompanied by a partial loss of chromatin domain boundaries and spreading of the inactive histone mark trimethylated lysine 27 in histone 3. Our results demonstrate that bivalent domains mark the promoters of genes that will become DNA methylated in adult tumor cells to enforce transcriptional silence. These bivalent domains not only remain upon drug induced gene reactivation, but also spread over adjacent CpG islands. These results may have important implications in understanding and managing epigenetic therapies of cancer.

  14. Age-dependent DNA methylation of genes that are suppressed in stem cells is a hallmark of cancer.

    PubMed

    Teschendorff, Andrew E; Menon, Usha; Gentry-Maharaj, Aleksandra; Ramus, Susan J; Weisenberger, Daniel J; Shen, Hui; Campan, Mihaela; Noushmehr, Houtan; Bell, Christopher G; Maxwell, A Peter; Savage, David A; Mueller-Holzner, Elisabeth; Marth, Christian; Kocjan, Gabrijela; Gayther, Simon A; Jones, Allison; Beck, Stephan; Wagner, Wolfgang; Laird, Peter W; Jacobs, Ian J; Widschwendter, Martin

    2010-04-01

    Polycomb group proteins (PCGs) are involved in repression of genes that are required for stem cell differentiation. Recently, it was shown that promoters of PCG target genes (PCGTs) are 12-fold more likely to be methylated in cancer than non-PCGTs. Age is the most important demographic risk factor for cancer, and we hypothesized that its carcinogenic potential may be referred by irreversibly stabilizing stem cell features. To test this, we analyzed the methylation status of over 27,000 CpGs mapping to promoters of approximately 14,000 genes in whole blood samples from 261 postmenopausal women. We demonstrate that stem cell PCGTs are far more likely to become methylated with age than non-targets (odds ratio = 5.3 [3.8-7.4], P < 10(-10)), independently of sex, tissue type, disease state, and methylation platform. We identified a specific subset of 69 PCGT CpGs that undergo hypermethylation with age and validated this methylation signature in seven independent data sets encompassing over 900 samples, including normal and cancer solid tissues and a population of bone marrow mesenchymal stem/stromal cells (P < 10(-5)). We find that the age-PCGT methylation signature is present in preneoplastic conditions and may drive gene expression changes associated with carcinogenesis. These findings shed substantial novel insights into the epigenetic effects of aging and support the view that age may predispose to malignant transformation by irreversibly stabilizing stem cell features.

  15. Impact of early developmental arsenic exposure on promotor CpG-island methylation of genes involved in neuronal plasticity.

    PubMed

    Martínez, Liborio; Jiménez, Verónica; García-Sepúlveda, Christian; Ceballos, Fátima; Delgado, Juan Manuel; Niño-Moreno, Perla; Doniz, Lesly; Saavedra-Alanís, Víctor; Castillo, Claudia G; Santoyo, Martha E; González-Amaro, Roberto; Jiménez-Capdeville, María E

    2011-04-01

    Epigenetic mechanisms are crucial to regulate the expression of different genes required for neuronal plasticity. Neurotoxic substances such as arsenic, which induces cognitive deficits in exposed children before any other manifestation of toxicity, could interfere with the epigenetic modulation of neuronal gene expression required for learning and memory. This study assessed in Wistar rats the effects that developmental arsenic exposure had on DNA methylation patterns in hippocampus and frontal cortex. Animals were exposed to arsenic in drinking water (3 and 36ppm) from gestation until 4 months of age, and DNA methylation in brain cells was determined by flow cytometry, immunohistochemistry and methylation-specific polymerase chain reaction (PCR) of the promoter regions of reelin (RELN) and protein phosphatase 1 (PP1) at 1, 2, 3 and 4 months of age. Immunoreactivity to 5 methyl-cytosine was significantly higher in the cortex and hippocampus of exposed animals compared to controls at 1 month, and DNA hypomethylation was observed the following months in the cortex at high arsenic exposure. Furthermore, we observed a significant increase in the non-methylated form of PP1 gene promoter at 2 and 3 months of age, either in cortex or hippocampus. In order to determine whether this exposure level is associated with memory deficits, a behavioral test was performed at the same age points, revealing progressive and dose-dependent deficits of fear memory. Our results demonstrate alterations of the methylation pattern of genes involved in neuronal plasticity in an animal model of memory deficit associated with arsenic exposure.

  16. Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas.

    PubMed

    Kleb, Brittany; Estécio, Marcos R H; Zhang, Jiexin; Tzelepi, Vassiliki; Chung, Woonbok; Jelinek, Jaroslav; Navone, Nora M; Tahir, Salahaldin; Marquez, Victor E; Issa, Jean-Pierre; Maity, Sankar; Aparicio, Ana

    2016-03-03

    Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor-derived xenografts (PDX) revealed that AR-negative SCPC (AR(-)SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR-positive castration-resistant adenocarcinomas (AR(+)ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR(-) and AR(+) PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR(-)SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR(-)SCPC cell lines. We conclude that the epigenome of AR(-) is distinct from that of AR(+) castration-resistant prostate carcinomas, and that the AR(-) phenotype can be reversed with epigenetic drugs.

  17. Activation of endocrine-related gene expression in placental choriocarcinoma cell lines following DNA methylation knock-down.

    PubMed

    Hogg, K; Robinson, W P; Beristain, A G

    2014-07-01

    Increasingly, placental DNA methylation is assessed as a factor in pregnancy-related complications, yet the transcriptional impact of such findings is not always clear. Using a proliferative in vitro placental model, the effect of DNA methylation loss on gene activation was evaluated at a number of genes selected for being differentially methylated in pre-eclampsia-associated placentae in vivo. We aimed to determine whether reduced DNA methylation at specific loci was associated with transcriptional changes at the corresponding gene, thus providing mechanistic underpinnings for previous clinical findings and to assess the degree of transcriptional response amongst our candidate genes. BeWo and JEG3 choriocarcinoma cells were exposed to 1 μM 5-Aza-2'-deoxycytidine (5-Aza-CdR) or vehicle control for 48 h, and re-plated and cultured for a further 72 h in normal media before cells were harvested for RNA and DNA. Bisulphite pyrosequencing confirmed that DNA methylation was reduced by ∼30-50% points at the selected loci studied in both cell lines. Gene activation, measured by qRT-PCR, was highly variable and transcript specific, indicating differential sensitivity to DNA methylation. Most notably, loss of DNA methylation at the leptin (LEP) promoter corresponded to a 200-fold and 40-fold increase in LEP expression in BeWo and JEG3 cells, respectively (P < 0.01). Transcripts of steroidogenic pathway enzymes CYP11A1 and HSD3B1 were up-regulated ∼40-fold in response to 5-Aza-CdR exposure in BeWo cells (P < 0.01). Other transcripts, including aromatase (CYP19), HSD11B2, inhibin (INHBA) and glucocorticoid receptor (NR3C1) were more moderately, although significantly, affected by loss of associated DNA methylation. These data present a mixed effect of DNA methylation changes at selected loci supporting cautionary interpretation of DNA methylation results in the absence of functional data.

  18. Effects of the social environment and stress on glucocorticoid receptor gene methylation: a systematic review

    PubMed Central

    Turecki, Gustavo; Meaney, Michael

    2015-01-01

    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 behaviour through epigenetic mechanisms. The hypothalamus-pituitary-adrenal (HPA) 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 Weaver et al published the initial findings 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 7 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. PMID:25687413

  19. 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.

  20. The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

    PubMed Central

    Tran, Stella; Wang, Ying; Lamba, Pankaj; Zhou, Xiang; Boehm, Ulrich; Bernard, Daniel J.

    2013-01-01

    Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5’ rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5’ flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5’ untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone. PMID:24098544

  1. Association between DNA Methylation of the BDNF Promoter Region and Clinical Presentation in Alzheimer's Disease

    PubMed Central

    Nagata, Tomoyuki; Kobayashi, Nobuyuki; Ishii, Jumpei; Shinagawa, Shunichiro; Nakayama, Ritsuko; Shibata, Nobuto; Kuerban, Bolati; Ohnuma, Tohru; Kondo, Kazuhiro; Arai, Heii; Yamada, Hisashi; Nakayama, Kazuhiko

    2015-01-01

    Background/Aims In the present study, we examined whether DNA methylation of the brain-derived neurotrophic factor (BDNF) promoter is associated with the manifestation and clinical presentation of Alzheimer's disease (AD). Methods Of 20 patients with AD and 20 age-matched normal controls (NCs), the DNA methylation of the BDNF promoter (measured using peripheral blood samples) was completely analyzed in 12 patients with AD and 6 NCs. The resulting methylation levels were compared statistically. Next, we investigated the correlation between the DNA methylation levels and the clinical presentation of AD. Results The total methylation ratio (in %) of the 20 CpG sites was significantly higher in the AD patients (5.08 ± 5.52%) than in the NCs (2.09 ± 0.81%; p < 0.05). Of the 20 CpG sites, the methylation level at the CpG4 site was significantly higher in the AD subjects than in the NCs (p < 0.05). Moreover, the methylation level was significantly and negatively correlated with some neuropsychological test subscores (registration, recall, and prehension behavior scores; p < 0.05). Conclusion These results suggest that the DNA methylation of the BDNF promoter may significantly influence the manifestation of AD and might be associated with its neurocognitive presentation. PMID:25873928

  2. Air pollution and gene-specific methylation in the Normative Aging Study: association, effect modification, and mediation analysis.

    PubMed

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

    2014-03-01

    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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Helicobacter pylori Infection Promotes Methylation and Silencing of Trefoil Factor 2, Leading to Gastric Tumor Development in Mice and Humans

    PubMed Central

    Peterson, Anthony J.; Menheniott, Trevelyan R.; O’Connor, Louise; Walduck, Anna K.; Fox, James G.; Kawakami, Kazuyuki; Minamoto, Toshinari; Ong, Eng Kok; Wang, Timothy C.; Judd, Louise M.; Giraud, Andrew S.

    2014-01-01

    BACKGROUND & AIMS Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. METHODS We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130F/F mouse model of gastric cancer. RESULTS In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130F/F/Tff2−/− mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130F/Fcontrols. The fundus of gp130F/F/Tff2−/− mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. CONCLUSIONS TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection. PMID:20801119

  8. 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

  9. Aberrant promoter CpG methylation as a molecular marker for disease monitoring in natural killer cell lymphomas.

    PubMed

    Siu, Lisa L P; Chan, John K C; Wong, Kit F; Choy, Carolyn; Kwong, Yok L

    2003-07-01

    Natural killer (NK) cell lymphomas lack suitable clonal markers for tumour cell detection, making the monitoring of minimal residual lymphoma difficult. Aberrant promoter CpG methylation occurs frequently in NK cell lymphomas. The objective of this study was to assess the potential of aberrant methylation as a surrogate tumour marker. Twenty-five primary tumours and 105 serial biopsies taken at various time points after treatment were examined using a methylation-specific polymerase chain reaction (MSP) for a panel of genes, comprising p73, p16, hMLH1, RARbeta and p15, previously shown to be methylated in NK cell lymphomas. All samples underwent independent morphological examination, supplemented by immunostaining for CD56 and in-situ hybridization for Epstein-Barr-virus-encoded RNA. Primary tumours showed the frequent methylation of the genes p73 (92%), p16 (71%), hMLH1 (61%), RARbeta (56%) and p15 (48%). MSP results in serial post-treatment biopsies were correlated with clinicopathological findings. Results were concordant in 89 follow-up samples (18 samples, histology positive/MSP positive; 71 samples, histology negative/MSP negative) and discordant in 16. Fifteen samples were histology negative/MSP positive, and tumour involvement was subsequently confirmed (positive re-biopsies or relapses at the same sites), indicating that MSP was more sensitive for minimal lymphoma detection. One sample was histology positive/MSP negative; a subsequent histological review and continuous clinical remission of the patient did not support tumour involvement. Our findings suggest that MSP for aberrantly methylated genes is a potentially valuable molecular marker for detecting either residual or relapsed disease in NK cell lymphoma patients.

  10. Firefly luciferase gene contains a cryptic promoter

    PubMed Central

    Vopálenský, Václav; Mašek, Tomáš; Horváth, Ondřej; Vicenová, Blanka; Mokrejš, Martin; Pospíšek, Martin

    2008-01-01

    A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10–16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferase-based assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now. PMID:18697919

  11. DNA-intercalators Causing Rapid Re-expression of Methylated and Silenced Genes in Cancer Cells

    PubMed Central

    Hossain, M. Zulfiquer; Healey, Megan A.; Lee, Calvin; Poh, Weijie; Yerram, Sashidhar R.; Patel, Kalpesh; Azad, Nilofer S.; Herman, James G.; Kern, Scott E.

    2013-01-01

    Epigenetic inactivation of tumor-suppressor and other regulatory genes plays a critical role in carcinogenesis. Transcriptional silencing is often maintained by DNA methyl transferase (DNMT)-mediated hypermethylation of CpG islands in promoter DNA. Nucleoside analogs including azacytidine and decitabine have been used to inhibit DNMT and re-activate genes, and are clinically used. Their shortcomings include a short half-life and a slow onset of action due to required nucleotide incorporation during DNA replication, which may limit clinical utility. It might be useful to begin to identify lead compounds having novel properties, specifically distinct and fast-acting gene desilencing. We previously identified chemicals augmenting gene expression in multiple reporter systems. We now report that a subset of these compounds that includes quinacrine re-expresses epigenetically silenced genes implicated in carcinogenesis. p16, TFPI2, the cadherins E-cadherin and CDH13, and the secreted frizzle-related proteins (SFRPs) SFRP1 and SFRP5 were desilenced in cancer cell lines. These lead compounds were fast-acting: re-expression occurred by 12-24 hours. Reactivation of silenced genes was accompanied by depletion of DNMT1 at the promoters of activated genes and demethylation of DNA. A model compound, 5175328, induced changes more rapidly than decitabine. These gene desilencing agents belonged to a class of acridine compounds, intercalated into DNA, and inhibited DNMT1 activity in vitro. Although to define the mechanism would be outside the scope of this initial report, this class may re-activate silenced genes in part by intercalating into DNA and subsequently inhibiting full DNMT1 activity. Rapid mechanisms for chemical desilencing of methylated genes therefore exist. PMID:23593653

  12. A prospective study of tumor suppressor gene methylation as a prognostic biomarker in surgically-resected stage I-IIIA non-small cell lung cancers

    PubMed Central

    Drilon, Alexander; Sugita, Hirofumi; Sima, Camelia S.; Zauderer, Marjorie; Rudin, Charles M.; Kris, Mark G.; Rusch, Valerie W.; Azzoli, Christopher G.

    2014-01-01

    Introduction While retrospective analyses support an association between early tumor recurrence and tumor suppressor gene (TSG) promoter methylation in early-stage non-small cell lung cancers (NSCLCs), few studies have investigated this question prospectively. Methods Primary tumor tissue from patients with resected pathologic stage I-IIIA NSCLCs was collected at the time of surgery and analyzed for promoter methylation via methylation-specific reverse-transcriptase polymerase chain reaction (MethyLight). The primary objective was to determine an association between promoter methylation of 10 individual TSGs (CDKN2A, CDH13, RASSF1, APC, MGMT, GSTP1, DAPK1, WIF1, SOCS3, and ADAMTS8) and recurrence-free survival (RFS), with the secondary objectives of determining association with overall survival (OS), and relation to clinical or pathologic features. Results 107 patients had sufficient tumor tissue for successful promoter methylation analysis. Majority of patients were former/current smokers (88%) with lung adenocarcinoma (78%) and pathologic stage I disease (66%). Median follow-up was 4 years. When controlled for pathologic stage, promoter methylation of the individual genes CDKN2A, CDH13, RASSF1, APC, MGMT, GSTP1, DAPK1, WIF1, and ADAMTS8 was not associated with RFS. Promoter methylation of the same genes was not associated with OS except for DAPK1 which was associated with improved OS (p=0.03). The total number of genes with methylated promoters did not correlate with RFS (p=0.89) or OS (p=0.55). Conclusions Contrary to data established by previous retrospective series, TSG promoter methylation (CDKN2A, CDH13, RASSF1,APC, MGMT, GSTP1, DAPK1, WIF1, and ADAMTS8) was not prognostic for early tumor recurrence in this prospective study of resected NSCLCs. PMID:25122424

  13. Mutation and methylation analysis of the chromodomain-helicase-DNA binding 5 gene in ovarian cancer.

    PubMed

    Gorringe, Kylie L; Choong, David Yh; Williams, Louise H; Ramakrishna, Manasa; Sridhar, Anita; Qiu, Wen; Bearfoot, Jennifer L; Campbell, Ian G

    2008-11-01

    Chromodomain, helicase, DNA binding 5 (CHD5) is a member of a subclass of the chromatin remodeling Swi/Snf proteins and has recently been proposed as a tumor suppressor in a diverse range of human cancers. We analyzed all 41 coding exons of CHD5 for somatic mutations in 123 primary ovarian cancers as well as 60 primary breast cancers using high-resolution melt analysis. We also examined methylation of the CHD5 promoter in 48 ovarian cancer samples by methylation-specific single-stranded conformation polymorphism and bisulfite sequencing. In contrast to previous studies, no mutations were identified in the breast cancers, but somatic heterozygous missense mutations were identified in 3 of 123 ovarian cancers. We identified promoter methylation in 3 of 45 samples with normal CHD5 and in 2 of 3 samples with CHD5 mutation, suggesting these tumors may have biallelic inactivation of CHD5. Hemizygous copy number loss at CHD5 occurred in 6 of 85 samples as assessed by single nucleotide polymorphism array. Tumors with CHD5 mutation or methylation were more likely to have mutation of KRAS or BRAF (P = .04). The aggregate frequency of CHD5 haploinsufficiency or inactivation is 16.2% in ovarian cancer. Thus, CHD5 may play a role as a tumor suppressor gene in ovarian cancer; however, it is likely that there is another target of the frequent copy number neutral loss of heterozygosity observed at 1p36.

  14. Role of CDH13 promoter methylation in the carcinogenesis, progression, and prognosis of colorectal cancer

    PubMed Central

    Ye, Meng; Huang, Tao; Li, Jinyun; Zhou, Chongchang; Yang, Ping; Ni, Chao; Chen, Si

    2017-01-01

    Abstract Background: H-cadherin (CDH13) is commonly downregulated through promoter methylation in various cancers. However, the role of CDH13 promoter methylation status in patients with colorectal cancer (CRC) remains to be clarified. Methods: Eligible articles were identified from online electronic database based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement criteria. The pooled odds ratio (OR) and the corresponding 95% confidence interval (95% CI) were calculated and analyzed. Results: Eventually, a total of nine studies were included in this meta-analysis, including 488 CRC, 298 adjacent, 144 normal, 68 premalignant tissues. The results demonstrated that CDH13 promoter methylation was notably higher in CRC than in normal, adjacent, and premalignant tissues (cancer tissues vs normal tissues: OR = 16.94, P < 0.001; cancer tissues vs adjacent tissues: OR = 20.06, P < 0.001; cancer tissues vs premalignant tissues: OR = 2.23, P = 0.038). CDH13 promoter methylation had a significantly increased risk for poorly differentiated CRC (OR = 4.07, P = 0.001). CDH13 promoter methylation was not associated with sex status, tumor stage, and lymph node status (all P > 0.05). One study with 85 CRC patients reported that CDH13 promoter methylation was correlated with poor prognosis in overall survival (OS). Conclusions: CDH13 promoter methylation may play an important role in the initiation and progression of CRC, and may be correlated with OS of patients with CRC. Additional studies with large sample sizes are needed to further confirm our findings in the future. PMID:28121942

  15. Promoter methylation of PCDH10 by HOTAIR regulates the progression of gastrointestinal stromal tumors

    PubMed Central

    Lee, Na Keum; Lee, Jung Hwa; Kim, Won Kyu; Yun, Seongju; Youn, Young Hoon; Park, Chan Hyuk; Choi, Yun Young; Kim, Hogeun; Lee, Sang Kil

    2016-01-01

    HOTAIR, a long non-coding RNA (lncRNA), plays a crucial role in tumor initiation and metastasis by interacting with the PRC2 complex and the modulation of its target genes. The role of HOTAIR in gastrointestinal stromal tumors (GISTs) is remains unclear. Herein we investigate the mechanism of HOTAIR in the genesis and promotion of GISTs. The expression of HOTAIR was found to be higher in surgically resected high-risk GISTs than that in low- and intermediate-risk GISTs. Using GIST-T1 and GIST882 cells, we demonstrated that HOTAIR repressed apoptosis, was associated with cell cycle progression, and controlled the invasion and migration of GIST cells. Using a gene expression microarray and lists of HOTAIR-associated candidate genes, we suggested that protocadherin 10 (PCDH10) is a key molecule. PCDH10 expression was significantly decreased in GIST-T1 and GIST882 cells, possibly as a consequence of hypermethylation. We observed that HOTAIR induced PCDH10 methylation in a SUZ12-dependent manner. In this study, we found that the malignant character of GISTs was initiated and amplified by PCDH10 in a process regulated by HOTAIR. In summary, our findings imply that PCDH10 and HOTAIR may be useful markers of disease progression and therapeutic targets. PMID:27659532

  16. The effect of smoking on MAOA promoter methylation in DNA prepared from lymphoblasts and whole blood.

    PubMed

    Philibert, Robert A; Beach, Steven R H; Gunter, Tracy D; Brody, Gene H; Madan, Anup; Gerrard, Meg

    2010-03-05

    Prior work using lymphoblast DNA prepared from 192 subjects from the Iowa Adoption Studies (IAS) demonstrated that decreased MAOA promoter methylation was associated with lifetime symptom count for nicotine dependence (ND) and provided suggestive evidence that the amount of methylation is genotype dependent. In the current investigation, we replicate and extend these prior findings in three ways using another 289 IAS subjects and the same methodologies. First, we show that methylation is dependent on current smoking status. Second, we introduce a factor analytic approach to DNA methylation, highlighting three distinct regions of the promoter that may function in somewhat different ways for males and females. Third, we directly compare the methylation signatures in DNA prepared from whole blood and lymphoblasts from a subset of these subjects and provide suggestive evidence favoring the use of lymphoblast DNA. We conclude that smoking reliably decreases MAOA methylation, but exact characterization of effects on level of methylation depend on genotype, smoking history, current smoking status, gender, and region of the promoter-associated CpG Island examined.

  17. 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

  18. Association of weight regain with specific methylation levels in the NPY and POMC promoters in leukocytes of obese men: a translational study.

    PubMed

    Crujeiras, Ana B; Campion, Javier; Díaz-Lagares, Angel; Milagro, Fermin I; Goyenechea, Estíbaliz; Abete, Itziar; Casanueva, Felipe F; Martínez, J Alfredo

    2013-09-10

    Specific methylation of appetite-related genes in leukocytes could serve as a useful biomarker to predict weight regain after an energy restriction program. We aimed to evaluate whether the pre-intervention DNA methylation patterns involved in the epigenetic control of appetite-regulatory genes in leukocytes are associated with the weight regain process. Eighteen men who lost ≥5% of body weight after an 8-week nutritional intervention were categorized as "regainers" (≥10% weight regain) and "non-regainers" (<10% weight regain) 32weeks after stopping dieting. At baseline, leukocytes were isolated and DNA was analyzed for epigenetic methylation patterns of appetite-related gene promoters by MALDI-TOF mass spectrometry. Regainers showed higher methylation levels than non-regainers in proopiomelanocortin (POMC) CpG sites +136bp and +138bp (fold change from non-regainers=26%; p=0.020) and lower methylation of the whole analyzed region of neuropeptide Y (NPY; fold change from non-regainers=-22%; p=0.033), as well as of several individual NPY-promoter CpG sites. Importantly, total baseline NPY methylation was associated with weight-loss regain (r=-0.76; p<0.001), baseline plasma ghrelin levels (r=0.60; p=0.011) and leptin/ghrelin ratio (r=-0.52; p=0.046). Lower methylation levels of POMC CpG sites +136bp and +138bp were associated with success in weight-loss maintenance (odds ratio=0.042 [95% CI 0.01-0.57]; p=0.018), whereas lower total methylation levels in NPY promoter were associated with higher risk of weight regain (odds ratio=14.0 [95% CI 1.13-172]; p=0.039). Therefore, the study of leukocyte methylation levels reflects a putative epigenetic regulation of NPY and POMC, which might be implicated in the weight regain process and be used as biomarkers for predicting weight regain after dieting.

  19. The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation.

    PubMed Central

    Sheldon, C C; Burn, J E; Perez, P P; Metzger, J; Edwards, J A; Peacock, W J; Dennis, E S

    1999-01-01

    A MADS box gene, FLF (for FLOWERING LOCUS F ), isolated from a late-flowering, T-DNA-tagged Arabidopsis mutant, is a semidominant gene encoding a repressor of flowering. The FLF gene appears to integrate the vernalization-dependent and autonomous flowering pathways because its expression is regulated by genes in both pathways. The level of FLF mRNA is downregulated by vernalization and by a decrease in genomic DNA methylation, which is consistent with our previous suggestion that vernalization acts to induce flowering through changes in gene activity that are mediated through a reduction in DNA methylation. The flf-1 mutant requires a greater than normal amount of an exogenous gibberellin (GA3) to decrease flowering time compared with the wild type or with vernalization-responsive late-flowering mutants, suggesting that the FLF gene product may block the promotion of flowering by GAs. FLF maps to a region on chromosome 5 near the FLOWERING LOCUS C gene, which is a semidominant repressor of flowering in late-flowering ecotypes of Arabidopsis. PMID:10072403

  20. 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.

  1. 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.

  2. Methylation-mediated gene silencing as biomarkers of gastric cancer: A review

    PubMed Central

    Nakamura, Jun; Tanaka, Tomokazu; Kitajima, Yoshihiko; Noshiro, Hirokazu; Miyazaki, Kohji

    2014-01-01

    Despite a decline in the overall incidence of gastric cancer (GC), the disease remains the second most common cause of cancer-related death worldwide and is thus a significant global health problem. The best means of improving the survival of GC patients is to screen for and treat early lesions. However, GC is often diagnosed at an advanced stage and is associated with a poor prognosis. Current diagnostic and therapeutic strategies have not been successful in decreasing the global burden of the disease; therefore, the identification of reliable biomarkers for an early diagnosis, predictive markers of recurrence and survival and markers of drug sensitivity and/or resistance is urgently needed. The initiation and progression of GC depends not only on genetic alterations but also epigenetic changes, such as DNA methylation and histone modification. Aberrant DNA methylation is the most well-defined epigenetic change in human cancers and is associated with inappropriate gene silencing. Therefore, an increasing number of genes methylated at the promoter region have been targeted as possible biomarkers for different purposes, including early detection, classification, the assessment of the tumor prognosis, the development of therapeutic strategies and patient follow-up. This review article summarizes the current understanding and recent evidence regarding DNA methylation markers in GC with a focus on the clinical potential of these markers. PMID:25232236

  3. Antipsychotic drugs attenuate aberrant DNA methylation of DTNBP1 (dysbindin) promoter in saliva and post-mortem brain of patients with schizophrenia and Psychotic bipolar disorder.

    PubMed

    Abdolmaleky, Hamid M; Pajouhanfar, Sara; Faghankhani, Masoomeh; Joghataei, Mohammad Taghi; Mostafavi, Ashraf; Thiagalingam, Sam

    2015-12-01

    Due to the lack of genetic association between individual genes and schizophrenia (SCZ) pathogenesis, the current consensus is to consider both genetic and epigenetic alterations. Here, we report the examination of DNA methylation status of DTNBP1 promoter region, one of the most credible candidate genes affected in SCZ, assayed in saliva and post-mortem brain samples. The Illumina DNA methylation profiling and bisulfite sequencing of representative samples were used to identify methylation status of the DTNBP1 promoter region. Quantitative methylation specific PCR (qMSP) was employed to assess methylation of DTNBP1 promoter CpGs flanking a SP1 binding site in the saliva of SCZ patients, their first-degree relatives and control subjects (30, 15, and 30/group, respectively) as well as in post-mortem brains of patients with SCZ and bipolar disorder (BD) versus controls (35/group). qRT-PCR was used to assess DTNBP1 expression. We found DNA hypermethylation of DTNBP1 promoter in the saliva of SCZ patients (∼12.5%, P = 0.036), particularly in drug-naïve patients (∼20%, P = 0.011), and a trend toward hypermethylation in their first-degree relatives (P = 0.085) versus controls. Analysis of post-mortem brain samples revealed an inverse correlation between DTNBP1 methylation and expression, and normalization of this epigenetic change by classic antipsychotic drugs. Additionally, BD patients with psychotic depression exhibited higher degree of methylation versus other BD patients (∼80%, P = 0.025). DTNBP1 promoter DNA methylation may become a key element in a panel of biomarkers for diagnosis, prevention, or therapy in SCZ and at risk individuals pending confirmatory studies with larger sample sizes to attain a higher degree of significance.

  4. 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

  5. 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

  6. DNA Methylation and Gene Regulation in Honeybees: From Genome-Wide Analyses to Obligatory Epialleles.

    PubMed

    Wedd, Laura; Maleszka, Ryszard

    2016-01-01

    In contrast to heavily methylated mammalian genomes, invertebrate genomes are only sparsely methylated in a 'mosaic' fashion with the majority of methylated CpG dinucleotides found across gene bodies. Importantly, this gene body methylation is frequently associated with active transcription, and studies in the honeybee have shown that there are strong links between gene body methylation and alternative splicing. Additional work also highlights that obligatory methylated epialleles influence transcriptional changes in a context-specific manner. Here we discuss the current knowledge in this emerging field and highlight both similarities and differences between DNA methylation systems in mammals and invertebrates. Finally, we argue that the relationship between genetic variation, differential DNA methylation, other epigenetic modifications and the transcriptome must be further explored to fully understand the role of DNA methylation in converting genomic sequences into phenotypes.

  7. 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

    2015-06-04

    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.

  8. Prognostic value of three different methods of MGMT promoter methylation analysis in a prospective trial on newly diagnosed glioblastoma.

    PubMed

    Christians, Arne; Hartmann, Christian; Benner, Axel; Meyer, Jochen; von Deimling, Andreas; Weller, Michael; Wick, Wolfgang; Weiler, Markus

    2012-01-01

    Hypermethylation in the promoter region of the MGMT gene encoding the DNA repair protein O(6)-methylguanine-DNA methyltransferase is among the most important prognostic factors for patients with glioblastoma and predicts response to treatment with alkylating agents like temozolomide. Hence, the MGMT status is widely determined in most clinical trials and frequently requested in routine diagnostics of glioblastoma. Since various different techniques are available for MGMT promoter methylation analysis, a generally accepted consensus as to the most suitable diagnostic method remains an unmet need. Here, we assessed methylation-specific polymerase chain reaction (MSP) as a qualitative and semi-quantitative method, pyrosequencing (PSQ) as a quantitative method, and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a semi-quantitative method in a series of 35 formalin-fixed, paraffin-embedded glioblastoma tissues derived from patients treated in a prospective clinical phase II trial that tested up-front chemoradiotherapy with dose-intensified temozolomide (UKT-05). Our goal was to determine which of these three diagnostic methods provides the most accurate prediction of progression-free survival (PFS). The MGMT promoter methylation status was assessable by each method in almost all cases (n = 33/35 for MSP; n = 35/35 for PSQ; n = 34/35 for MS-MLPA). We were able to calculate significant cut-points for the continuous methylation signals at each CpG site analysed by PSQ (range, 11.5 to 44.9%) and at one CpG site assessed by MS-MLPA (3.6%) indicating that a dichotomisation of continuous methylation data as a prerequisite for comparative survival analyses is feasible. Our results show that, unlike MS-MLPA, MSP and PSQ provide a significant improvement of predicting PFS compared with established clinical prognostic factors alone (likelihood ratio tests: p<0.001). Conclusively, taking into consideration prognostic value, cost

  9. Expression of the TIMP2 gene is not regulated by promoter hypermethylation in the Caski cell line.

    PubMed

    Parashar, Gaurav; Capalash, Neena

    2012-05-01

    Promoter hypermethylation has been linked to loss of expression of tumor suppressor genes in various types of tumors. A strong reciprocal correlation between promoter hypermethylation and expression of the TIMP2 gene was observed in the Caski cell line. The TIMP2 promoter was found to be methylated within the 1919 and 1987 region (-325 to -257), relative to the transcription start site through methylation-specific PCR in the HeLa, SiHa and Caski cervical cancer cell lines. However, a reverse transcription PCR analysis of the TIMP2 gene confirmed a normal expression in the HeLa and SiHa cell lines with a high expression in the Caski cell line, indicating that expression of the TIMP2 gene is independent of methylation of CpG sites located within the -325 to -257 region of the TIMP2 promoter, relative to the transcription start site.

  10. 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

  11. Promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in human cancer cell lines, multidrug-resistant cell models and tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients

    PubMed Central

    Spitzwieser, Melanie; Pirker, Christine; Koblmüller, Bettina; Pfeiler, Georg; Hacker, Stefan; Berger, Walter; Heffeter, Petra; Cichna-Markl, Margit

    2016-01-01

    Overexpression of ABCB1, ABCC1 and ABCG2 in tumor tissues is considered a major cause of limited efficacy of anticancer drugs. Gene expression of ABC transporters is regulated by multiple mechanisms, including changes in the DNA methylation status. Most of the studies published so far only report promoter methylation levels for either ABCB1 or ABCG2, and data on the methylation status for ABCC1 are scarce. Thus, we determined the promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in 19 human cancer cell lines. In order to contribute to the elucidation of the role of DNA methylation changes in acquisition of a multidrug resistant (MDR) phenotype, we also analyzed the promoter methylation patterns in drug-resistant sublines of the cancer cell lines GLC-4, SW1573, KB-3-1 and HL-60. In addition, we investigated if aberrant promoter methylation levels of ABCB1, ABCC1 and ABCG2 occur in tumor and tumor-surrounding tissues from breast cancer patients. Our data indicates that hypomethylation of the ABCC1 promoter is not cancer type-specific but occurs in cancer cell lines of different origins. Promoter methylation was found to be an important mechanism in gene regulation of ABCB1 in parental cancer cell lines and their drug-resistant sublines. Overexpression of ABCC1 in MDR cell models turned out to be mediated by gene amplification, not by changes in the promoter methylation status of ABCC1. In contrast to the promoters of ABCC1 and ABCG2, the promoter of ABCB1 was significantly higher methylated in tumor tissues than in tumor-adjacent and tumor-distant tissues from breast cancer patients. PMID:27689338

  12. Isolation of Methyl Parathion-Degrading Strain M6 and Cloning of the Methyl Parathion Hydrolase Gene

    PubMed Central

    Zhongli, Cui; Shunpeng, Li; Guoping, Fu

    2001-01-01

    A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp. strain M6 was able to hydrolyze methyl parathion to p-nitrophenol. A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the mpd gene was determined. The gene could be effectively expressed in Esherichia coli. PMID:11571204

  13. Impact of the location of CpG methylation within the GSTP1 gene on its specificity as a DNA marker for hepatocellular carcinoma.

    PubMed

    Jain, Surbhi; Chen, Sitong; Chang, Kung-Chao; Lin, Yih-Jyh; Hu, Chi-Tan; Boldbaatar, Batbold; Hamilton, James P; Lin, Selena Y; Chang, Ting-Tsung; Chen, Shun-Hua; Song, Wei; Meltzer, Stephen J; Block, Timothy M; Su, Ying-Hsiu

    2012-01-01

    Hypermethylation of the glutathione S-transferase π 1 (GSTP1) gene promoter region has been reported to be a potential biomarker to distinguish hepatocellular carcinoma (HCC) from other liver diseases. However, reports regarding how specific a marker it is have ranged from 100% to 0%. We hypothesized that, to a large extent, the variation of specificity depends on the location of the CpG sites analyzed. To test this hypothesis, we compared the methylation status of the GSTP1 promoter region of the DNA isolated from HCC, cirrhosis, hepatitis, and normal liver tissues by bisulfite-PCR sequencing. We found that the 5' region of the position -48 nt from the transcription start site of the GSTP1 gene is selectively methylated in HCC, whereas the 3' region is methylated in all liver tissues examined, including normal liver and the HCC tissue. Interestingly, when DNA derived from fetal liver and 11 nonhepatic normal tissue was also examined by bisulfite-PCR sequencing, we found that methylation of the 3' region of the promoter appeared to be liver-specific. A methylation-specific PCR assay targeting the 5' region of the promoter was developed and used to quantify the methylated GSTP1 gene in various diseased liver tissues including HCC. When we used an assay targeting the 3' region, we found that the methylation of the 5'-end of the GSTP1 promoter was significantly more specific than that of the 3'-end (97.1% vs. 60%, p<0.0001 by Fisher's exact test) for distinguishing HCC (n = 120) from hepatitis (n = 35) and cirrhosis (n = 35). Encouragingly, 33.8% of the AFP-negative HCC contained the methylated GSTP1 gene. This study clearly demonstrates the importance of the location of CpG site methylation for HCC specificity and how liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC.

  14. Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages

    PubMed Central

    Navasa, Nicolás; Martin-Ruiz, Itziar; Atondo, Estíbaliz; Sutherland, James D.; Angel Pascual-Itoiz, Miguel; Carreras-González, Ana; Izadi, Hooman; Tomás-Cortázar, Julen; Ayaz, Furkan; Martin-Martin, Natalia; Torres, Iviana M; Barrio, Rosa; Carracedo, Arkaitz; Olivera, Elias R.; Rincón, Mercedes; Anguita, Juan

    2015-01-01

    MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions. PMID:26419808

  15. 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

  16. CG methylation.

    PubMed

    Vinson, Charles; Chatterjee, Raghunath

    2012-12-01

    A striking feature of mammalian genomes is the paucity of the CG dinucleotide. There are approximately 20,000 regions termed CpG islands where CGs cluster. This represents 5% of all CGs and 1% of the genome. CpG islands are typically unmethylated and are often promoters for housekeeping genes. The remaining 95% of CG dinucleotides are disposed throughout 99% of the genome and are typically methylated and found in half of all promoters. CG methylation facilitates binding of the C/EBP family of transcription factors, proteins critical for differentiation of many tissues. This allows these proteins to localize in the methylated CG poor regions of the genome where they may produce advantageous changes in gene expression at nearby or more distant regions of the genome. In this review, our growing understanding of the consequences of CG methylation will be surveyed.

  17. Alternative promoters of gene MAGE4a

    SciTech Connect

    De Plaen, E.; Naerhuyzen, B.; De Smet, C.

    1997-03-01

    Gene MAGE-4 (HGMW-approved symbol MAGE4) is expressed in several types of tumors, but not in normal tissues, except testis and placenta. The 5{prime} end of this gene contains eight homologous exons spread over a 5.8-kb region. These exons are alternatively spliced to a unique second exon and a unique third exon, which encodes a protein of 317 amino acids. The analysis of transcripts found in testis, placenta, and a sarcoma cell line showed that each of the alternative first exons is used in at least one of these tissues. Various regions of the promoter of the fifth alternative exon (1.5) were cloned in a luciferase reporter plasmid, and the constructs were transfected in a sarcoma cell line that expresses MAGE-4. Two Ets motifs located between positions -70 and -29 relative to the transcription start site were found to drive 55% of the promoter activity. A region containing an Sp1 consensus binding site located upstream of the two Ets motifs was found to be responsible for 44% of the transcriptional activity. MAGE-4a promoters 1.4 and 1.6, which also contain the Sp1 and the two Ets binding motifs, supported a level of transcription comparable to that of promoter 1.5, whereas promoter 1.1, which contains only one Ets binding site, was sixfold less active. In line with observations made with gene MAGE-1 (HGMW-approved symbol MAGE1), we found that promoter 1.5 stimulated a high level of transcription in a melanoma cell line that does not express MAGE-4. This suggests that the tumor-specific expression of MAGE genes is not determined by the presence of specific transcription factors. 26 refs., 7 figs., 2 tabs.

  18. Determining the effect of DNA methylation on gene expression in cancer cells.

    PubMed

    Lee, Chai-Jin; Evans, Jared; Kim, Kwangsoo; Chae, Heejoon; Kim, Sun

    2014-01-01

    DNA methylation, a DNA modification by adding methyl group to cytosine, has an important role in the regulation of gene expression. DNA methylation is known to be associated with gene transcription by interfering with DNA-binding proteins, such as transcription factors. DNA methylation is closely related to tumorigenesis, and the methylation state of some genes can be used as a biomarker for tumorigenesis. Aberrant DNA methylation of genomic regions, including CpG islands, CpG shores, and first exons, is related to the altered gene expression pattern characteristics of all human cancers. Subheading 1 surveys recent developments on DNA methylation and gene expressions in cancer. Then we provide analysis of DNA methylation and gene expression in 30 breast cancer cell lines representing different tumor phenotypes. This study conducted an integrated analysis to identify the relationship between DNA methylation in various genomic regions and expression levels of downstream genes, using MethylCapseq data (affinity purification followed by next-generation sequencing of eluted DNA) and Affymetrix gene expression microarray data. The goal of this study was to assess genome-wide methylation profiles associated with different molecular subtypes of human breast cancer (luminal, basal A, and basal B) and to comprehensively investigate the effect of DNA methylation on gene expression in breast cancer phenotypes. This showed that methylation of genomic regions near transcription start sites, CpG island, CpG shore, and first exon was strongly associated with gene repression, and the effects of the regions on gene expression patterns were different for different molecular subtypes of breast cancer. The results further indicated that aberrant methylation of specific genomic regions was significantly associated with different breast cancer subtypes.

  19. Loss of the polycomb mark from bivalent promoters leads to activation of cancer-promoting genes in colorectal tumors.

    PubMed

    Hahn, Maria A; Li, Arthur X; Wu, Xiwei; Yang, Richard; Drew, David A; Rosenberg, Daniel W; Pfeifer, Gerd P

    2014-07-01

    In colon tumors, the transcription of many genes becomes deregulated by poorly defined epigenetic mechanisms that have been studied mainly in established cell lines. In this study, we used frozen human colon tissues to analyze patterns of histone modification and DNA cytosine methylation in cancer and matched normal mucosa specimens. DNA methylation is strongly targeted to bivalent H3K4me3- and H3K27me3-associated promoters, which lose both histone marks and acquire DNA methylation. However, we found that loss of the Polycomb mark H3K27me3 from bivalent promoters was accompanied often by activation of genes associated with cancer progression, including numerous stem cell regulators, oncogenes, and proliferation-associated genes. Indeed, we found many of these same genes were also activated in patients with ulcerative colitis where chronic inflammation predisposes them to colon cancer. Based on our findings, we propose that a loss of Polycomb repression at bivalent genes combined with an ensuing selection for tumor-driving events plays a major role in cancer progression.

  20. 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.

  1. Gene body methylation is conserved between plant orthologs and is of evolutionary consequence

    PubMed Central

    Takuno, Shohei; Gaut, Brandon S.

    2013-01-01

    DNA methylation is a common feature of eukaryotic genomes and is especially common in noncoding regions of plants. Protein coding regions of plants are often methylated also, but the extent, function, and evolutionary consequences of gene body methylation remain unclear. Here we investigate gene body methylation using an explicit comparative evolutionary approach. We generated bisulfite sequencing data from two tissues of Brachypodium distachyon and compared genic methylation patterns to those of rice (Oryza sativa ssp. japonica). Gene body methylation was strongly conserved between orthologs of the two species and affected a biased subset of long, slowly evolving genes. Because gene body methylation is conserved over evolutionary time, it shapes important features of plant genome evolution, such as the bimodality of G+C content among grass genes. Our results superficially contradict previous observations of high cytosine methylation polymorphism within Arabidopsis thaliana genes, but reanalyses of these data are consistent with conservation of methylation within gene regions. Overall, our results indicate that the methylation level is a long-term property of individual genes and therefore of evolutionary consequence. PMID:23319627

  2. Gene expression and nucleotide composition are associated with genic methylation level in Oryza sativa

    PubMed Central

    2014-01-01

    Background The methylation of cytosines at CpG dinucleotides, which plays an important role in gene expression regulation, is one of the most studied epigenetic modifications. Thus far, the detection of DNA methylation has been determined mostly by experimental methods, which are not only prone to bench effects and artifacts but are also time-consuming, expensive, and cannot be easily scaled up to many samples. It is therefore useful to develop computational prediction methods for DNA methylation. Our previous studies highlighted the existence of correlations between the GC content of the third codon position (GC3), methylation, and gene expression. We thus designed a model to predict methylation in Oryza sativa based on genomic sequence features and gene expression data. Results We first derive equations to describe the relationship between gene methylation levels, GC3, expression, length, and other gene compositional features. We next assess gene compositional features involving sixmers and their association with methylation levels and other gene level properties. By applying our sixmer-based approach on rice gene expression data we show that it can accurately predict methylation (Pearson’s correlation coefficient r = 0.79) for the majority (79%) of the genes. Matlab code with our model is included. Conclusions Gene expression variation can be used as predictors of gene methylation levels. PMID:24447369

  3. Gene body methylation shows distinct patterns associated with different gene origins and duplication modes and has a heterogeneous relationship with gene expression in Oryza sativa (rice).

    PubMed

    Wang, Yupeng; Wang, Xiyin; Lee, Tae-Ho; Mansoor, Shahid; Paterson, Andrew H

    2013-04-01

    Whole-genome duplication (WGD) has been recurring and single-gene duplication is also widespread in angiosperms. Recent whole-genome DNA methylation maps indicate that gene body methylation (i.e. of coding regions) has a functional role. However, whether gene body methylation is related to gene origins and duplication modes has yet to be reported. In rice (Oryza sativa), we computed a body methylation level (proportion of methylated CpG within coding regions) for each gene in five tissues. Body methylation levels follow a bimodal distribution, but show distinct patterns associated with transposable element-related genes; WGD, tandem, proximal and transposed duplicates; and singleton genes. For pairs of duplicated genes, divergence in body methylation levels increases with physical distance and synonymous (Ks) substitution rates, and WGDs show lower divergence than single-gene duplications of similar Ks levels. Intermediate body methylation tends to be associated with high levels of gene expression, whereas heavy body methylation is associated with lower levels of gene expression. The biological trends revealed here are consistent across five rice tissues, indicating that genes of different origins and duplication modes have distinct body methylation patterns, and body methylation has a heterogeneous relationship with gene expression and may be related to survivorship of duplicated genes.

  4. 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.

  5. 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.

  6. Association between methylation of the glucocorticoid receptor gene, childhood maltreatment, and clinical severity in borderline personality disorder.

    PubMed

    Martín-Blanco, Ana; Ferrer, Marc; Soler, Joaquim; Salazar, Juliana; Vega, Daniel; Andión, Oscar; Sanchez-Mora, Cristina; Arranz, Maria Jesús; Ribases, Marta; Feliu-Soler, Albert; Pérez, Víctor; Pascual, Juan Carlos

    2014-10-01

    The hypothalamus-pituitary-adrenal axis (HPA) is essential in the regulation of stress responses. Increased methylation of the promoter region of the glucocorticoid receptor gene (NR3C1) has been described both in subjects with history of childhood trauma and in patients with Borderline Personality Disorder (BPD). However, no data on the possible association between a higher methylation of this gene and clinical severity is available. The aim of this study was to evaluate the association between NR3C1 methylation status, the history of childhood trauma, and current clinical severity in subjects with BPD. A sample of 281 subjects with BPD (diagnosed by SCID-II and DIB-R semi-structured diagnostic interviews) was recruited. Clinical variables included previous hospitalizations, self-injurious behavior, and self-reported history of childhood trauma. DNA was extracted from peripheral blood. The results indicated a significant positive correlation between NR3C1 methylation status and childhood maltreatment (specifically physical abuse). In addition, a positive correlation between methylation status and clinical severity (DIB-R total score and hospitalizations) was observed. These findings suggest that NR3C1 methylation in subjects with BPD may be associated not only with childhood trauma but also with clinical severity, adding new evidence to the involvement of gene-environment interactions in this disorder.

  7. 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

    2007-08-03

    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.

  8. General implications for CpG hot spot mutations: methylation patterns of the human iduronate-2-sulfatase gene locus.

    PubMed

    Tomatsu, Shunji; Orii, Koji O; Bi, Y; Gutierrez, Monica A; Nishioka, Tatsuo; Yamaguchi, Seiji; Kondo, Naomi; Orii, Tadao; Noguchi, Akihiko; Sly, William S

    2004-06-01

    The methylation pattern at CpG sites of a housekeeping gene correlates with the likelihood of mutation. Mucopolysaccharidosis (MPS) type II, an X-linked disorder, results from the deficiency of iduronate-2-sulfatase (IDS). In these patients, over 35% of independent point mutations at the IDS gene locus were found at CpG sites as transitional events. To gain insight into the relationship between methylation status and CpG hot spot mutations, we investigated patterns of cytosine methylation in the entire IDS gene, except for introns 4-8. Bisulfite genomic sequencing was performed on the normal leukocyte DNA. Our data show that: 1) cytosine methylation at the CpG sites was extensive, except for those present from the promoter region to a portion of intron 3; 2) a sharp boundary of methylated-nonmethylated regions was observed at the 5'-flanking region, whereas a gradual change in methylation was observed in the 2.0-kb segment in the 3'-flanking region; 3) the boundary of the 5'-flanking region contained multiple Sp1 sites and the TATA box; 4) the CpG sites in exons 1 and 2 were hypomethylated and were associated only with rare transitional mutations, while the CpG sites in exon 3 were also hypomethylated, yet were associated with a high rate of transitional mutations; 5) there was no striking sex difference in the methylation patterns in active alleles; and, 6) the methylation in both strands was symmetrical, except at the boundary of methylated-unmethylated regions.

  9. Longitudinal Study of DNA Methylation of Inflammatory Genes and Cancer Risk

    PubMed Central

    Joyce, Brian Thomas; Gao, Tao; Liu, Lei; Zheng, Yinan; Liu, Siran; Zhang, Wei; Penedo, Frank; Dai, Qi; Schwartz, Joel; Baccarelli, Andrea A.; Hou, Lifang

    2015-01-01

    Background Chronic inflammation plays a key role in cancer etiology. DNA methylation modification, one of the epigenetic mechanisms regulating gene expression, is considered a hallmark of cancer. Human and animal models have identified numerous links between DNA methylation and inflammatory biomarkers. Our objective was to prospectively and longitudinally examine associations between methylation of four inflammatory genes and cancer risk. Methods We included 795 Normative Aging Study participants with blood drawn 1-4 times from 1999-2012 (median follow up 10.6 years). Promoter DNA methylation of IL-6, ICAM-1, IFN, and TLR2 in blood leukocytes was measured using pyrosequencing at multiple CpG sites and averaged by gene for data analysis. We used Cox regression models to examine prospective associations of baseline and time-dependent methylation with cancer risk, and compared mean methylation differences over time between cancer cases and cancer-free participants. Results Baseline IFN hypermethylation was associated with all-cancer (HR=1.49, p=0.04) and prostate cancer incidence (HR=1.69, p=0.02). Baseline ICAM-1 and IL-6 hypermethylation were associated with prostate cancer incidence (HR=1.43, p=0.02; HR=0.70, p=0.03 respectively). In our time-dependent analyses, IFN hypermethylation was associated with all-cancer (HR=1.79, p=0.007) and prostate cancer (HR=1.57, p=0.03) incidence; and ICAM-1 and IL-6 hypermethylation were associated with prostate cancer incidence (HR=1.39, p=0.02; HR=0.69, p=0.03 respectively). We detected significant ICAM-1 hypermethylation in cancer cases (p=0.0003) 10-13 years pre-diagnosis. Conclusion Hypermethylation of IFN and ICAM-1 may play important roles in early carcinogenesis, particularly that of prostate cancer. Impact These methylation changes could inform the development of early detection biomarkers and potential treatments of inflammation-related carcinogenesis. PMID:26265203

  10. c-Myc inhibits TP53INP1 expression via promoter methylation in esophageal carcinoma

    SciTech Connect

    Weng, Wenhao; Yang, Qinyuan; Huang, Miaolong; Qiao, Yongxia; Xie, Yuan; Yu, Yongchun; Jing, An; Li, Zhi

    2011-02-11

    Research highlights: {yields} TP53INP1 expression is down-regulated in esophageal carcinoma and is associated with CGI-131 methylation. {yields} Inhibition of CGI-131 methylation upregulates TP53INP1 expression in ESCC cell lines. {yields} Ectopic expression of TP53INP1 inhibits growth of ESCC cells by inducing apoptosis and inhibiting cell cycle progression. {yields} c-Myc binds to the promoter of TP53INP1 in vivo and vitro and recruits DNMT3A to TP53INP1 promoter for CGI-131 methylation. -- Abstract: Tumor protein p53-induced nuclear protein 1 (TP53INP1) is a well known stress-induced protein that plays a role in both cell cycle arrest and p53-mediated apoptosis. Loss of TP53INP1 expression has been reported in human melanoma, breast carcinoma, and gastric cancer. However, TP53INP1 expression and its regulatory mechanism in esophageal squamous cell carcinoma (ESCC) remain unclear. Our findings are in agreement with previous reports in that the expression of TP53INP1 was downregulated in 28% (10/36 cases) of ESCC lesions, and this was accompanied by significant promoter methylation. Overexpression of TP53INP1 induced G1 cell cycle arrest and increased apoptosis in ESCC cell lines (EC-1, EC-109, EC-9706). Furthermore, our study showed that the oncoprotein c-Myc bound to the core promoter of TP53INP1 and recruited DNA methyltransferase 3A to methylate the local promoter region, leading to the inhibition of TP53INP1 expression. Our findings revealed that TP53INP1 is a tumor suppressor in ESCC and that c-Myc-mediated DNA methylation-associated silencing of TP53INP1 contributed to the pathogenesis of human ESCC.

  11. 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

  12. Detection of aberrant methylated SEPT9 and NTRK3 genes in sporadic colorectal cancer patients as a potential diagnostic biomarker

    PubMed Central

    Behrouz Sharif, Shahin; Hashemzadeh, Shahriar; Mousavi Ardehaie, Reza; Eftekharsadat, Amirtaher; Ghojazadeh, Mortaza; Mehrtash, Amir Hossein; Estiar, Mehrdad Asghari; Teimoori-Toolabi, Ladan; Sakhinia, Ebrahim

    2016-01-01

    Colorectal cancer (CRC) is one of the most common malignancies, and the third leading cause of cancer mortality worldwide. Timely detection of CRC in patients with earlier stages provides the highest rate of survival. Epigenetic alterations are important in the occurrence and progression of CRC, and represent the primary modifications of cancer cells. Therefore, detection of these alterations in CRC cases are thought to hold great promise as diagnostic biomarkers. It has been shown that the SEPT9 and NTRK3 genes are aberrantly methylated and their detection can be used as biomarkers for early diagnosis of CRC. The present study analyzed promoter methylation status of these genes in CRC patients. Genomic DNA was extracted from 45 CRC and paired adjacent healthy tissues and undergone bisulfite conversion, and the methylation status of NTRK3 and SEPT9 were defined using the MS-HRM assay. Our results showed that there are statistically significant differences in methylation status of NTRK3 and specially SEPT9 between CRC and adjacent normal tissues (P<0.001). High sensitivity and specificity for a specific location in SEPT9 gene promoter as a diagnostic biomarker was observed. SEPT9 promoter hypermethylation may serve as a promising biomarker for the detection of CRC development. However, to validate the biomarker potential of NTRK3 there is a requirement for further investigation. PMID:28105243

  13. Hypermethylation of CpG islands in the mouse asparagine synthetase gene: relationship to asparaginase sensitivity in lymphoma cells. Partial methylation in normal cells.

    PubMed

    Peng, H; Shen, N; Qian, L; Sun, X L; Koduru, P; Goodwin, L O; Issa, J P; Broome, J D

    2001-09-14

    We have sequenced the promoter region of the murine asparagine synthetase gene and examined its methylation profile in the CpG islands of L-asparaginase-sensitive 6C3HED cells (asparagine auxotrophs) and resistant variants (prototrophs). In the former, complete methylation of the CpG island is correlated with failure of expression of mRNA: cells of the latter possess both methylated and unmethylated alleles, as do cells of the intrinsically asparagine-independent lines L1210 and EL4. A similar phenomenon was seen in normal splenic cells of adult mice. This was age related: no methylation was found in weanlings, but up to 45% of gene copies in animals 18 weeks or older were methylated. It was also tissue related, with methylation occurring rarely in liver cells. The relationship of these changes to oncogenesis is considered.

  14. Restoration of CpG Methylation in The Egf Promoter Region during Rat Liver Regeneration

    PubMed Central

    Deming, Li; Ziwei, Li; Xueqiang, Guo; Cunshuan, Xu

    2015-01-01

    Epidermal growth factor (EGF) is an important factor for healing after tissue damage in diverse experimental models. It plays an important role in liver regeneration (LR). The objective of this experiment is to investigate the methylation variation of 10 CpG sites in the Egf promoter region and their relevance to Egf expression during rat liver regenera- tion. As a follow up of our previous study, rat liver tissue was collected after rat 2/3 partial hepatectomy (PH) during the re-organization phase (from days 14 to days 28). Liver DNA was extracted and modified by sodium bisulfate. The methylation status of 10 CpG sites in Egf promoter region was determined using bisulfite sequencing polymerase chain reaction (PCR), as BSP method. The results showed that 3 (sites 3, 4 and 9) out of 10 CpG sites have strikingly methylation changes during the re-organization phase compared to the regeneration phase (from 2 hours to 168 hours, P=0.002, 0.048 and 0.018, respectively). Our results showed that methylation modification of CpGs in the Egf promoter region could be restored to the status before PH operation and changes of methylation didn’t affect Egf mRNA expression during the re-organization phase. PMID:26464832

  15. Fibrillarin methylates H2A in RNA polymerase I trans-active promoters in Brassica oleracea

    PubMed Central

    Loza-Muller, Lloyd; Rodríguez-Corona, Ulises; Sobol, Margarita; Rodríguez-Zapata, Luis C.; Hozak, Pavel; Castano, Enrique

    2015-01-01

    Fibrillarin is a well conserved methyltransferase involved in several if not all of the more than 100 methylations sites in rRNA which are essential for proper ribosome function. It is mainly localized in the nucleoli and Cajal bodies inside the cell nucleus where it exerts most of its functions. In plants, fibrillarin binds directly the guide RNA together with Nop56, Nop58, and 15.5ka proteins to form a snoRNP complex that selects the sites to be methylated in pre-processing of ribosomal RNA. Recently, the yeast counterpart NOP1 was found to methylate histone H2A in the nucleolar regions. Here we show that plant fibrillarin can also methylate histone H2A. In Brassica floral meristem cells the methylated histone H2A is mainly localized in the nucleolus but unlike yeast or human cells it also localize in the periphery of the nucleus. In specialized transport cells the pattern is altered and it exhibits a more diffuse staining in the nucleus for methylated histone H2A as well as for fibrillarin. Here we also show that plant fibrillarin is capable of interacting with H2A and carry out its methylation in the rDNA promoter. PMID:26594224

  16. 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

  17. Altered expression of miRNAs and methylation of their promoters are correlated in neuroblastoma.

    PubMed

    Maugeri, Marco; Barbagallo, Davide; Barbagallo, Cristina; Banelli, Barbara; Di Mauro, Stefania; Purrello, Francesco; Magro, Gaetano; Ragusa, Marco; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

    2016-12-13

    Neuroblastoma is the most common human extracranial solid tumor during infancy. Involvement of several miRNAs in its pathogenesis has been ascertained. Interestingly, most of their encoding genes reside in hypermethylated genomic regions: thus, their tumor suppressor function is normally disallowed in these tumors. To date, the therapeutic role of the demethylating agent 5'-Aza-2 deoxycytidine (5'-AZA) and its effects on miRNAome modulation in neuroblastoma have not been satisfactorily explored. Starting from a high-throughput expression profiling of 754 miRNAs and based on a proper selection, we focused on miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p as candidate miRNAs for our analysis. They resulted downregulated in four neuroblastoma cell lines with respect to normal adrenal gland. MiRNAs 29a-3p and 34b-3p also resulted downregulated in vivo in a murine neuroblastoma progression model. Unlike the amount of methylation of their encoding gene promoters, all these miRNAs were significantly overexpressed following treatment with 5'-AZA. Transfection with candidate miRNAs mimics significantly decreased neuroblastoma cells proliferation rate. A lower expression of miR-181c was significantly associated to a worse overall survival in a public dataset of 498 neuroblastoma samples (http://r2.amc.nl). Our data strongly suggest that CDK6, DNMT3A, DNMT3B are targets of miR-29a-3p, while CCNE2 and E2F3 are targets of miR-34b-3p. Based on all these data, we propose that miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p are disallowed tumor suppressor genes in neuroblastoma and suggest them as new therapeutic targets in neuroblastoma.

  18. DNMT3B7 expression related to MENT expression and its promoter methylation in human lymphomas.

    PubMed

    Alkebsi, Lobna; Handa, Hiroshi; Sasaki, Yoshiko; Osaki, Yohei; Yanagisawa, Kunio; Ogawa, Yoshiaki; Yokohama, Akihiko; Hattori, Hikaru; Koiso, Hiromi; Saitoh, Takayuki; Mitsui, Takeki; Tsukamoto, Norifumi; Nojima, Yoshihisa; Murakami, Hirokazu

    2013-12-01

    DNA methyltransferase (DNMT) 3B7 is the most expressed DNMT3B splice variant. It was reported that the loss of DNMT3B function led to overexpression of the MEthylated in Normal Thymocyes (MENT) and accelerated mouse lymphomagenesis. We investigated the DNMT3B7 expression and its relationship to MENT expression and promoter methylation in human lymphomas. DNMT3B7 and MENT expression were significantly (p<0.0001, p<0.01) higher in lymphomas than in non-malignant. Expression of DNMT3B7 and MENT were associated with MENT promoter hypomethylation. DNMT3B7 overexpression might interfere with the normal DNA methylation mechanism required for silencing the MENT proto-oncogene, and may accelerate human lymphomagenesis.

  19. The "Methyl-CpG Binding Domain protein 2" plays a repressive role in relation to the promoter CpG content in the normal human cell line MRC5.

    PubMed

    Perriaud, Laury; Lachuer, Joel; Dante, Robert

    2014-01-01

    In cancer cells, methylation-dependent gene silencing is at least partly mediated by the "Methyl-CpG-Binding Domain protein 2" (MBD2 protein), via the recruitment of chromatin remodeling complexes. However this repressive role was poorly investigated in normal cells. To identify the genes repressed by MBD2 in these cells, we have determined the impact of MBD2 depletion on gene expression in human embryonic MRC5 fibroblasts, using RNA inference combined with microarray analysis. The up-regulation of some randomly selected genes was confirmed and a direct association between gene repression and MBD2 binding on methylated promoters associated to these genes was subsequently established. This control of gene expression appears to depend on the CpG content of promoters as MBD2 depletion was not sufficient to induce the expression of silent genes associated with High-CpG promoters, but it was required to achieve the methyl-dependent transcriptional locking of the genes associated with promoters exhibiting intermediate CpG content. Therefore, MBD2 seems to play a selective role in gene repression depending on the CpG content of the promoter regions.

  20. 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 help