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Sample records for aberrant dna methylation

  1. Aberrant DNA methylation in cloned ovine embryos

    Institute of Scientific and Technical Information of China (English)

    LIU Lei; HOU Jian; LEI TingHua; BAI JiaHua; GUAN Hong; AN XiaoRong

    2008-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of cloned ovine embryos. The em-bryos derived from in vitro fertilization were also examined for reference purpose. The results showed that: (1) during the pre-implantation development, cloned embryos displayed a similar demethylation profile to the fertilized embryos; that is, the methylation level decreased to the lowest at 8-cell stage, and then increased again at morulae stage. However, methylation level was obviously higher in cloned embryos than in stage-matched fertilized embryos, especially at 8-cell stage and afterwards; (2) at blastocyst stage, the methylation pattern in cloned embryos was different from that in fertilized em-bryos. In cloned blastocyst, inner cell mass (ICM) exhibited a comparable level to trophectoderm cells (TE), while in in-vitro fertilized blastocyst the methylation level of ICM was lower than that of TE, which is not consistent with that reported by other authors. These results indicate that DNA methylation is abnormally reprogrammed in cloned embryos, implying that aberrant DNA methylation reprogramming may be one of the factors causing cloned embryos developmental failure.

  2. Aberrant DNA methylation in cervical carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Hui-Juan Yang

    2013-01-01

    Persistent infection with high-risk types of human papillomavirus(HPV) is known to cause cervical cancer; however,additional genetic and epigenetic alterations are required for progression from precancerous disease to invasive cancer.DNA methylation is an early and frequent molecular alteration in cervical carcinogenesis.In this review,we summarize DNA methylation within the HPV genome and human genome and identify its clinical implications.Methylation of the HPV long control region (LCR) and L1 gene is common during cervical carcinogenesis and increases with the severity of the cervical neoplasm.The L1 gene of HPV16 and HPV18 is consistently hypermethylated in invasive cervical cancers and can potentially be used as a clinical marker of cancer progression.Moreover,promoters of tumor suppressor genes (TSGs) involved in many cellular pathways are methylated in cervical precursors and invasive cancers.Some are associated with squamous cell carcinomas,and others are associated with adenocarcinomas.Identification of methylated TSGs in Pap smear could be an adjuvant test in cervical cancer screening for triage of women with high-risk HPV,atypical squamous cells of undetermined significance,or low grade squamous intraepithelial lesion (LSIL).However,consistent panels must be validated for this approach to be translated to the clinic.Furthermore,reversion of methylated TSGs using demethylating drugs may be an alternative anticancer treatment,but demethylating drugs without toxic carcinogenic and mutagenic properties must be identified and validated.

  3. Aberrantly methylated DNA as a biomarker in breast cancer

    DEFF Research Database (Denmark)

    Kristiansen, Søren; Jørgensen, Lars Mønster; Guldberg, Per;

    2013-01-01

    hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients......Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA...... occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into...

  4. miRNA gene promoters are frequent targets of aberrant DNA methylation in human breast cancer.

    Science.gov (United States)

    Vrba, Lukas; Muñoz-Rodríguez, José L; Stampfer, Martha R; Futscher, Bernard W

    2013-01-01

    miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

  5. Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  6. Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

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    Chih-Jie Shen

    2014-01-01

    Full Text Available Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region −2615 to −2239 nucleotides (nt may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome.

  7. Aberrant DNA Methylation: Implications in Racial Health Disparity.

    Directory of Open Access Journals (Sweden)

    Xuefeng Wang

    Full Text Available Incidence and mortality rates of colorectal carcinoma (CRC are higher in African Americans (AAs than in Caucasian Americans (CAs. Deficient micronutrient intake due to dietary restrictions in racial/ethnic populations can alter genetic and molecular profiles leading to dysregulated methylation patterns and the inheritance of somatic to germline mutations.Total DNA and RNA samples of paired tumor and adjacent normal colon tissues were prepared from AA and CA CRC specimens. Reduced Representation Bisulfite Sequencing (RRBS and RNA sequencing were employed to evaluate total genome methylation of 5'-regulatory regions and dysregulation of gene expression, respectively. Robust analysis was conducted using a trimming-and-retrieving scheme for RRBS library mapping in conjunction with the BStool toolkit.DNA from the tumor of AA CRC patients, compared to adjacent normal tissues, contained 1,588 hypermethylated and 100 hypomethylated differentially methylated regions (DMRs. Whereas, 109 hypermethylated and 4 hypomethylated DMRs were observed in DNA from the tumor of CA CRC patients; representing a 14.6-fold and 25-fold change, respectively. Specifically; CHL1, 4 anti-inflammatory genes (i.e., NELL1, GDF1, ARHGEF4, and ITGA4, and 7 miRNAs (of which miR-9-3p and miR-124-3p have been implicated in CRC were hypermethylated in DNA samples from AA patients with CRC. From the same sample set, RNAseq analysis revealed 108 downregulated genes (including 14 ribosomal proteins and 34 upregulated genes (including POLR2B and CYP1B1 [targets of miR-124-3p] in AA patients with CRC versus CA patients.DNA methylation profile and/or products of its downstream targets could serve as biomarker(s addressing racial health disparity.

  8. Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver.

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    Wolfgang Tränkenschuh

    Full Text Available BACKGROUND: Gene silencing due to aberrant DNA methylation is a frequent event in hepatocellular carcinoma (HCC and also in hepatocellular adenoma (HCA. However, very little is known about epigenetic defects in fibrolamellar carcinoma (FLC, a rare variant of hepatocellular carcinoma that displays distinct clinical and morphological features. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the methylation status of the APC, CDH1, cyclinD2, GSTπ1, hsa-mir-9-1, hsa-mir-9-2, and RASSF1A gene in a series of 15 FLC and paired normal liver tissue specimens by quantitative high-resolution pyrosequencing. Results were compared with common HCC arising in non-cirrhotic liver (n = 10. Frequent aberrant hypermethylation was found for the cyclinD2 (19% and the RASSF1A (38% gene as well as for the microRNA genes mir-9-1 (13% and mir-9-2 (33%. In contrast to common HCC the APC and CDH1 (E-cadherin genes were found devoid of any DNA methylation in FLC, whereas the GSTπ1 gene showed comparable DNA methylation in tumor and surrounding tissue at a moderate level. Changes in global DNA methylation level were measured by analyzing methylation status of the highly repetitive LINE-1 sequences. No evidence of global hypomethylation could be found in FLCs, whereas HCCs without cirrhosis showed a significant reduction in global methylation level as described previously. CONCLUSIONS: FLCs display frequent and distinct gene-specific hypermethylation in the absence of significant global hypomethylation indicating that these two epigenetic aberrations are induced by different pathways and that full-blown malignancy can develop in the absence of global loss of DNA methylation. Only quantitative DNA methylation detection methodology was able to identify these differences.

  9. Aberrant DNA methylation patterns in cultured mouse embryos

    Institute of Scientific and Technical Information of China (English)

    HOU Jian; CUI Xiuhong; LEI Tinghua; LIU Lei; AN Xiaorong; CHEN Yongfu

    2005-01-01

    Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofiuorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.

  10. The key culprit in the pathogenesis of systemic lupus erythematosus: Aberrant DNA methylation.

    Science.gov (United States)

    Wu, Haijing; Zhao, Ming; Tan, Lina; Lu, Qianjin

    2016-07-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease with multiple organ involvement. It is characterized by abundant autoantibodies that form immune complex with autoantigens and deposit in organs and cause tissue damage by inducing inflammation. The pathogenesis of SLE has been intensively studied but remains unclear. B and T lymphocyte abnormalities, dysregulation of apoptosis, defects in the clearance of apoptotic materials, and various genetic and epigenetic factors are believed to contribute to the initiation and development of SLE. The up-to-date research findings point to the relationship between abnormal DNA methylation and SLE, which has attracted considerable interest worldwide. Besides the global hypomethylation on lupus T and B cells, the gene specific and site-specific methylation has been identified and documented to be responsible for SLE. The purpose of this review was to present and summarize the association between aberrant DNA methylation of immune cells and SLE, the possible mechanisms of immune dysfunction caused by DNA methylation, and to better understand the roles of aberrant DNA methylation in the initiation and development of SLE and to provide an insight into the related diagnosis biomarkers and therapeutic options in SLE.

  11. The role for oxidative stress in aberrant DNA methylation in Alzheimer's disease.

    Science.gov (United States)

    Fleming, Jessica L; Phiel, Christopher J; Toland, Amanda Ewart

    2012-11-01

    Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without highly effective therapies. The etiology of AD is heterogeneous with amyloid-beta plaques, neurofibrillary tangles, oxidative stress, and aberrant DNA methylation all implicated in the disease pathogenesis. DNA methylation is a well-established process for regulating gene expression and has been found to regulate a growing number of important genes involved in AD development and progression. Additionally, aberrations in one-carbon metabolism are a common finding in AD patients with individuals exhibiting low S-adenosylmethionine and high homocysteine levels as well as low folate and vitamin B. Oxidative stress is considered one of the earliest events in AD pathogenesis and is thought to contribute largely to neuronal cell death. Emerging evidence suggests an interaction exists between oxidative stress and DNA methylation; however, the mechanism(s) remain unclear. This review summarizes known and potential genes implicated in AD that are regulated by DNA methylation and oxidative stress. We also highlight the evidence for the role of oxidative damage contributing to DNA hypomethylation in AD patients through several mechanisms as well as implications for disease understanding and therapeutic development. PMID:21605062

  12. Aberrant methylation patterns in cancer

    OpenAIRE

    Hudler, Petra; Videtič, Alja

    2016-01-01

    Epigenetic mechanisms, such as DNA methylation, DNA hydroxymethylation, post-translational modifications (PTMs) of histone proteins affecting nucleosome remodelling, and regulation by small and large non-coding RNAs (ncRNAs) work in concert with cis and trans acting elements to drive appropriate gene expression. Advances in detection methods and development of dedicated platforms and methylation arrays resulted in an explo - sion of information on aberrantly methylated sequences linking devia...

  13. ∆DNMT3B4-del Contributes to Aberrant DNA Methylation Patterns in Lung Tumorigenesis

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    Mark Z. Ma

    2015-10-01

    Full Text Available Aberrant DNA methylation is a hallmark of cancer but mechanisms contributing to the abnormality remain elusive. We have previously shown that ∆DNMT3B is the predominantly expressed form of DNMT3B. In this study, we found that most of the lung cancer cell lines tested predominantly expressed DNMT3B isoforms without exons 21, 22 or both 21 and 22 (a region corresponding to the enzymatic domain of DNMT3B termed DNMT3B/∆DNMT3B-del. In normal bronchial epithelial cells, DNMT3B/ΔDNMT3B and DNMT3B/∆DNMT3B-del displayed equal levels of expression. In contrast, in patients with non-small cell lung cancer NSCLC, 111 (93% of the 119 tumors predominantly expressed DNMT3B/ΔDNMT3B-del, including 47 (39% tumors with no detectable DNMT3B/∆DNMT3B. Using a transgenic mouse model, we further demonstrated the biological impact of ∆DNMT3B4-del, the ∆DNMT3B-del isoform most abundantly expressed in NSCLC, in global DNA methylation patterns and lung tumorigenesis. Expression of ∆DNMT3B4-del in the mouse lungs resulted in an increased global DNA hypomethylation, focal DNA hypermethylation, epithelial hyperplastia and tumor formation when challenged with a tobacco carcinogen. Our results demonstrate ∆DNMT3B4-del as a critical factor in developing aberrant DNA methylation patterns during lung tumorigenesis and suggest that ∆DNMT3B4-del may be a target for lung cancer prevention.

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

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    Orozco Javier I

    2011-10-01

    Full Text Available Abstract 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.

  15. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    International Nuclear Information System (INIS)

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells

  16. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent d...

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

    Science.gov (United States)

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

    2016-01-01

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

  18. Detection of aberrant methylation in fecal DNA as a molecular screening tool for colorectal cancer and precancerous lesions

    Institute of Scientific and Technical Information of China (English)

    Zhao-Hui Huang; Li-Hua Li; Fan Yang; Jin-Fu Wang

    2007-01-01

    AIM: To investigate the feasibility of detecting methylated fecal DNA as a screening tool for colorectal carcinoma (CRC) and precancerous lesions.METHODS: Methylated secreted frizzled-related protein gene 2 (SFRP2), hyperplastic polyposis protein gene (HPP1) and O6-methylguanine-DNA methyltransferase gene (MGMT) in stools from 52 patients with CRC, 35 patients with benign colorectal diseases and 24 normal individuals were analyzed using methylation-specific PCR.RESULTS: Methylated SFRP2, HPP1 and MGMT were detected in 94.2%, 71.2%, 48.1% of CRC patients and 52.4%, 57.1%, 28.6% of adenoma patients, respectively. The overall prevalence of fecal DNA with at least one methylated gene was 96.2% and 81.8% in patients with CRC and precancerous lesions, respectively. In contrast, only one of the 24 normal individuals revealed methylated DNA. These results indicated a 93.7% sensitivity and a 77.1% specificity of the assay for detecting CRC and precancerous lesions.CONCLUSION: Methylation testing of fecal DNA using a panel of epigenetic markers may be a simple and promising non-invasive screening method for CRC and precancerous lesions.

  19. Aberrant DNA methylation associated with MTHFR C677T genetic polymorphism in cutaneous squamous cell carcinoma in renal transplant patients.

    LENUS (Irish Health Repository)

    Laing, M E

    2010-08-01

    Changes in genomic DNA methylation associated with cancer include global DNA hypomethylation and gene-specific hyper- or hypomethylation. We have previously identified a genetic variant in the MTHFR gene involved in the methylation pathway which confers risk for the development of squamous cell carcinoma (SCC) in renal transplant patients. This genetic variant has also been discovered to confer SCC risk in nontransplant patients with low folate status.

  20. Aberrantly Methylated MGMT, hMLH1 and hMSH2 in Tumor and Serum DNA of Gliomas Patients

    Institute of Scientific and Technical Information of China (English)

    Changqing Zheng; Shouping Ji; Feng Gong; Anming Li; Junli Tai; Subuo Li; Yingli Wang; Hongyu Chang; Hongwei Gao; Yangpei Zhang

    2009-01-01

    OBJECTIVE This study is to investigate the prevalence of promoter CpG island methylation of O6-methylguananine-DNA methyltransferase (MGMT), mismatch repair genes (hMLH1 and hMSH2) in both tumor and serum samples of gliomas.METHODS Methylation-specific PCR (MSP) was employed to detect promoter CpG island methylation of the MGMT, hMLH1 and hMSH2 genes in 39 samples taken from surgery and 32 samples of pretreatment serum all from the patients with gliomas.RESULTS Promoter CpG island methylation of MGMT, hMLH1 and hMSH2 was detected and the results were 46.2%, 10.3% and 20.5%, respectively in tumor DNA of the cases with gliomas,and 40.6%, 9.4% and 18.8%, respectively in serum DNA of the cases. The methylation pattern in primary tumor and serum was found to be concordant in matched tissue and serum samples of 21 patients. In the cases with positive result of methylation for MGMT, hMLH1 and hMSH2 in tumor tissues, the results of detection for those in the paired serum sample were 77.8% (7/9),66.7% (2/3) and 75.0 % (3/4), respectively. False positive results were not obtained in any of the patients who did not exhibit methylation. No association was found between the promoter methylation of MGMT, hMLH1, and hMSH2 genes in primary gliomas and gender, age, localization, grade of malignant or tumor stage.CONCLUSION Promoter CpG island methylation is a frequent event in gliomagenesis. Methylation analysis appears to be a promising predictive factor of the prognosis for the glioma patients treated with alkylating drugs and a noninvasive tumor marker in serum DNA.

  1. Aberrant DNA Methylation of P16, MGMT, and hMLH1 Genes in Combination with MTHFR C677T Genetic Polymorphism in gastric cancer

    OpenAIRE

    Song, Binbin; Ai, Jiang; Kong, Xianghong; Liu, Dexin; Li, Jun

    2013-01-01

    Objective: We aimed to explore the association of P16, MGMT and HMLH1 with gastric cancer and their relation with Methylenetetrahydrofolate reductase (MTHFR). Methods: 322 gastric patients who were confirmed with pathological diagnosis were included in our study. Aberrant DNA methylation of P16, MGMT and HMLH1 and polymorphisms of MTHFR C677T and A1298C were detected using PCR-RFLP. Results: The proportions of DNA hypermethylation in P16, MGMT and hMLH1 genes in gastric cancer tissues were 75...

  2. The Aberrant DNA Methylation Profile of Human Induced Pluripotent Stem Cells Is Connected to the Reprogramming Process and Is Normalized During In Vitro Culture.

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    Lenka Tesarova

    Full Text Available The potential clinical applications of human induced pluripotent stem cells (hiPSCs are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs. We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications.

  3. Detection of aberrant methylation of a six-gene panel in serum DNA for diagnosis of breast cancer.

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    Shan, Ming; Yin, Huizi; Li, Junnan; Li, Xiaobo; Wang, Dong; Su, Yonghui; Niu, Ming; Zhong, Zhenbin; Wang, Ji; Zhang, Xianyu; Kang, Wenli; Pang, Da

    2016-04-01

    Detection of breast cancer at an early stage is the key for successful treatment and improvement of outcome. However the limitations of mammography are well recognized, especially for those women with premenopausal breast cancer. Novel approaches to breast cancer screening are necessary, especially in the developing world where mammography is not feasible. In this study, we examined the promoter methylation of six genes (SFN, P16, hMLH1, HOXD13, PCDHGB7 and RASSF1a) in circulating free DNA (cfDNA) extracted from serum. We used a high-throughput DNA methylation assay (MethyLight) to examine serum from 749 cases including breast cancer patients, patients with benign breast diseases and healthy women. The six-gene methylation panel test achieved 79.6% and 82.4% sensitivity with a specificity of 72.4% and 78.1% in diagnosis of breast cancer when compared with healthy and benign disease controls, respectively. Moreover, the methylation panel positive group showed significant differences in the following independent variables: (a) involvement of family history of tumors; (b) a low proliferative index, ki-67; (c) high ratios in luminal subtypes. Additionally the panel also complemented some breast cancer cases which were neglected by mammography or ultrasound. These data suggest that epigenetic markers in serum have potential for diagnosis of breast cancer. PMID:26918343

  4. Detection of aberrant methylation of a six-gene panel in serum DNA for diagnosis of breast cancer.

    Science.gov (United States)

    Shan, Ming; Yin, Huizi; Li, Junnan; Li, Xiaobo; Wang, Dong; Su, Yonghui; Niu, Ming; Zhong, Zhenbin; Wang, Ji; Zhang, Xianyu; Kang, Wenli; Pang, Da

    2016-04-01

    Detection of breast cancer at an early stage is the key for successful treatment and improvement of outcome. However the limitations of mammography are well recognized, especially for those women with premenopausal breast cancer. Novel approaches to breast cancer screening are necessary, especially in the developing world where mammography is not feasible. In this study, we examined the promoter methylation of six genes (SFN, P16, hMLH1, HOXD13, PCDHGB7 and RASSF1a) in circulating free DNA (cfDNA) extracted from serum. We used a high-throughput DNA methylation assay (MethyLight) to examine serum from 749 cases including breast cancer patients, patients with benign breast diseases and healthy women. The six-gene methylation panel test achieved 79.6% and 82.4% sensitivity with a specificity of 72.4% and 78.1% in diagnosis of breast cancer when compared with healthy and benign disease controls, respectively. Moreover, the methylation panel positive group showed significant differences in the following independent variables: (a) involvement of family history of tumors; (b) a low proliferative index, ki-67; (c) high ratios in luminal subtypes. Additionally the panel also complemented some breast cancer cases which were neglected by mammography or ultrasound. These data suggest that epigenetic markers in serum have potential for diagnosis of breast cancer.

  5. Quantitative DNA Methylation Profiling in Cancer.

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    Ammerpohl, Ole; Haake, Andrea; Kolarova, Julia; Siebert, Reiner

    2016-01-01

    Epigenetic mechanisms including DNA methylation are fundamental for the regulation of gene expression. Epigenetic alterations can lead to the development and the evolution of malignant tumors as well as the emergence of phenotypically different cancer cells or metastasis from one single tumor cell. Here we describe bisulfite pyrosequencing, a technology to perform quantitative DNA methylation analyses, to detect aberrant DNA methylation in malignant tumors.

  6. Reasons of carcinogenesis indicate a big-bang inside: a hypothesis for the aberration of DNA methylation.

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    Roy, A; Roy Chattopadhyay, N

    2013-07-01

    Cancer involves various sets of altered gene functions which embrace all the three basic mechanisms of regulation of gene expression. However, no common mechanism is inferred till date for this versatile disease and thus no full proof remedy can be offered. Here we show that the basic mechanisms are interlinked and indicate towards one of those mechanisms as being the superior one; the methylation of cytosines in specific DNA sequences, for the initiation and maintenance of carcinogenesis. The analyses of the previous reports and the nucleotide sequences of the DNA methyltransferases strongly support the assumption that the mutation(s) in the DNA-binding site(s) of DNA-methyltransferases acts as a master regulator; though it continues the cycle from mutation to repair to methylation. We anticipate that our hypothesis will start a line of study for the proposal of a treatment regime for cancers by introducing wild type methyltransferases in the diseased cells and/or germ cells, and/or by targeting ligands to the altered binding domain(s) where a mutation in the concerned enzyme(s) is seen. PMID:23623297

  7. Aberrant DNA Methylation of P16, MGMT, and hMLH1 Genes in Combination with MTHFR C677T Genetic Polymorphism in gastric cancer

    Science.gov (United States)

    Song, Binbin; Ai, Jiang; Kong, Xianghong; Liu, Dexin; Li, Jun

    2013-01-01

    Objective: We aimed to explore the association of P16, MGMT and HMLH1 with gastric cancer and their relation with Methylenetetrahydrofolate reductase (MTHFR). Methods: 322 gastric patients who were confirmed with pathological diagnosis were included in our study. Aberrant DNA methylation of P16, MGMT and HMLH1 and polymorphisms of MTHFR C677T and A1298C were detected using PCR-RFLP. Results: The proportions of DNA hypermethylation in P16, MGMT and hMLH1 genes in gastric cancer tissues were 75.2% (242/322), 27.6% (89/322) and 5.3% (17/322), respectively. In the remote normal-appearing tissues, 29.5% (95/322) and 16.1%(52/322) showed hypermethylation in P16 and MGMT genes, respectively. We found a significantly higher proportion of DNA hypermethylation of P16 in patients with N1 TNM stage in cancer tissues and remote normal-appearing tissues (P<0.05). Similarly, we found DNA hypermethylation of MGMT had significantly higher proportion in N1 and M1 TNM stage (P<0.05). Individuals with homozygotes (TT) of MTHFR C677T had significant risk of DNA hypermethylation of MGMT in cancer tissues [OR (95% CI)=4.27(1.76-7.84)], and a significant risk was also found in those carrying MTHFR 677CT/TT genotype [OR (95% CI)= 3.27(1.21-4.77)]. Conclusion: We found the aberrant hypermethylation of cancer-related genes, such as P16, MGMT and HMLH1, could be predictive biomarkers for detection of gastric cancer. PMID:24550949

  8. Whole blood DNA aberrant methylation in pancreatic adenocarcinoma shows association with the course of the disease: a pilot study.

    Directory of Open Access Journals (Sweden)

    Albertas Dauksa

    Full Text Available Pancreatic tumors are usually diagnosed at an advanced stage in the progression of the disease, thus reducing the survival chances of the patients. Non-invasive early detection would greatly enhance therapy and survival rates. Toward this aim, we investigated in a pilot study the power of methylation changes in whole blood as predictive markers for the detection of pancreatic tumors. We investigated methylation levels at selected CpG sites in the CpG rich regions at the promoter regions of p16, RARbeta, TNFRSF10C, APC, ACIN1, DAPK1, 3OST2, BCL2 and CD44 in the blood of 30 pancreatic tumor patients and in the blood of 49 matching controls. In addition, we studied LINE-1 and Alu repeats using degenerate amplification approach as a surrogate marker for genome-wide methylation. The site-specific methylation measurements at selected CpG sites were done by the SIRPH method. Our results show that in the patient's blood, tumor suppressor genes were slightly but significantly higher methylated at several CpG sites, while repeats were slightly less methylated compared to control blood. This was found to be significantly associated with higher risk for pancreatic ductal adenocarcinoma. Additionally, high methylation levels at TNFRSCF10C were associated with positive perineural spread of tumor cells, while higher methylation levels of TNFRSF10C and ACIN1 were significantly associated with shorter survival. This pilot study shows that methylation changes in blood could provide a promising method for early detection of pancreatic tumors. However, larger studies must be carried out to explore the clinical usefulness of a whole blood methylation based test for non-invasive early detection of pancreatic tumors.

  9. DNA Methylation in Thyroid Tumorigenesis

    International Nuclear Information System (INIS)

    Thyroid cancer is the most common endocrine cancer with 1,690 deaths each year. There are four main types of which the papillary and follicular types together account for >90% followed by medullary cancers with 3% to 5% and anaplastic carcinomas making up <3%. Epigenetic events of DNA hypermethylation are emerging as promising molecular targets for cancer detection. Our immediate and long term goal is to identify DNA methylation markers for early detection of thyroid cancer. This pilot study comprised of 21 patients to include 11 papillary thyroid cancers (PTC), 2 follicular thyroid cancers (FTC), 5 normal thyroid cases, and 3 hyperthyroid cases. Aberrant promoter methylation was examined in 24 tumor suppressor genes using the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and in the NIS gene using methylation-specific PCR (MSP). The frequently methylated genes were CASP8 (17/21), RASSF1 (16/21) and NIS (9/21). In the normal samples, CASP8, RASSF1 and NIS were methylated in 5/5, 4/5 and 1/5 respectively. In the hyperthyroid samples, CASP8, RASSF1 and NIS were methylated in 3/3, 2/3 and 1/3 respectively. In the thyroid cancers, CASP8, RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13 respectively. CASP8, RASSF1 and NIS were also methylated in concurrently present normal thyroid tissue in 3/11, 4/11 and 3/11 matched thyroid cancer cases (matched for presence of both normal thyroid tissue and thyroid cancer), respectively. Our data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid tumorigenesis regardless of cell type

  10. Apoptosis and DNA Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Huan X.; Hackett, James A. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Nestor, Colm [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Dunican, Donncha S.; Madej, Monika; Reddington, James P. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Pennings, Sari [Queen' s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ (United Kingdom); Harrison, David J. [Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Meehan, Richard R., E-mail: Richard.Meehan@hgu.mrc.ac.uk [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom)

    2011-04-01

    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  11. Apoptosis and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Richard R. Meehan

    2011-04-01

    Full Text Available Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  12. DNA methylation in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Iris Tischoff; Andrea Tannapfel

    2008-01-01

    As for many other tumors, development of hepatocellular carcinoma (HCC) must be understood as a multistep process with accumulation of genetic and epigenetic alterations in regulatory genes, leading to activation of oncogenes and inactivation or loss of tumor suppressor genes (TSG). In the last decades, in addition to genetic alterations, epigenetic inactivation of (tumor suppressor) genes by promoter hypermethylation has been recognized as an important and alternative mechanism in tumorigenesis. In HCC, aberrant methylation of promoter sequences occurs not only in advanced tumors, it has been also observed in premalignant conditions just as chronic viral hepatitis B or C and cirrhotic liver. This review discusses the epigenetic alterations in hepatocellular carcinoma focusing DNA methylation.

  13. Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Victoria Gonzalo

    Full Text Available BACKGROUND: Colorectal cancer (CRC multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. METHODOLOGY/PRINCIPAL FINDINGS: We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2, RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008 and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047 as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006. Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17, SFRP1 (r = 0.83, 0.06, HPP1 (r = 0.64, p = 0.17, 3OST2 (r = 0.83, p = 0.06 and GATA4 (r = 0.6, p = 0.24. Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant

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

    Directory of Open Access Journals (Sweden)

    Guzmán Leda

    2012-07-01

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

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

    OpenAIRE

    Erin M Siegel; 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 ...

  16. Methylated DNA in Borrelia species.

    OpenAIRE

    Hughes, C A; Johnson, R C

    1990-01-01

    The DNA of Borrelia species was examined for the presence of methylated GATC sequences. The relapsing-fever Borrelia sp., B. coriaceae, and only 3 of 22 strains of B. burgdorferi contained adenine methylation systems. B. anserina lacked an adenine methylation system. Fundamental differences in DNA methylation exist among members of the genus Borrelia.

  17. Event extraction for DNA methylation

    OpenAIRE

    Ohta Tomoko; Pyysalo Sampo; Miwa Makoto; Tsujii Jun’ichi

    2011-01-01

    Abstract Background We consider the task of automatically extracting DNA methylation events from the biomedical domain literature. DNA methylation is a key mechanism of epigenetic control of gene expression and implicated in many cancers, but there has been little study of automatic information extraction for DNA methylation. Results We present an annotation scheme for DNA methylation following the representation of the BioNLP shared task on event extraction, select a set of 200 abstracts inc...

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

    Directory of Open Access Journals (Sweden)

    Yasuko eKikuchi

    2013-12-01

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

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

    OpenAIRE

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

    2010-01-01

    BACKGROUND: Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-...

  20. Frequency of aberrant promoter methylation of p15INK4b and O6-methylguanine-DNA methyltransferase genes in b-cell non-Hodgkin lymphoma: A pilot study

    Directory of Open Access Journals (Sweden)

    Kraguljac-Kurtović Nada

    2010-01-01

    Full Text Available The methylation status of the target promoter sequences of p15INK4B (p15 and O6-methylguanine-DNA methyltransferase (MGMT genes was studied by methylation-specific PCR in 10 adult patients with de novo B-cell non- Hodgkin lymphoma (B-NHL. The aberrant hypermethylation of the p15 gene was more frequent (50% compared to the hypermethylation of the MGMT gene (30%, and was detected in different types of B-NHL in both genes. Hypermethylation of the MGMT gene occurred exclusively in association with the hypermethylation of the p15 gene. All lymphoma patients with hypermethylation of the p15 and/or MGMT genes had a higher clinical stage of the disease (IV - V. We show the association of anemia and/or thrombocytopenia with the hypermethylation of the p15 gene, ascribing the p15 gene as a potential prognostic marker in B-NHL. Comethylation of MGMT with the p15 gene represents a novel finding and presents both genes as candidates for future studies of the hypermethylation profiles of B-NHL.

  1. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  2. A convenient method to generate methylated and un-methylated control DNA in methylation studies

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    Mehdi Manoochehri

    2013-09-01

    Full Text Available Methylated and un-methylated control DNA is an important part of DNA methylation studies. Although human and mouse DNA methylation control sets are commercially available, in case of methylation studies on other species such as animals, plants, and bacteria, control sets need to be prepared. In this paper a simple method of generating methylated and un-methylated control DNA is described. Whole genome amplification and enzymatic methylation were performed to generate un-methylated and methylated DNA. The generated DNA were confirmed using methylation sensitive/dependant enzymes, and methylation specific PCR. Control reaction assays confirmed the generated methylated and un-methylated DNA.

  3. Comparative methylome analysis in solid tumors reveals aberrant methylation at chromosome 6p in nasopharyngeal carcinoma

    International Nuclear Information System (INIS)

    Altered patterns of DNA methylation are key features of cancer. Nasopharyngeal carcinoma (NPC) has the highest incidence in Southern China. Aberrant methylation at the promoter region of tumor suppressors is frequently reported in NPC; however, genome-wide methylation changes have not been comprehensively investigated. Therefore, we systematically analyzed methylome data in 25 primary NPC tumors and nontumor counterparts using a high-throughput approach with the Illumina HumanMethylation450 BeadChip. Comparatively, we examined the methylome data of 11 types of solid tumors collected by The Cancer Genome Atlas (TCGA). In NPC, the hypermethylation pattern was more dominant than hypomethylation and the majority of de novo methylated loci were within or close to CpG islands in tumors. The comparative methylome analysis reveals hypermethylation at chromosome 6p21.3 frequently occurred in NPC (false discovery rate; FDR=1.33 × 10−9), but was less obvious in other types of solid tumors except for prostate and Epstein–Barr virus (EBV)-positive gastric cancer (FDR<10−3). Bisulfite pyrosequencing results further confirmed the aberrant methylation at 6p in an additional patient cohort. Evident enrichment of the repressive mark H3K27me3 and active mark H3K4me3 derived from human embryonic stem cells were found at these regions, indicating both DNA methylation and histone modification function together, leading to epigenetic deregulation in NPC. Our study highlights the importance of epigenetic deregulation in NPC. Polycomb Complex 2 (PRC2), responsible for H3K27 trimethylation, is a promising therapeutic target. A key genomic region on 6p with aberrant methylation was identified. This region contains several important genes having potential use as biomarkers for NPC detection

  4. Regulated DNA Methylation and the Circadian Clock: Implications in Cancer

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    Tammy M. Joska

    2014-09-01

    Full Text Available Since the cloning and discovery of DNA methyltransferases (DNMT, there has been a growing interest in DNA methylation, its role as an epigenetic modification, how it is established and removed, along with the implications in development and disease. In recent years, it has become evident that dynamic DNA methylation accompanies the circadian clock and is found at clock genes in Neurospora, mice and cancer cells. The relationship among the circadian clock, cancer and DNA methylation at clock genes suggests a correlative indication that improper DNA methylation may influence clock gene expression, contributing to the etiology of cancer. The molecular mechanism underlying DNA methylation at clock loci is best studied in the filamentous fungi, Neurospora crassa, and recent data indicate a mechanism analogous to the RNA-dependent DNA methylation (RdDM or RNAi-mediated facultative heterochromatin. Although it is still unclear, DNA methylation at clock genes may function as a terminal modification that serves to prevent the regulated removal of histone modifications. In this capacity, aberrant DNA methylation may serve as a readout of misregulated clock genes and not as the causative agent. This review explores the implications of DNA methylation at clock loci and describes what is currently known regarding the molecular mechanism underlying DNA methylation at circadian clock genes.

  5. DNA methylation in metabolic disorders

    DEFF Research Database (Denmark)

    Barres, Romain; Zierath, Juleen R

    2011-01-01

    DNA methylation is a major epigenetic modification that controls gene expression in physiologic and pathologic states. Metabolic diseases such as diabetes and obesity are associated with profound alterations in gene expression that are caused by genetic and environmental factors. Recent reports...... have provided evidence that environmental factors at all ages could modify DNA methylation in somatic tissues, which suggests that DNA methylation is a more dynamic process than previously appreciated. Because of the importance of lifestyle factors in metabolic disorders, DNA methylation provides...... a mechanism by which environmental factors, including diet and exercise, can modify genetic predisposition to disease. This article considers the current evidence that defines a role for DNA methylation in metabolic disorders....

  6. Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza-2-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line.METHODS: Methylated DNA sequences in genome were enriched with methylated CpG islands amplification (MCA)to undergo representational difference analysis (RDA),with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR (MSP) and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer.Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent.RESULTS: Nineteen differentially methylated sequences were obtained and located at 5' end, exons, introns and 3' end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, aS the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signals except with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent.CONCLUSION: Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical

  7. The DNA methylation events in normal and cloned rabbit embryos

    Institute of Scientific and Technical Information of China (English)

    TaoChen; Yan-LingZhang; YanJiang; Shu-ZhenLiu; HeideSchatten; Da-YuanChen; Qing-YuanSun

    2005-01-01

    To study the DNA methylation events in normal and cloned rabbit embryos, we investigated the methylation status of a satellite seqnence and the promoter region of a single-copy gene using bisulfite-sequencing technology. During normal rabbit embryo development, both sequences maintained hypermethylation status until the 8- to 16-cell stage when progressive demethylation took place. In cloned embryos, the single-copy gene promoter sequence was rapidly demethylated and preco-ciously de novo methylated, while the satellite sequence mainrained the donor-type methylation status in all examined stages. Our results indicate that unique sequences as well as satellitesequences may have aberrant methylation patterns in cloned embryos.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Erin M Siegel

    Full Text Available 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.

  11. Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Nordlund, Jessica; Bäcklin, Christofer L; Wahlberg, Per;

    2013-01-01

    BACKGROUND: Although aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic...

  12. Genome-wide analysis of DNA methylation in hepatoblastoma tissues

    Science.gov (United States)

    Cui, Ximao; Liu, Baihui; Zheng, Shan; Dong, Kuiran; Dong, Rui

    2016-01-01

    DNA methylation has a crucial role in cancer biology. In the present study, a genome-wide analysis of DNA methylation in hepatoblastoma (HB) tissues was performed to verify differential methylation levels between HB and normal tissues. As alpha-fetoprotein (AFP) has a critical role in HB, AFP methylation levels were also detected using pyrosequencing. Normal and HB liver tissue samples (frozen tissue) were obtained from patients with HB. Genome-wide analysis of DNA methylation in these tissues was performed using an Infinium HumanMethylation450 BeadChip, and the results were confirmed with reverse transcription-quantitative polymerase chain reaction. The Infinium HumanMethylation450 BeadChip demonstrated distinctively less methylation in HB tissues than in non-tumor tissues. In addition, methylation enrichment was observed in positions near the transcription start site of AFP, which exhibited lower methylation levels in HB tissues than in non-tumor liver tissues. Lastly, a significant negative correlation was observed between AFP messenger RNA expression and DNA methylation percentage, using linear Pearson's R correlation coefficients. The present results demonstrate differential methylation levels between HB and normal tissues, and imply that aberrant methylation of AFP in HB could reflect HB development. Expansion of these findings could provide useful insight into HB biology.

  13. High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion

    Science.gov (United States)

    Feinweber, Carmen; Knothe, Claudia; Lötsch, Jörn; Thomas, Dominique; Geisslinger, Gerd; Parnham, Michael J.; Resch, Eduard

    2016-01-01

    DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50–80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer. PMID:27749902

  14. Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype

    OpenAIRE

    Van der Auwera, I; Laere, S.J.; Van den Bosch, S M; Van den Eynden, G. G.; Trinh, B X; van Dam, P A; Colpaert, C G; van Engeland, M; Van Marck, E A; Vermeulen, P B; Dirix, L Y

    2008-01-01

    Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal b...

  15. The role of DNA methylation in cancer development.

    Directory of Open Access Journals (Sweden)

    Michał W Luczak

    2006-09-01

    Full Text Available Epigenetic modifications include DNA methylation and covalent modification of histones. These alterations are reversible but very stable and exert a significant impact on the regulation of gene expression. Changes in methylation of promoter or first exon may mimic the effect of mutations of various tumor suppressor genes (TSGs or protooncogenes. Carcinogenesis can also result from aberrations in genomic DNA methylation that include hypermethylation and hypomethylation of promoter or first exon of cancer-related genes. Hypermethylation of promoter of various TSGs causes their transcriptional silencing. However, hypomethylation of regulatory DNA sequences activates transcription of protooncogenes, retrotransposons, as well as genes encoding proteins involved in genomic instability and malignant cell metastasis. The methylation of genomic DNA in malignant cells is catalyzed by DNA methyltransferases DNMT1 and DNMT3B, revealing significantly elevated expression in different types of cancers. The reversibility of hypermethylation can be used as target of therapeutic treatment in cancer. DNMT 1 and DNMT3B inhibitors including 5-Aza-2'-deoxycytidine and antisense oligonucleotides have been applied in clinical trials of such treatment. Identification of aberrations of DNA methylation in cancer cells is a new field of investigation in carcinogenesis. We believe that epigenetic cancer diagnostic and therapy will be achieved in the next decades.

  16. Putting muscle in DNA methylation

    Institute of Scientific and Technical Information of China (English)

    James P Reddington; Richard R Meehan

    2011-01-01

    Over 25 years ago seminal experiments from the labs of Peter Jones and Harold Weintraub demonstrated that alteration in the DNA modification state underlie the myogenic conversion of fibroblast cell lines [1,2].This paved the way for the identification of myogenic helix-loop-helix (HLH) proteins in muscle differentiation,but the mechanism by which DNA methylation regulates muscle differentiation has remained elusive [3].

  17. Clinical potential of DNA methylation in organ transplantation.

    Science.gov (United States)

    Peters, Fleur S; Manintveld, Olivier C; Betjes, Michiel G H; Baan, Carla C; Boer, Karin

    2016-07-01

    Identification of patients at risk for post-transplant complications is a major challenge, but it will improve clinical care and patient health after organ transplantation. The poor predictive value of the current biomarkers highlights the need to explore novel and innovative methods, such as epigenetics, for the discovery of new biomarkers. Cell differentiation and function of immune cells is dependent on epigenetic mechanisms, which regulate gene expression without altering the original DNA sequence. These epigenetic mechanisms are dynamic, potentially heritable, change with age, and can be regulated and influenced by environmental conditions. One of the most well-known epigenetic mechanisms is DNA methylation, which comprises the methylation of a cytosine (C) next to a guanine (G; CpG dinucleotides). Aberrant DNA methylation is increasingly associated with disease, including immune-mediated diseases, and these alterations precede the clinical phenotype. The impact of DNA methylation profiles on transplant acceptance and rejection as well as on other post-transplant complications is unknown. In this study we examine the current evidence of the functional role of recipient and donor DNA methylation on outcome after organ transplantation. Changes in DNA methylation may predict the risk of developing post-transplant complications, such as infections, malignancies and allograft rejection. We speculate that identification of these changes in DNA methylation contributes to earlier diagnosis and prevention of post-transplant complications, leading to improved patient care. PMID:27085975

  18. DNA methylation and carcinogenesis.

    Science.gov (United States)

    Lichtenstein, A V; Kisseljova, N P

    2001-03-01

    In the world of easy things truth is opposed to lie; in the world of complicated things one profound truth is opposed to another not less profound than the first. Neils Bohr The hypothesis of the exclusively genetic origin of cancer ("cancer is a disease of genes, a tumor without any damage to the genome does not exist") dominated in the oncology until recently. A considerable amount of data confirming this hypothesis was accumulated during the last quarter of the last century. It was demonstrated that the accumulation of damage of specific genes lies at the origin of a tumor and its following progression. The damage gives rise to structural changes in the respective proteins and, consequently, to inappropriate mitogenic stimulation of cells (activation of oncogenes) or to the inactivation of tumor suppressor genes that inhibit cell division, or to the combination of both (in most cases). According to an alternative (epigenetic) hypothesis that was extremely unpopular until recently, a tumor is caused not by a gene damage, but by an inappropriate function of genes ("cancer is a disease of gene regulation and differentiation"). However, recent studies led to the convergence of these hypotheses that initially seemed to be contradictory. It was established that both factors--genetic and epigenetic--lie at the origin of carcinogenesis. The relative contribution of each varies significantly in different human tumors. Suppressor genes and genes of repair are inactivated in tumors due to their damage or methylation of their promoters (in the latter case an "epimutation", an epigenetic equivalent of a mutation, occurs, producing the same functional consequences). It is becoming evident that not only the mutagens, but various factors influencing cell metabolism, notably methylation, should be considered as carcinogens.

  19. A novel method for detecting 7-methyl guanine reveals aberrant methylation levels in Huntington disease

    OpenAIRE

    Thomas, Beena; Matson, Samantha; Chopra, Vanita; Sun, Liping; Sharma, Swati; Hersch, Steven; Rosas, H. Diana; Scherzer, Clemens; Ferrante, Robert; Matson, Wayne

    2013-01-01

    Guanine methylation is a ubiquitous process affecting DNA and various RNA species. N-7 guanine methylation (7-MG), though relatively less studied, could have a significant role in normal transcriptional regulation as well as in the onset and development of pathological conditions. The lack of a sensitive method to accurately quantify trace amounts of altered bases like 7-MG, has been a major deterrent in delineating its biological function(s). Here we report the development of methods to dete...

  20. miRNAting control of DNA methylation

    Indian Academy of Sciences (India)

    Ashwani Jha; Ravi Shankar

    2014-06-01

    DNA methylation is a type of epigenetic modification where a methyl group is added to the cytosine or adenine residue of a given DNA sequence. It has been observed that DNA methylation is achieved by some collaborative agglomeration of certain proteins and non-coding RNAs. The assembly of IDN2 and its homologous proteins with siRNAs recruits the enzyme DRM2, which adds a methyl group at certain cytosine residues within the DNA sequence. In this study, it was found that de novo DNA methylation might be regulated by miRNAs through systematic targeting of the genes involved in DNA methylation. A comprehensive genome-wide and system-level study of miRNA targeting, transcription factors, DNA-methylation-causing genes and their target genes has provided a clear picture of an interconnected relationship of all these factors which regulate DNA methylation in Arabidopsis. The study has identified a DNA methylation system that is controlled by four different genes: IDN2, IDNl1, IDNl2 and DRM2. These four genes along with various critical transcription factors appear to be controlled by five different miRNAs. Altogether, DNA methylation appears to be a finely tuned process of opposite control systems of DNA-methylation-causing genes and certain miRNAs pitted against each other.

  1. Electronic transport in methylated fragments of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L., E-mail: umbertofulco@gmail.com; Albuquerque, E. L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Freire, V. N. [Departamento de Física, Universidade Federal do Ceará, 60455-760 Fortaleza, CE (Brazil); Caetano, E. W. S. [Instituto Federal de Educação, Ciência e Tecnologia do Ceará, 60040-531 Fortaleza, CE (Brazil); Moura, F. A. B. F. de; Lyra, M. L. [Instituto de Física, Universidade Federal de Alagoas, 57072-900 Maceió-AL (Brazil)

    2015-11-16

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  2. DNA methylation detection based on difference of base content

    Science.gov (United States)

    Sato, Shinobu; Ohtsuka, Keiichi; Honda, Satoshi; Sato, Yusuke; Takenaka, Shigeori

    2016-04-01

    Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.

  3. Brahmarasayana protects against Ethyl methanesulfonate or Methyl methanesulfonate induced chromosomal aberrations in mouse bone marrow cells

    Directory of Open Access Journals (Sweden)

    Guruprasad Kanive

    2012-08-01

    Full Text Available Abstract Background Ayurveda, the traditional Indian system of medicine has given great emphasis to the promotion of health. Rasayana is one of the eight branches of Ayurveda which refers to rejuvenant therapy. It has been reported that rasayanas have immuno-modulatory, antioxidant and antitumor functions, however, the genotoxic potential and modulation of DNA repair of many rasayanas have not been evaluated. Methods The present study assessed the role of Brahmarasayana (BR on Ethyl methanesulfonate (EMS-and Methyl methanesulfonate (MMS-induced genotoxicity and DNA repair in in vivo mouse test system. The mice were orally fed with BR (5 g or 8 mg / day for two months and 24 h later EMS or MMS was given intraperitoneally. The genotoxicity was analyzed by chromosomal aberrations, sperm count, and sperm abnormalities. Results The results have revealed that BR did not induce significant chromosomal aberrations when compared to that of the control animals (p >0.05. On the other hand, the frequencies of chromosomal aberrations induced by EMS (240 mg / kg body weight or MMS (125 mg / kg body weight were significantly higher (p Conclusion The effect of BR, as it relates to antioxidant activity was not evident in liver tissue however rasayana treatment was observed to increase constitutive DNA base excision repair and reduce clastogenicity. Whilst, the molecular mechanisms of such repair need further exploration, this is the first report to demonstrate these effects and provides further evidence for the role of brahmarasayana in the possible improvement of quality of life.

  4. Quantitation of DNA methylation by melt curve analysis

    Directory of Open Access Journals (Sweden)

    Jones Michael E

    2009-04-01

    Full Text Available Abstract Background Methylation of DNA is a common mechanism for silencing genes, and aberrant methylation is increasingly being implicated in many diseases such as cancer. There is a need for robust, inexpensive methods to quantitate methylation across a region containing a number of CpGs. We describe and validate a rapid, in-tube method to quantitate DNA methylation using the melt data obtained following amplification of bisulfite modified DNA in a real-time thermocycler. Methods We first describe a mathematical method to normalise the raw fluorescence data generated by heating the amplified bisulfite modified DNA. From this normalised data the temperatures at which melting begins and finishes can be calculated, which reflect the less and more methylated template molecules present respectively. Also the T50, the temperature at which half the amplicons are melted, which represents the summative methylation of all the CpGs in the template mixture, can be calculated. These parameters describe the methylation characteristics of the region amplified in the original sample. Results For validation we used synthesized oligonucleotides and DNA from fresh cells and formalin fixed paraffin embedded tissue, each with known methylation. Using our quantitation we could distinguish between unmethylated, partially methylated and fully methylated oligonucleotides mixed in varying ratios. There was a linear relationship between T50 and the dilution of methylated into unmethylated DNA. We could quantitate the change in methylation over time in cell lines treated with the demethylating drug 5-aza-2'-deoxycytidine, and the differences in methylation associated with complete, clonal or no loss of MGMT expression in formalin fixed paraffin embedded tissues. Conclusion We have validated a rapid, simple in-tube method to quantify methylation which is robust and reproducible, utilizes easily designed primers and does not need proprietary algorithms or software. The

  5. First evidence of DNA methylation in insect Tribolium castaneum: environmental regulation of DNA methylation within heterochromatin.

    Science.gov (United States)

    Feliciello, Isidoro; Parazajder, Josip; Akrap, Ivana; Ugarković, Durđica

    2013-05-01

    DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.

  6. Absolute Quantitation of DNA Methylation of 28 Candidate Genes in Prostate Cancer Using Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Nataڑa Vasiljeviš

    2011-01-01

    Full Text Available Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa. We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH samples using the pyrosequencing (PSQ method to identify genes with diagnostic and prognostic potential.

  7. Prioritizing cancer-related genes with aberrant methylation based on a weighted protein-protein interaction network

    Directory of Open Access Journals (Sweden)

    Lv Jie

    2011-10-01

    Full Text Available Abstract Background As an important epigenetic modification, DNA methylation plays a crucial role in the development of mammals and in the occurrence of complex diseases. Genes that interact directly or indirectly may have the same or similar functions in the biological processes in which they are involved and together contribute to the related disease phenotypes. The complicated relations between genes can be clearly represented using network theory. A protein-protein interaction (PPI network offers a platform from which to systematically identify disease-related genes from the relations between genes with similar functions. Results We constructed a weighted human PPI network (WHPN using DNA methylation correlations based on human protein-protein interactions. WHPN represents the relationships of DNA methylation levels in gene pairs for four cancer types. A cancer-associated subnetwork (CASN was obtained from WHPN by selecting genes associated with seed genes which were known to be methylated in the four cancers. We found that CASN had a more densely connected network community than WHPN, indicating that the genes in CASN were much closer to seed genes. We prioritized 154 potential cancer-related genes with aberrant methylation in CASN by neighborhood-weighting decision rule. A function enrichment analysis for GO and KEGG indicated that the optimized genes were mainly involved in the biological processes of regulating cell apoptosis and programmed cell death. An analysis of expression profiling data revealed that many of the optimized genes were expressed differentially in the four cancers. By examining the PubMed co-citations, we found 43 optimized genes were related with cancers and aberrant methylation, and 10 genes were validated to be methylated aberrantly in cancers. Of 154 optimized genes, 27 were as diagnostic markers and 20 as prognostic markers previously identified in literature for cancers and other complex diseases by searching Pub

  8. Folic acid, polymorphism of methyl-group metabolism genes, and DNA methylation in relation to GI carcinogenesis.

    Science.gov (United States)

    Fang, Jing Yuan; Xiao, Shu Dong

    2003-01-01

    DNA methylation is the main epigenetic modification after replication in humans. DNA (cytosine-5)-methyltransferase (DNMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to C5 of cytosine within CpG dinucleotide sequences in the genomic DNA of higher eukaryotes. There is considerable evidence that aberrant DNA methylation plays an integral role in carcinogenesis. Folic acid or folate is crucial for normal DNA synthesis and can regulate DNA methylation, and through this, it affects cellular SAM levels. Folate deficiency results in DNA hypomethylation. Epidemiological studies have indicated that folic acid protects against gastrointestinal (GI) cancers. Methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are the enzymes involved in folate metabolism and are thought to influence DNA methylation. MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level. Two common MTHFR polymorphisms, 677CT (or 677TT) and A1298C, and an MS polymorphism, A-->G at 2756, have been identified. Most studies support an inverse association between folate status and the rate of colorectal adenomas and carcinomas. During human GI carcinogenesis, MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level, as well as aberrant methylation.

  9. Influence of DNA methylation on transgene expression

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene b-glu- curonidase (GUS) gene (uidA) was introduced into tobaccos via Agrobacterium-mediated transformation method, and the foreign uidA gene became inactive in some transgenic tobaccos. No mRNA of uidA was detected in these plants by Northern blotting analysis, and DNA methylation of promoter region was found. The results indicated that gene silencing might be caused by DNA methylation of promoter.

  10. Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

    Science.gov (United States)

    Pal, Arnab; Srivastava, Tapasya; Sharma, Manish K; Mehndiratta, Mohit; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

    2010-11-01

    Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells.

  11. Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors

    International Nuclear Information System (INIS)

    Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above

  12. An Integrated Workflow for DNA Methylation Analysis

    Institute of Scientific and Technical Information of China (English)

    Pingchuan Li; Feray Demirci; Gayathri Mahalingam; Caghan Demirci; Mayumi Nakano; Blake C.Meyers

    2013-01-01

    The analysis of cytosine methylation provides a new way to assess and describe epigenetic regulation at a whole-genome level in many eukaryotes.DNA methylation has a demonstrated role in the genome stability and protection,regulation of gene expression and many other aspects of genome function and maintenance.BS-seq is a relatively unbiased method for profiling the DNA methylation,with a resolution capable of measuring methylation at individual cytosines.Here we describe,as an example,a workflow to handle DNA methylation analysis,from BS-seq library preparation to the data visualization.We describe some applications for the analysis and interpretation of these data.Our laboratory provides public access to plant DNA methylation data via visualization tools available at our "Next-Gen Sequence" websites (http://mpss.udel.edu),along with small RNA,RNA-seq and other data types.

  13. Radiation effects on DNA methylation in mice

    International Nuclear Information System (INIS)

    Effects of ionizing radiation on DNA methylation in liver, brain and spleen were examined by high performance liquid chromatography (HPLC). The total methylated cytosine level in the genome was reduced within 8 hours after 3.8 Gy of irradiation in liver of adult mice. But no appreciable effect was observed in brain and spleen. When mice were irradiated at newborn, liver DNA revealed no change in methylated cytosine level. Even though slight effects of radiation were detected in he methylation of the c-myc and c-fos genes, they were only temporary and no long-term effects were observed. These data suggest that the effect of radiation on DNA methylation in vivo is not prevailing a DNA damage, but rather influenced much through biological parameters. (author)

  14. Turning over DNA methylation in the mind

    Directory of Open Access Journals (Sweden)

    Ryan eLister

    2015-07-01

    Full Text Available Cytosine DNA methylation is a stable epigenetic modification with established roles in regulating transcription, imprinting, female X-chromosome inactivation, and silencing of transposons. Dynamic gain or loss of DNA methylation reshapes the genomic landscape of cells during early differentiation, and in post-mitotic mammalian brain cells these changes continue to accumulate throughout the phases of cortical maturation in childhood and adolescence. There is also evidence for dynamic changes in the methylation status of specific genomic loci during the encoding of new memories, and these epigenome dynamics could play a causal role in memory formation. However, the mechanisms that may dynamically regulate DNA methylation in neurons during memory formation and expression, and the function of such epigenomic changes in this context, are unclear. Here we discuss the possible roles of DNA methylation in encoding and retrieval of memory.

  15. DNA Methylation of BDNF Gene in Schizophrenia

    Science.gov (United States)

    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

  16. Androgen receptor function links human sexual dimorphism to DNA methylation.

    Directory of Open Access Journals (Sweden)

    Ole Ammerpohl

    Full Text Available Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

  17. Dietary and lifestyle factors of DNA methylation.

    Science.gov (United States)

    Lim, Unhee; Song, Min-Ae

    2012-01-01

    Lifestyle factors, such as diet, smoking, physical activity, and body weight management, are known to constitute the majority of cancer causes. Epigenetics has been widely proposed as a main mechanism that mediates the reversible effects of dietary and lifestyle factors on carcinogenesis. This chapter reviews human studies on potential dietary and lifestyle determinants of DNA methylation. Apart from a few prospective investigations and interventions of limited size and duration, evidence mostly comes from cross-sectional observational studies and supports some associations. Studies to date suggest that certain dietary components may alter genomic and gene-specific DNA methylation levels in systemic and target tissues, affecting genomic stability and transcription of tumor suppressors and oncogenes. Most data and supportive evidence exist for folate, a key nutritional factor in one-carbon metabolism that supplies the methyl units for DNA methylation. Other candidate bioactive food components include alcohol and other key nutritional factors of one-carbon metabolism, polyphenols and flavonoids in green tea, phytoestrogen, and lycopene. Some data also support a link of DNA methylation with physical activity and energy balance. Effects of dietary and lifestyle exposures on DNA methylation may be additionally modified by common genetic variants, environmental carcinogens, and infectious agents, an aspect that remains largely unexplored. In addition, growing literature supports that the environmental conditions during critical developmental stages may influence later risk of metabolic disorders in part through persistent programming of DNA methylation. Further research of these modifiable determinants of DNA methylation will improve our understanding of cancer etiology and may present certain DNA methylation markers as attractive surrogate endpoints for prevention research. Considering the plasticity of epigenetic marks and correlated nature of lifestyle factors, more

  18. DNA Methylation Biomarkers: Cancer and Beyond

    Directory of Open Access Journals (Sweden)

    Thomas Mikeska

    2014-09-01

    Full Text Available Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.

  19. Targeting DNA Methylation for Epigenetic Therapy

    Science.gov (United States)

    Yang, Xiaojing; Lay, Fides; Han, Han; Jones, Peter A.

    2010-01-01

    DNA methylation patterns are established during embryonic development and faithfully copied through somatic cell divisions. Based on our understanding of DNA methylation and other interrelated epigenetic modifications, a comprehensive view of the epigenetic landscape and cancer epigenome is evolving. The cancer methylome is highly disrupted, making DNA methylation an excellent target for anti-cancer therapies. During the last few decades, an increasing number of drugs targeting DNA methylation have been developed in an effort to increase efficacy, stability and to decrease toxicity. The earliest and the most successful epigenetic drug to date, 5-Azacytidine, is currently recommended as the first-line treatment for high risk myelodysplastic syndromes (MDS) patients. Encouraging results from clinical trials have prompted further efforts to elucidate epigenetic alterations in cancer and subsequently develop new epigenetic therapies. This review delineates the latest cancer epigenetic models, recent discovery of hypomethylation agents and their application in the clinic. PMID:20846732

  20. Dysregulation of microRNA expression drives aberrant DNA hypermethylation in basal-like breast cancer.

    Science.gov (United States)

    Sandhu, Rupninder; Rivenbark, Ashley G; Mackler, Randi M; Livasy, Chad A; Coleman, William B

    2014-02-01

    Basal-like breast cancers frequently express aberrant DNA hypermethylation associated with concurrent silencing of specific genes secondary to DNMT3b overexpression and DNMT hyperactivity. DNMT3b is known to be post-transcriptionally regulated by microRNAs. The objective of the current study was to determine the role of microRNA dysregulation in the molecular mechanism governing DNMT3b overexpression in primary breast cancers that express aberrant DNA hypermethylation. The expression of microRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a and miR-148b) or are predicted to regulate DNMT3b (miR‑26a, miR-26b, miR-203 and miR-222) were evaluated among 70 primary breast cancers (36 luminal A-like, 13 luminal B-like, 5 HER2‑enriched, 16 basal-like) and 18 normal mammoplasty tissues. Significantly reduced expression of miR-29c distinguished basal-like breast cancers from other breast cancer molecular subtypes. The expression of aberrant DNA hypermethylation was determined in a subset of 33 breast cancers (6 luminal A-like, 6 luminal B-like, 5 HER2-enriched and 16 basal-like) through examination of methylation‑sensitive biomarker gene expression (CEACAM6, CDH1, CST6, ESR1, GNA11, MUC1, MYB, TFF3 and SCNN1A), 11/33 (33%) cancers exhibited aberrant DNA hypermethylation including 9/16 (56%) basal-like cancers, but only 2/17 (12%) non-basal-like cancers (luminal A-like, n=1; HER2-enriched, n=1). Breast cancers with aberrant DNA hypermethylation express diminished levels of miR-29a, miR-29b, miR-26a, miR-26b, miR-148a and miR-148b compared to cancers lacking aberrant DNA hypermethylation. A total of 7/9 (78%) basal-like breast cancers with aberrant DNA hypermethylation exhibit diminished levels of ≥6 regulatory miRs. The results show that i) reduced expression of miR-29c is characteristic of basal-like breast cancers, ii) miR and methylation-sensitive gene expression patterns identify two subsets of basal-like breast cancers, and iii) the subset of basal

  1. Blood diagnostic biomarkers for major depressive disorder using multiplex DNA methylation profiles: discovery and validation.

    Science.gov (United States)

    Numata, Shusuke; Ishii, Kazuo; Tajima, Atsushi; Iga, Jun-ichi; Kinoshita, Makoto; Watanabe, Shinya; Umehara, Hidehiro; Fuchikami, Manabu; Okada, Satoshi; Boku, Shuken; Hishimoto, Akitoyo; Shimodera, Shinji; Imoto, Issei; Morinobu, Shigeru; Ohmori, Tetsuro

    2015-01-01

    Aberrant DNA methylation in the blood of patients with major depressive disorder (MDD) has been reported in several previous studies. However, no comprehensive studies using medication-free subjects with MDD have been conducted. Furthermore, the majority of these previous studies has been limited to the analysis of the CpG sites in CpG islands (CGIs) in the gene promoter regions. The main aim of the present study is to identify DNA methylation markers that distinguish patients with MDD from non-psychiatric controls. Genome-wide DNA methylation profiling of peripheral leukocytes was conducted in two set of samples, a discovery set (20 medication-free patients with MDD and 19 controls) and a replication set (12 medication-free patients with MDD and 12 controls), using Infinium HumanMethylation450 BeadChips. Significant diagnostic differences in DNA methylation were observed at 363 CpG sites in the discovery set. All of these loci demonstrated lower DNA methylation in patients with MDD than in the controls, and most of them (85.7%) were located in the CGIs in the gene promoter regions. We were able to distinguish patients with MDD from the control subjects with high accuracy in the discriminant analysis using the top DNA methylation markers. We also validated these selected DNA methylation markers in the replication set. Our results indicate that multiplex DNA methylation markers may be useful for distinguishing patients with MDD from non-psychiatric controls.

  2. DNA methylation dynamics in human induced pluripotent stem cells over time.

    Directory of Open Access Journals (Sweden)

    Koichiro Nishino

    2011-05-01

    Full Text Available Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs. Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell and five human embryonic stem cell (ESC lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the "convergence" of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs.

  3. Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

    Energy Technology Data Exchange (ETDEWEB)

    Novak, Petr; Jensen, Taylor J.; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2009-04-20

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.

  4. Modifying the comet assay for measuring global DNA methylation in a variety of tissue cells / Johannes Frederik Wentzel

    OpenAIRE

    Wentzel, Johannes Frederik

    2010-01-01

    It is becoming abundantly clear that DNA methylation plays a crucial role in gene regulation and that aberrant regulation of DNA methylation influences the development of certain diseases such as cancer. Although a wide variety of methylation analysis techniques are available today, they are still relatively expensive and a large number of them is platform specific. The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive and simple technique which is traditionally use...

  5. Aberrant promoter CpG methylation and its translational applications in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Ting-Xiu Xiang; Ying Yuan; Li-Li Li; Zhao-Hui Wang; Liang-Ying Dan; Yan Chen; Guo-Sheng Ren; Qian Tao

    2013-01-01

    Breast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations.Recent studies revealed that abnormal gene expression induced by epigenetic changes,including aberrant promoter methylation and histone modification,plays a critical role in human breast carcinogenesis.Silencing of tumor suppressor genes (TSGs) by promoter CpG methylation facilitates cells growth and survival advantages and further results in tumor initiation and progression,thus directly contributing to breast tumorigenesis.Usually,aberrant promoter methylation of TSGs,which can be reversed by pharmacological reagents,occurs at the early stage of tumorigenesis and therefore may serve as a potential tumor marker for early diagnosis and therapeutic targeting of breast cancer.In this review,we summarize the epigenetic changes of multiple TSGs involved in breast pathogenesis and their potential clinical applications as tumor markers for early detection and treatment of breast cancer.

  6. Adult porcine genome-wide DNA methylation patterns support pigs as a biomedical model

    NARCIS (Netherlands)

    Schachtschneider, K.M.; Madsen, O.; Park, C.; Rund, L.A.; Groenen, M.A.M.; Schook, L.B.

    2015-01-01

    Background: Pigs (Sus scrofa) provide relevant biomedical models to dissect complex diseases due to their anatomical, genetic, and physiological similarities with humans. Aberrant DNA methylation has been linked to many of these diseases and is associated with gene expression; however, the functiona

  7. Base-pair resolution DNA methylation sequencing reveals profoundly divergent epigenetic landscapes in acute myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Altuna Akalin

    Full Text Available We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML, we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions.

  8. Expression and aberrant promoter methylation of Wnt inhibitory factor-1 in human astrocytomas

    Directory of Open Access Journals (Sweden)

    Wu Jun

    2010-03-01

    Full Text Available Abstract Background Wnt inhibitory factor-1(WIF-1 acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the expression and promoter methylation status of WIF-1 gene in human astrocytomas. Methods The tissue samples consisted of 53 astrocytomas and 6 normal brain tissues. The expression levels of WIF-1 were determined by immunohistochemistry and semiquantitative RT-PCR. The results were analyzed in correlation with clinicopathological data. Methylation status of WIF-1 gene promoter was investigated using methylation specific PCR. The relationship between methylation and expression of the genes was analyzed. Results The average expression levels of WIF-1 protein and mRNA in astrocytomas were decreased significantly compared with normal control tissues. The protein and mRNA expression of WIF-1 gene in astrocytomas was decreased with the increase of pathological grade. Furthermore, WIF-1 promoter methylation was observed by MS-PCR in astrocytomas which showed significant reduction of WIF-1 expression. The WIF-1 promoter hypermethylation was associated with reduced expression of WIF-1 expression. Conclusion Our results demonstrate that the WIF-1 gene is frequently down-regulated or silenced in astrocytomas by aberrant promoter methylation. This may be an important mechanism in astrocytoma carcinogenesis.

  9. Editing DNA Methylation in the Mammalian Genome.

    Science.gov (United States)

    Liu, X Shawn; Wu, Hao; Ji, Xiong; Stelzer, Yonatan; Wu, Xuebing; Czauderna, Szymon; Shu, Jian; Dadon, Daniel; Young, Richard A; Jaenisch, Rudolf

    2016-09-22

    Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation. PMID:27662091

  10. Methylation of cell-free circulating DNA in the diagnosis of cancer

    Science.gov (United States)

    Warton, Kristina; Samimi, Goli

    2015-01-01

    A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. Unlike mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. Since DNA methylation is reflected within circDNA, detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA) for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker. PMID:25988180

  11. Prognostic DNA Methylation Markers for Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Siri H. Strand

    2014-09-01

    Full Text Available Prostate cancer (PC is the most commonly diagnosed neoplasm and the third most common cause of cancer-related death amongst men in the Western world. PC is a clinically highly heterogeneous disease, and distinction between aggressive and indolent disease is a major challenge for the management of PC. Currently, no biomarkers or prognostic tools are able to accurately predict tumor progression at the time of diagnosis. Thus, improved biomarkers for PC prognosis are urgently needed. This review focuses on the prognostic potential of DNA methylation biomarkers for PC. Epigenetic changes are hallmarks of PC and associated with malignant initiation as well as tumor progression. Moreover, DNA methylation is the most frequently studied epigenetic alteration in PC, and the prognostic potential of DNA methylation markers for PC has been demonstrated in multiple studies. The most promising methylation marker candidates identified so far include PITX2, C1orf114 (CCDC181 and the GABRE~miR-452~miR-224 locus, in addition to the three-gene signature AOX1/C1orf114/HAPLN3. Several other biomarker candidates have also been investigated, but with less stringent clinical validation and/or conflicting evidence regarding their possible prognostic value available at this time. Here, we review the current evidence for the prognostic potential of DNA methylation markers in PC.

  12. Prognostic DNA methylation markers for prostate cancer.

    Science.gov (United States)

    Strand, Siri H; Orntoft, Torben F; Sorensen, Karina D

    2014-01-01

    Prostate cancer (PC) is the most commonly diagnosed neoplasm and the third most common cause of cancer-related death amongst men in the Western world. PC is a clinically highly heterogeneous disease, and distinction between aggressive and indolent disease is a major challenge for the management of PC. Currently, no biomarkers or prognostic tools are able to accurately predict tumor progression at the time of diagnosis. Thus, improved biomarkers for PC prognosis are urgently needed. This review focuses on the prognostic potential of DNA methylation biomarkers for PC. Epigenetic changes are hallmarks of PC and associated with malignant initiation as well as tumor progression. Moreover, DNA methylation is the most frequently studied epigenetic alteration in PC, and the prognostic potential of DNA methylation markers for PC has been demonstrated in multiple studies. The most promising methylation marker candidates identified so far include PITX2, C1orf114 (CCDC181) and the GABRE~miR-452~miR-224 locus, in addition to the three-gene signature AOX1/C1orf114/HAPLN3. Several other biomarker candidates have also been investigated, but with less stringent clinical validation and/or conflicting evidence regarding their possible prognostic value available at this time. Here, we review the current evidence for the prognostic potential of DNA methylation markers in PC. PMID:25238417

  13. Expression and aberrant promoter methylation of Wnt inhibitory factor-1 in human astrocytomas

    OpenAIRE

    Wu Jun; Liu Jinfang; Chen Fenghua; Fang Jiasheng; Wang Ying; Yang Zhuanyi; Wang Yanjin

    2010-01-01

    Abstract Background Wnt inhibitory factor-1(WIF-1) acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the expression and promoter methylation status of WIF-1 gene in human astrocytomas. Methods The tissue samples consisted of 53 astrocytomas and 6 normal brain tissues. The expression levels of WIF-1 were det...

  14. Aberrant gene methylation implicated in the progression of monoclonal gammopathy of undetermined significance to multiple myeloma

    OpenAIRE

    Chim, Chor‐Sang; Liang, Raymond; Leung, Man‐Hin; Kwong, Yok‐Lam

    2007-01-01

    Malignant transformation is a multistep process that may involve dysregulation of oncogenes and tumour suppressor genes, and monoclonal gammopathy of undetermined significance (MGUS) is believed to be a precursor of multiple myeloma. To investigate whether aberrant promoter methylation might be involved in the evolution of MGUS to multiple myeloma, we examined the p16, protein tyrosine phosphatase, non-receptor type 6 (SHP1), death-associated protein (DAP) kinase, E-cadherin and oestrogen rec...

  15. Classification of Epstein-Barr virus-positive gastric cancers by definition of DNA methylation epigenotypes.

    Science.gov (United States)

    Matsusaka, Keisuke; Kaneda, Atsushi; Nagae, Genta; Ushiku, Tetsuo; Kikuchi, Yasuko; Hino, Rumi; Uozaki, Hiroshi; Seto, Yasuyuki; Takada, Kenzo; Aburatani, Hiroyuki; Fukayama, Masashi

    2011-12-01

    Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, opportunistic lymphomas in immunocompromised hosts, and a fraction of gastric cancers. Aberrant promoter methylation accompanies human gastric carcinogenesis, though the contribution of EBV to such somatic methylation changes has not been fully clarified. We analyzed promoter methylation in gastric cancer cases with Illumina's Infinium BeadArray and used hierarchical clustering analysis to classify gastric cancers into 3 subgroups: EBV(-)/low methylation, EBV(-)/high methylation, and EBV(+)/high methylation. The 3 epigenotypes were characterized by 3 groups of genes: genes methylated specifically in the EBV(+) tumors (e.g., CXXC4, TIMP2, and PLXND1), genes methylated both in EBV(+) and EBV(-)/high tumors (e.g., COL9A2, EYA1, and ZNF365), and genes methylated in all of the gastric cancers (e.g., AMPH, SORCS3, and AJAP1). Polycomb repressive complex (PRC) target genes in embryonic stem cells were significantly enriched among EBV(-)/high-methylation genes and commonly methylated gastric cancer genes (P = 2 × 10(-15) and 2 × 10(-34), respectively), but not among EBV(+) tumor-specific methylation genes (P = 0.2), suggesting a different cause for EBV(+)-associated de novo methylation. When recombinant EBV was introduced into the EBV(-)/low-methylation epigenotype gastric cancer cell, MKN7, 3 independently established subclones displayed increases in DNA methylation. The promoters targeted by methylation were mostly shared among the 3 subclones, and the new methylation changes caused gene repression. In summary, DNA methylation profiling classified gastric cancer into 3 epigenotypes, and EBV(+) gastric cancers showed distinct methylation patterns likely attributable to EBV infection.

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

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2006-01-01

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

  17. DNA methylation and healthy human aging.

    Science.gov (United States)

    Jones, Meaghan J; Goodman, Sarah J; Kobor, Michael S

    2015-12-01

    The process of aging results in a host of changes at the cellular and molecular levels, which include senescence, telomere shortening, and changes in gene expression. Epigenetic patterns also change over the lifespan, suggesting that epigenetic changes may constitute an important component of the aging process. The epigenetic mark that has been most highly studied is DNA methylation, the presence of methyl groups at CpG dinucleotides. These dinucleotides are often located near gene promoters and associate with gene expression levels. Early studies indicated that global levels of DNA methylation increase over the first few years of life and then decrease beginning in late adulthood. Recently, with the advent of microarray and next-generation sequencing technologies, increases in variability of DNA methylation with age have been observed, and a number of site-specific patterns have been identified. It has also been shown that certain CpG sites are highly associated with age, to the extent that prediction models using a small number of these sites can accurately predict the chronological age of the donor. Together, these observations point to the existence of two phenomena that both contribute to age-related DNA methylation changes: epigenetic drift and the epigenetic clock. In this review, we focus on healthy human aging throughout the lifetime and discuss the dynamics of DNA methylation as well as how interactions between the genome, environment, and the epigenome influence aging rates. We also discuss the impact of determining 'epigenetic age' for human health and outline some important caveats to existing and future studies. PMID:25913071

  18. DNA methylation changes are a late event in acute promyelocytic leukemia and coincide with loss of transcription factor binding

    DEFF Research Database (Denmark)

    Schoofs, Till; Rohde, Christian; Hebestreit, Katja;

    2013-01-01

    . Transcription factor-binding sites (eg, the c-myc-binding sites) were associated with low methylation. However, SUZ12- and REST-binding sites identified in embryonic stem cells were preferentially DNA hypermethylated in APL cells. Unexpectedly, PML-RARα-binding sites were also protected from aberrant DNA...

  19. Regulation and function of DNA methylation in plants and animals

    KAUST Repository

    He, Xinjian

    2011-02-15

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. © 2011 IBCB, SIBS, CAS All rights reserved.

  20. DNA methylation markers for breast cancer prognosis

    OpenAIRE

    Dedeurwaerder, Sarah; Fuks, François

    2012-01-01

    Currently, most of the prognostic and predictive gene expression signatures emerging for breast cancer concern the tumor component. In Dedeurwaerder et al. we show that DNA methylation profiling of breast tumors is a particularly sensitive means of capturing features of the immune component of breast tumors. Most importantly, correlation is observed between T-cell marker genes and breast cancer clinical outcome.

  1. Global DNA methylation of ischemic stroke subtypes.

    Directory of Open Access Journals (Sweden)

    Carolina Soriano-Tárraga

    Full Text Available Ischemic stroke (IS, a heterogeneous multifactorial disorder, is among the leading causes of mortality and long-term disability in the western world. Epidemiological data provides evidence for a genetic component to the disease, but its epigenetic involvement is still largely unknown. Epigenetic mechanisms, such as DNA methylation, change over time and may be associated with aging processes and with modulation of the risk of various pathologies, such as cardiovascular disease and stroke. We analyzed 2 independent cohorts of IS patients. Global DNA methylation was measured by luminometric methylation assay (LUMA of DNA blood samples. Univariate and multivariate regression analyses were used to assess the methylation differences between the 3 most common IS subtypes, large-artery atherosclerosis (LAA, small-artery disease (SAD, and cardio-aortic embolism (CE. A total of 485 IS patients from 2 independent hospital cohorts (n = 281 and n = 204 were included, distributed across 3 IS subtypes: LAA (78/281, 59/204, SAD (97/281, 53/204, and CE (106/281, 89/204. In univariate analyses, no statistical differences in LUMA levels were observed between the 3 etiologies in either cohort. Multivariate analysis, adjusted by age, sex, hyperlipidemia, and smoking habit, confirmed the lack of differences in methylation levels between the analyzed IS subtypes in both cohorts. Despite differences in pathogenesis, our results showed no global methylation differences between LAA, SAD, and CE subtypes of IS. Further work is required to establish whether the epigenetic mechanism of methylation might play a role in this complex disease.

  2. Information Thermodynamics of Cytosine DNA Methylation.

    Science.gov (United States)

    Sanchez, Robersy; Mackenzie, Sally A

    2016-01-01

    Cytosine DNA methylation (CDM) is a stable epigenetic modification to the genome and a widespread regulatory process in living organisms that involves multicomponent molecular machines. Genome-wide cytosine methylation patterning participates in the epigenetic reprogramming of a cell, suggesting that the biological information contained within methylation positions may be amenable to decoding. Adaptation to a new cellular or organismal environment also implies the potential for genome-wide redistribution of CDM changes that will ensure the stability of DNA molecules. This raises the question of whether or not we would be able to sort out the regulatory methylation signals from the CDM background ("noise") induced by thermal fluctuations. Here, we propose a novel statistical and information thermodynamic description of the CDM changes to address the last question. The physical basis of our statistical mechanical model was evaluated in two respects: 1) the adherence to Landauer's principle, according to which molecular machines must dissipate a minimum energy ε = kBT ln2 at each logic operation, where kB is the Boltzmann constant, and T is the absolute temperature and 2) whether or not the binary stretch of methylation marks on the DNA molecule comprise a language of sorts, properly constrained by thermodynamic principles. The study was performed for genome-wide methylation data from 152 ecotypes and 40 trans-generational variations of Arabidopsis thaliana and 93 human tissues. The DNA persistence length, a basic mechanical property altered by CDM, was estimated with values from 39 to 66.9 nm. Classical methylome analysis can be retrieved by applying information thermodynamic modelling, which is able to discriminate signal from noise. Our finding suggests that the CDM signal comprises a language scheme properly constrained by molecular thermodynamic principles, which is part of an epigenomic communication system that obeys the same thermodynamic rules as do current

  3. Information Thermodynamics of Cytosine DNA Methylation.

    Science.gov (United States)

    Sanchez, Robersy; Mackenzie, Sally A

    2016-01-01

    Cytosine DNA methylation (CDM) is a stable epigenetic modification to the genome and a widespread regulatory process in living organisms that involves multicomponent molecular machines. Genome-wide cytosine methylation patterning participates in the epigenetic reprogramming of a cell, suggesting that the biological information contained within methylation positions may be amenable to decoding. Adaptation to a new cellular or organismal environment also implies the potential for genome-wide redistribution of CDM changes that will ensure the stability of DNA molecules. This raises the question of whether or not we would be able to sort out the regulatory methylation signals from the CDM background ("noise") induced by thermal fluctuations. Here, we propose a novel statistical and information thermodynamic description of the CDM changes to address the last question. The physical basis of our statistical mechanical model was evaluated in two respects: 1) the adherence to Landauer's principle, according to which molecular machines must dissipate a minimum energy ε = kBT ln2 at each logic operation, where kB is the Boltzmann constant, and T is the absolute temperature and 2) whether or not the binary stretch of methylation marks on the DNA molecule comprise a language of sorts, properly constrained by thermodynamic principles. The study was performed for genome-wide methylation data from 152 ecotypes and 40 trans-generational variations of Arabidopsis thaliana and 93 human tissues. The DNA persistence length, a basic mechanical property altered by CDM, was estimated with values from 39 to 66.9 nm. Classical methylome analysis can be retrieved by applying information thermodynamic modelling, which is able to discriminate signal from noise. Our finding suggests that the CDM signal comprises a language scheme properly constrained by molecular thermodynamic principles, which is part of an epigenomic communication system that obeys the same thermodynamic rules as do current

  4. DNA methylation and microRNAs in cancer

    OpenAIRE

    Li, Xiang-Quan; Guo, Yuan-Yuan(Department of Physics, Shanxi University, Taiyuan, Shanxi 030006, China); Wei,, J.B.

    2012-01-01

    DNA methylation is a type of epigenetic modification in the human genome, which means that gene expression is regulated without altering the DNA sequence. Methylation and the relationship between methylation and cancer have been the focus of molecular biology researches. Methylation represses gene expression and can influence embryogenesis and tumorigenesis. In different tissues and at different stages of life, the level of methylation of DNA varies, implying a fundamental but distinct role f...

  5. Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA.

    Science.gov (United States)

    Osakabe, Akihisa; Adachi, Fumiya; Arimura, Yasuhiro; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi

    2015-10-01

    DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.

  6. Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1).

    Science.gov (United States)

    Takeshita, Kohei; Suetake, Isao; Yamashita, Eiki; Suga, Michihiro; Narita, Hirotaka; Nakagawa, Atsushi; Tajima, Shoji

    2011-05-31

    Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291-1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes. PMID:21518897

  7. COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.

    Science.gov (United States)

    Castellanos-Rizaldos, Elena; Milbury, Coren A; Karatza, Elli; Chen, Clark C; Makrigiorgos, G Mike; Merewood, Anne

    2014-01-01

    Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.

  8. COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.

    Science.gov (United States)

    Castellanos-Rizaldos, Elena; Milbury, Coren A; Karatza, Elli; Chen, Clark C; Makrigiorgos, G Mike; Merewood, Anne

    2014-01-01

    Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes. PMID:24728321

  9. Role of TET enzymes in DNA methylation, development, and cancer

    DEFF Research Database (Denmark)

    Rasmussen, Kasper Dindler; Helin, Kristian

    2016-01-01

    The pattern of DNA methylation at cytosine bases in the genome is tightly linked to gene expression, and DNA methylation abnormalities are often observed in diseases. The ten eleven translocation (TET) enzymes oxidize 5-methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation...

  10. DNA methylation in tissues of Chamaedorea elegans

    Institute of Scientific and Technical Information of China (English)

    LU Yongquan; QING Jia; LI Haiying; TONG Zaikang

    2012-01-01

    DNA methylation plays a crucial role in regulating plant development and tissue differentiation.In this study,we compared the methylation levels in leaf,root,and stem in Chamaedorea elegans by using the technique of methylation-sensitive amplified fragment length polymorphism AFLP.Over 19% (42/220) bases were uniformly methylated in these tissues.The percentages of polymorphism resulting from varied methylation in mature leaf (L1),young leaf (L2),baby leaf (L3),stem (S),young root (R1) and lignified root (R2) were 29.5%,29.0%,27.1%,30.7%,63.0% and 28.3%,respectively.The numbers of polymorphic loci detected in the leaves of three developmental stages were similar,ranging from 20 to 30.In contrast,roots at the two developmental stages differed greatly,with 145 polymorphic loci detected in R1 and 27 in R2.Our results suggest that the methylation level in leaves slightly increases with aging,while that in roots decreases dramatically with aging.

  11. MTHFD1 controls DNA methylation in Arabidopsis

    Science.gov (United States)

    Groth, Martin; Moissiard, Guillaume; Wirtz, Markus; Wang, Haifeng; Garcia-Salinas, Carolina; Ramos-Parra, Perla A.; Bischof, Sylvain; Feng, Suhua; Cokus, Shawn J.; John, Amala; Smith, Danielle C.; Zhai, Jixian; Hale, Christopher J.; Long, Jeff A.; Hell, Ruediger; Díaz de la Garza, Rocío I.; Jacobsen, Steven E.

    2016-01-01

    DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases. PMID:27291711

  12. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Pedersen, Marianne Terndrup;

    2011-01-01

    throughout the genome of embryonic stem cells, with the majority of binding sites located at transcription start sites (TSSs) of CpG-rich promoters and within genes. The hmC modification is found in gene bodies and in contrast to mC is also enriched at CpG-rich TSSs. We provide evidence further that TET1 has...... a role in transcriptional repression. TET1 binds a significant proportion of Polycomb group target genes. Furthermore, TET1 associates and colocalizes with the SIN3A co-repressor complex. We propose that TET1 fine-tunes transcription, opposes aberrant DNA methylation at CpG-rich sequences and thereby...

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

    Science.gov (United States)

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

    2011-12-01

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

  14. Dynamic regulation of DNA methylation during mammalian development.

    Science.gov (United States)

    Guibert, Sylvain; Forné, Thierry; Weber, Michael

    2009-10-01

    DNA methylation occurs on cytosines, is catalyzed by DNA methyltransferases (DNMTs), and is present at high levels in all vertebrates. DNA methylation plays essential roles in maintaining genome integrity, but its implication in orchestrating gene-expression patterns remained a matter of debate for a long time. Recent efforts to map DNA methylation at the genome level helped to get a better picture of the distribution of this mark and revealed that DNA methylation is more dynamic between cell types than previously anticipated. In particular, these datasets showed that DNA methylation is targeted to important developmental genes and might act as a barrier to prevent accidental cellular reprogramming. In this review, we will discuss the distribution and function of DNA methylation in mammalian genomes, with particular emphasis on the waves of global DNA methylation reprogramming occurring in early embryos and primordial germ cells. PMID:22122638

  15. Comparative (Computational Analysis of the DNA Methylation Status of Trinucleotide Repeat Expansion Diseases

    Directory of Open Access Journals (Sweden)

    Mohammadmersad Ghorbani

    2013-01-01

    Full Text Available Previous studies have examined DNA methylation in different trinucleotide repeat diseases. We have combined this data and used a pattern searching algorithm to identify motifs in the DNA surrounding aberrantly methylated CpGs found in the DNA of patients with one of the three trinucleotide repeat (TNR expansion diseases: fragile X syndrome (FRAXA, myotonic dystrophy type I (DM1, or Friedreich’s ataxia (FRDA. We examined sequences surrounding both the variably methylated (VM CpGs, which are hypermethylated in patients compared with unaffected controls, and the nonvariably methylated CpGs which remain either always methylated (AM or never methylated (NM in both patients and controls. Using the J48 algorithm of WEKA analysis, we identified that two patterns are all that is necessary to classify our three regions CCGG* which is found in VM and not in AM regions and AATT* which distinguished between NM and VM + AM using proportional frequency. Furthermore, comparing our software with MEME software, we have demonstrated that our software identifies more patterns than MEME in these short DNA sequences. Thus, we present evidence that the DNA sequence surrounding CpG can influence its susceptibility to be de novo methylated in a disease state associated with a trinucleotide repeat.

  16. Differential DNA methylation profiles in gynecological cancers and correlation with clinico-pathological data

    Directory of Open Access Journals (Sweden)

    Tsang Percy CK

    2006-08-01

    Full Text Available Abstract Background Epigenetic gene silencing is one of the major causes of carcinogenesis. Its widespread occurrence in cancer genome could inactivate many cellular pathways including DNA repair, cell cycle control, apoptosis, cell adherence, and detoxification. The abnormal promoter methylation might be a potential molecular marker for cancer management. Methods For rapid identification of potential targets for aberrant methylation in gynecological cancers, methylation status of the CpG islands of 34 genes was determined using pooled DNA approach and methylation-specific PCR. Pooled DNA mixture from each cancer type (50 cervical cancers, 50 endometrial cancers and 50 ovarian cancers was made to form three test samples. The corresponding normal DNA from the patients of each cancer type was also pooled to form the other three control samples. Methylated alleles detected in tumors, but not in normal controls, were indicative of aberrant methylation in tumors. Having identified potential markers, frequencies of methylation were further analyzed in individual samples. Markers identified are used to correlate with clinico-pathological data of tumors using χ2 or Fisher's exact test. Results APC and p16 were hypermethylated across the three cancers. MINT31 and PTEN were hypermethylated in cervical and ovarian cancers. Specific methylation was found in cervical cancer (including CDH1, DAPK, MGMT and MINT2, endometrial cancer (CASP8, CDH13, hMLH1 and p73, and ovarian cancer (BRCA1, p14, p15, RIZ1 and TMS1. The frequencies of occurrence of hypermethylation in 4 candidate genes in individual samples of each cancer type (DAPK, MGMT, p16 and PTEN in 127 cervical cancers; APC, CDH13, hMLH1 and p16 in 60 endometrial cancers; and BRCA1, p14, p16 and PTEN in 49 ovarian cancers were examined for further confirmation. Incidence varied among different genes and in different cancer types ranging from the lowest 8.2% (PTEN in ovarian cancer to the highest 56

  17. DNA Methylation as a Biomarker for Preeclampsia

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Cindy M.; Ralph, Jody L.; Wright, Michelle L.; Linggi, Bryan E.; Ohm, Joyce E.

    2014-10-01

    Background: Preeclampsia contributes significantly to pregnancy-associated morbidity and mortality as well as future risk of cardiovascular disease in mother and offspring, and preeclampsia in offspring. The lack of reliable methods for early detection limits the opportunities for prevention, diagnosis, and timely treatment. Purpose: The purpose of this study was to explore distinct DNA methylation patterns associated with preeclampsia in both maternal cells and fetal-derived tissue that represent potential biomarkers to predict future preeclampsia and inheritance in children. Method: A convenience sample of nulliparous women (N = 55) in the first trimester of pregnancy was recruited for this prospective study. Genome-wide DNA methylation was quantified in first-trimester maternal peripheral white blood cells and placental chorionic tissue from normotensive women and those with preeclampsia (n = 6/group). Results: Late-onset preeclampsia developed in 12.7% of women. Significant differences in DNA methylation were identified in 207 individual linked cytosine and guanine (CpG) sites in maternal white blood cells collected in the first trimester (132 sites with gain and 75 sites with loss of methylation), which were common to approximately 75% of the differentially methylated CpG sites identified in chorionic tissue of fetal origin. Conclusion: This study is the first to identify maternal epigenetic targets and common targets in fetal-derived tissue that represent putative biomarkers for early detection and heritable risk of preeclampsia. Findings may pave the way for diagnosis of preeclampsia prior to its clinical presentation and acute damaging effects, and the potential for prevention of the detrimental long-term sequelae.

  18. DNA methylation: a new twist in the tail

    Institute of Scientific and Technical Information of China (English)

    Gavin Kelsey

    2011-01-01

    DNA methylation is the epigenetic mark with the longest history and that we probably understand best, yet we still have no adequate account for why specific DNA sequences are selected to become methylated.Gene-specific DNA methylation is fundamental to processes such as developmental silencing of genes, classical epigenetic phenomena such as genomic imprinting, and occurs pathologically in the silencing of tumor suppressor genes in cancer.Fully understanding the mechanisms of methylation is thus of huge importance.In mammals,the acquisition of DNA methylation is determined by one of two de novo DNA methyltransferase enzymes, Dnmt3a and Dnmt3b.

  19. Analysis of DNA Cytosine Methylation on Cotton under Salt Stress

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yun-le; YE Wu-wei; WANG Jun-juan; FAN Bao-xiang

    2008-01-01

    @@ DNA methylation,especially methylation of cytosine in eukaryotic organisms,has been implicated in gene regulation,genomic imprinting,the timing of DNA replication,and determination of chromatin structure.It was reported that 6.5% of the whole cytosine residues in the nuclear DNA in higher plants were methylated.The methylation of cytosine in plant nuclear DNA occurs usually in both CpG and CpNG sequences,and the methylation state can be maintained through the cycles of DNA replication and is likely to play an integral role in regulating gene expression.

  20. DNA methylation in spermatogenesis and male infertility

    Science.gov (United States)

    Cui, Xiangrong; Jing, Xuan; Wu, Xueqing; Yan, Meiqin; Li, Qiang; Shen, Yan; Wang, Zhenqiang

    2016-01-01

    Infertility is a significant problem for human reproduction, with males and females equally affected. However, the molecular mechanisms underlying male infertility remain unclear. Spermatogenesis is a highly complex process involving mitotic cell division, meiosis cell division and spermiogenesis; during this period, unique and extensive chromatin and epigenetic modifications occur to bring about specific epigenetic profiles in spermatozoa. It has recently been suggested that the dysregulation of epigenetic modifications, in particular the methylation of sperm genomic DNA, may serve an important role in the development of numerous diseases. The present study is a comprehensive review on the topic of male infertility, aiming to elucidate the association between sperm genomic DNA methylation and poor semen quality in male infertility. In addition, the current status of the genetic and epigenetic determinants of spermatogenesis in humans is discussed.

  1. Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

    Science.gov (United States)

    Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

    2015-10-01

    DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR

  2. DNA Methylation: Insights into Human Evolution.

    OpenAIRE

    Irene Hernando-Herraez; Raquel Garcia-Perez; Sharp, Andrew J; Tomas Marques-Bonet

    2015-01-01

    A fundamental initiative for evolutionary biologists is to understand the molecular basis underlying phenotypic diversity. A long-standing hypothesis states that species-specific traits may be explained by differences in gene regulation rather than differences at the protein level. Over the past few years, evolutionary studies have shifted from mere sequence comparisons to integrative analyses in which gene regulation is key to understanding species evolution. DNA methylation is an important ...

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

    Directory of Open Access Journals (Sweden)

    Prakash Neeraj

    2010-11-01

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

  4. Dissolved humic substances initiate DNA-methylation in cladocerans.

    Science.gov (United States)

    Menzel, Stefanie; Bouchnak, Rihab; Menzel, Ralph; Steinberg, Christian E W

    2011-10-01

    DNA-methylation is one pathway of epigenetic programming of gene expression and can be responsive to environmental challenges such as methylating agents in the food. Here we report on the DNA-methylation in the cladocerans Daphnia magna and Moina macrocopa exposed to humic substances, ubiquitous biogeochemicals. The methylation of DNA can alter the stress response, presumably including exposure to synthetic xenobiotic chemicals. PMID:21963594

  5. Allele-Specific DNA Methylation Detection by Pyrosequencing®

    DEFF Research Database (Denmark)

    Sommer Kristensen, Lasse; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide......-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon....

  6. Relationship of DNA lesions and their repair to chromosomal aberration production

    International Nuclear Information System (INIS)

    Recent work on the roles of specific kinds of DNA lesions and their enzymatic repair systems in the production of chromosomal aberrations seems consistent with a simple molecular model of chromosomal aberrations formation. Evidence from experiments with the human repair-deficient genetic diseases xeroderma pigmentosom, ataxia telangiectasia, and Fanconi's anemia is reviewed in the light of the contributions to aberration production of single and double polynucleotide strand breaks, base damage, polynucleotide strand crosslinks, and pyrimidine cyclobutane dimers

  7. Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers.

    Science.gov (United States)

    Olkhov-Mitsel, Ekaterina; Bapat, Bharati

    2012-10-01

    DNA methylation, consisting of the addition of a methyl group at the fifth-position of cytosine in a CpG dinucleotide, is one of the most well-studied epigenetic mechanisms in mammals with important functions in normal and disease biology. Disease-specific aberrant DNA methylation is a well-recognized hallmark of many complex diseases. Accordingly, various studies have focused on characterizing unique DNA methylation marks associated with distinct stages of disease development as they may serve as useful biomarkers for diagnosis, prognosis, prediction of response to therapy, or disease monitoring. Recently, novel CpG dinucleotide modifications with potential regulatory roles such as 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine have been described. These potential epigenetic marks cannot be distinguished from 5-methylcytosine by many current strategies and may potentially compromise assessment and interpretation of methylation data. A large number of strategies have been described for the discovery and validation of DNA methylation-based biomarkers, each with its own advantages and limitations. These strategies can be classified into three main categories: restriction enzyme digestion, affinity-based analysis, and bisulfite modification. In general, candidate biomarkers are discovered using large-scale, genome-wide, methylation sequencing, and/or microarray-based profiling strategies. Following discovery, biomarker performance is validated in large independent cohorts using highly targeted locus-specific assays. There are still many challenges to the effective implementation of DNA methylation-based biomarkers. Emerging innovative methylation and hydroxymethylation detection strategies are focused on addressing these gaps in the field of epigenetics. The development of DNA methylation- and hydroxymethylation-based biomarkers is an exciting and rapidly evolving area of research that holds promise for potential applications in diverse clinical

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

    Science.gov (United States)

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

  9. Aberrant promoter methylation and expression of UTF1 during cervical carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Samuel Guenin

    Full Text Available Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1 promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2'-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression.

  10. Association of the patterns of globalDNA methylation and expression analysis ofDNA methyltransferases in impaired spermatogenic patients

    Institute of Scientific and Technical Information of China (English)

    DeepikaJaiswal; SameerTrivedi; NeerajK Agrawal; KiranSingh

    2015-01-01

    Objective:To analyse global DNA methylation along with DNA methyltransferases (DNMTs) expression at transcript level in patients with impaired spermatogenesis to dissect its role in pathophysiology of human male infertility.Methods:The content of global methylated cytosine (mC) was determined using ELISA system (Imprint Methylated DNA Quantification Kit, Sigma-Aldrich) in 31 testicular biopsies showing impaired spermatogenesis and 8 with normal spermatogenesis. Real-time reverse transcription-polymerase chain reaction was done to analyze DNMTs (DNMT1, DNMT3A, DNMT3B and DNMT3l) mRNA levels in biopsies with different testicular phenotypes.Results:There was significant increase in levels of global methylation in different impaired testicular phenotypes as compared to normal. Expression analysis revealed significantly increased expression of DNMT1 and its positive correlation with global DNA methylation.Conclusion:In conclusion, increased levels of global methylation in impaired cases might be the one of the contributing factors for aberrant gene expression in infertile patients.

  11. Human papilloma virus, DNA methylation and microRNA expression in cervical cancer (Review)

    OpenAIRE

    JIMÉNEZ-WENCES, HILDA; Peralta-Zaragoza, Oscar; Fernández-Tilapa, Gloria

    2014-01-01

    Cancer is a complex disease caused by genetic and epigenetic abnormalities that affect gene expression. The progression from precursor lesions to invasive cervical cancer is influenced by persistent human papilloma virus (HPV) infection, which induces changes in the host genome and epigenome. Epigenetic alterations, such as aberrant miRNA expression and changes in DNA methylation status, favor the expression of oncogenes and the silencing of tumor-suppressor genes. Given that some miRNA genes...

  12. Analysis of DNA methylation in different maize tissues

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism (AFLP) to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP (methylation sensitive amplification polymorphism) ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5′-CCGG-3′ sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.

  13. DNA Methylation, Behavior and Early Life Adversity

    Institute of Scientific and Technical Information of China (English)

    Moshe Szyf

    2013-01-01

    The impact of early physical and social environments on life-long phenotypes is well known.Moreover,we have documented evidence for gene-enviromnent interactions where identical gene variants are associated with different phenotypes that are dependent on early life adversity.What are the mechanisms that embed these early life experiences in the genome? DNA methylation is an enzymaticallycatalyzed modification of DNA that serves as a mechanism by which similar sequences acquire cell type identity during cellular differentiation and embryogenesis in the same individual.The hypothesis that will be discussed here proposes that the same mechanism confers environmental-exposure specific identity upon DNA providing a mechanism for embedding environmental experiences in the genome,thus affecting long-term phenotypes.Particularly important is the environment early in life including both the prenatal and postnatal social environments.

  14. Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

    2007-02-07

    The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

  15. Methylation of cell-free circulating DNA in the diagnosis of cancer

    Directory of Open Access Journals (Sweden)

    Goli eSamimi

    2015-04-01

    Full Text Available A range of molecular alterations found in tumor cells, such as DNA mutations and methylation changes, is also reflected in cell-free circulating DNA (circDNA released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. DNA methylation is a common molecular alteration found in many cancer types. Unlike DNA mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. DNA methylation is reflected within circDNA and therefore detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker.

  16. Recognition of methylated DNA through methyl-CpG binding domain proteins

    DEFF Research Database (Denmark)

    Zou, Xueqing; Ma, Wen; Solov'yov, Ilia;

    2012-01-01

    DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although...... the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines...

  17. Epigenetic editing using programmable zinc ginger proteins : inherited silencing of endogenous gene expression by targeted DNA methylation

    NARCIS (Netherlands)

    Stolzenburg, Sabine

    2014-01-01

    Cancer development is not only the result of genetic mutations but also stems from modifications in the epigenetic code leading to an aberrant expression of genes relevant for cancer. The most studied epigenetic mark is DNA methylation of cytosines in the promoters of genes, which is associated with

  18. DNA methylation plays a crucial role during early Nasonia development

    NARCIS (Netherlands)

    Zwier, M. V.; Verhulst, E. C.; Zwahlen, R. D.; Beukeboom, L. W.; van de Zande, L.

    2012-01-01

    Although the role of DNA methylation in insect development is still poorly understood, the number and role of DNA methyltransferases in insects vary strongly between species. DNA methylation appears to be widely present among the social hymenoptera and functional studies in Apis have suggested a cru

  19. Analysis of DNA Cytosine Methylation on Cotton under Salt Stress

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    DNA methylation,especially methylation of cytosine in eukaryotic organisms,has been implicated in gene regulation,genomic imprinting,the timing of DNA replication,and determination of chromatin structure.It was reported that 6.5% of the whole cytosine residues in the nuclear DNA in higher

  20. Detection and Clinical Significance of DLC1 Gene Methylation in Serum DNA from Colorectal Cancer Patients

    Institute of Scientific and Technical Information of China (English)

    Ping-ping Wu; Ji-hong Zou; Ri-ning Tang; Yao Yao; Cheng-zhong You

    2011-01-01

    Objective:Deleted in liver cancer 1 (DLC1) is a new candidate tumor suppressor gene,whose down-regulation or even silence will result from promoter hypermethylation in various human cancers including colorectal cancer (CRC).The aim of this study is to evaluate the diagnostic role of DLC1 gene methylation in the serum DNA from CRC patients.Methods:This study enrolled 85 CRC patients and 45 patients with benign colorectal diseases.Methylation-specific polymerase chain reaction (MSP) was used to determine the promoter methylation status of DLC1 gene in serum DNA.The combination of DLC1 methylation and conventional tumor markers was further analyzed.Results:Hypermethylation of DLC1 was detected in 42.4% (36/85) of CRC serums,while seldom in the benign controls (8.9%,4/45) (P<0.001).The aberrant DLC1 methylation in serum DNA was not associated with patients' clinicopathological features and elevated CEA/CA19-9 levels.Furthermore,the combinational analysis of CEA,CA19-9 and DLC1 methylation showed a higher sensitivity and no reduced diagnostic specificity than CEA and CA19-9 combination for CRC diagnosis.Conclusion:The serum DLC1 methylation may be a promising biomarker for the early detection of CRC,which will further increase the diagnostic efficiency in combination with CEA and CA19-9.

  1. A DNA methylation fingerprint of 1628 human samples

    OpenAIRE

    Fernandez, A. F.; Assenov, Y.; Martin-Subero, J.I. (José Ignacio); Balint, B.; Siebert, R.; Taniguchi, H; Yamamoto, H.; M. Hidalgo; Tan, A.-C.; Galm, O; Ferrer, I.; Sanchez-Cespedes, M.; Villanueva, A; Carmona, J; Sanchez-Mut, J. V.

    2012-01-01

    Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns reve...

  2. Methylated DNA Immunoprecipitation Analysis of Mammalian Endogenous Retroviruses.

    Science.gov (United States)

    Rebollo, Rita; Mager, Dixie L

    2016-01-01

    Endogenous retroviruses are repetitive sequences found abundantly in mammalian genomes which are capable of modulating host gene expression. Nevertheless, most endogenous retrovirus copies are under tight epigenetic control via histone-repressive modifications and DNA methylation. Here we describe a common method used in our laboratory to detect, quantify, and compare mammalian endogenous retrovirus DNA methylation. More specifically we describe methylated DNA immunoprecipitation (MeDIP) followed by quantitative PCR. PMID:26895065

  3. Aberrant Hepatic Methionine Metabolism and Gene Methylation in the Pathogenesis and Treatment of Alcoholic Steatohepatitis

    Directory of Open Access Journals (Sweden)

    Charles H. Halsted

    2012-01-01

    Full Text Available The pathogenesis of alcoholic steatohepatitis (ASH involves ethanol-induced aberrations in hepatic methionine metabolism that decrease levels of S-adenosylmethionine (SAM, a compound which regulates the synthesis of the antioxidant glutathione and is the principal methyl donor in the epigenetic regulation of genes relevant to liver injury. The present paper describes the effects of ethanol on the hepatic methionine cycle, followed by evidence for the central role of reduced SAM in the pathogenesis of ASH according to clinical data and experiments in ethanol-fed animals and in cell models. The efficacy of supplemental SAM in the prevention of ASH in animal models and in the clinical treatment of ASH will be discussed.

  4. Aberrant Hepatic Methionine Metabolism and Gene Methylation in the Pathogenesis and Treatment of Alcoholic Steatohepatitis

    Science.gov (United States)

    Halsted, Charles H.; Medici, Valentina

    2012-01-01

    The pathogenesis of alcoholic steatohepatitis (ASH) involves ethanol-induced aberrations in hepatic methionine metabolism that decrease levels of S-adenosylmethionine (SAM), a compound which regulates the synthesis of the antioxidant glutathione and is the principal methyl donor in the epigenetic regulation of genes relevant to liver injury. The present paper describes the effects of ethanol on the hepatic methionine cycle, followed by evidence for the central role of reduced SAM in the pathogenesis of ASH according to clinical data and experiments in ethanol-fed animals and in cell models. The efficacy of supplemental SAM in the prevention of ASH in animal models and in the clinical treatment of ASH will be discussed. PMID:22007317

  5. Human papilloma virus, DNA methylation and microRNA expression in cervical cancer (Review).

    Science.gov (United States)

    Jiménez-Wences, Hilda; Peralta-Zaragoza, Oscar; Fernández-Tilapa, Gloria

    2014-06-01

    Cancer is a complex disease caused by genetic and epigenetic abnormalities that affect gene expression. The progression from precursor lesions to invasive cervical cancer is influenced by persistent human papilloma virus (HPV) infection, which induces changes in the host genome and epigenome. Epigenetic alterations, such as aberrant miRNA expression and changes in DNA methylation status, favor the expression of oncogenes and the silencing of tumor-suppressor genes. Given that some miRNA genes can be regulated through epigenetic mechanisms, it has been proposed that alterations in the methylation status of miRNA promoters could be the driving mechanism behind their aberrant expression in cervical cancer. For these reasons, we assessed the relationship among HPV infection, cellular DNA methylation and miRNA expression. We conclude that alterations in the methylation status of protein-coding genes and various miRNA genes are influenced by HPV infection, the viral genotype, the physical state of the viral DNA, and viral oncogenic risk. Furthermore, HPV induces deregulation of miRNA expression, particularly at loci near fragile sites. This deregulation occurs through the E6 and E7 proteins, which target miRNA transcription factors such as p53. PMID:24737381

  6. Forensic DNA methylation profiling from evidence material for investigative leads.

    Science.gov (United States)

    Lee, Hwan Young; Lee, Soong Deok; Shin, Kyoung-Jin

    2016-07-01

    DNA methylation is emerging as an attractive marker providing investigative leads to solve crimes in forensic genetics. The identification of body fluids that utilizes tissue-specific DNA methylation can contribute to solving crimes by predicting activity related to the evidence material. The age estimation based on DNA methylation is expected to reduce the number of potential suspects, when the DNA profile from the evidence does not match with any known person, including those stored in the forensic database. Moreover, the variation in DNA implicates environmental exposure, such as cigarette smoking and alcohol consumption, thereby suggesting the possibility to be used as a marker for predicting the lifestyle of potential suspect. In this review, we describe recent advances in our understanding of DNA methylation variations and the utility of DNA methylation as a forensic marker for advanced investigative leads from evidence materials. [BMB Reports 2016; 49(7): 359-369]. PMID:27099236

  7. Forensic DNA methylation profiling from evidence material for investigative leads.

    Science.gov (United States)

    Lee, Hwan Young; Lee, Soong Deok; Shin, Kyoung-Jin

    2016-07-01

    DNA methylation is emerging as an attractive marker providing investigative leads to solve crimes in forensic genetics. The identification of body fluids that utilizes tissue-specific DNA methylation can contribute to solving crimes by predicting activity related to the evidence material. The age estimation based on DNA methylation is expected to reduce the number of potential suspects, when the DNA profile from the evidence does not match with any known person, including those stored in the forensic database. Moreover, the variation in DNA implicates environmental exposure, such as cigarette smoking and alcohol consumption, thereby suggesting the possibility to be used as a marker for predicting the lifestyle of potential suspect. In this review, we describe recent advances in our understanding of DNA methylation variations and the utility of DNA methylation as a forensic marker for advanced investigative leads from evidence materials. [BMB Reports 2016; 49(7): 359-369].

  8. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism.

    Science.gov (United States)

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  9. High-definition DNA methylation profiles from breast and ovarian carcinoma cell lines with differing doxorubicin resistance.

    Directory of Open Access Journals (Sweden)

    Michael Boettcher

    Full Text Available Acquired drug resistance represents a frequent obstacle which hampers efficient chemotherapy of cancers. The contribution of aberrant DNA methylation to the development of drug resistant tumor cells has gained increasing attention over the past decades. Hence, the objective of the presented study was to characterize DNA methylation changes which arise from treatment of tumor cells with the chemotherapeutic drug doxorubicin. DNA methylation levels from CpG islands (CGIs linked to twenty-eight genes, whose expression levels had previously been shown to contribute to resistance against DNA double strand break inducing drugs or tumor progression in different cancer types were analyzed. High-definition DNA methylation profiles which consisted of methylation levels from 800 CpG sites mapping to CGIs around the transcription start sites of the selected genes were determined. In order to investigate the influence of CGI methylation on the expression of associated genes, their mRNA levels were investigated via qRT-PCR. It was shown that the employed method is suitable for providing highly accurate methylation profiles, comparable to those obtained via clone sequencing, the gold standard for high-definition DNA methylation studies. In breast carcinoma cells with acquired resistance against the double strand break inducing drug doxorubicin, changes in methylation of specific cytosines from CGIs linked to thirteen genes were detected. Moreover, similarities between methylation profiles obtained from breast and ovarian carcinoma cell lines with acquired doxorubicin resistance were found. The expression levels of a subset of analyzed genes were shown to be linked to the methylation levels of the analyzed CGIs. Our results provide detailed DNA methylation information from two separate model systems for acquired doxorubicin resistance and suggest the occurrence of similar methylation changes in both systems upon exposure to the drug.

  10. Genome-wide DNA methylation analysis predicts an epigenetic switch for GATA factor expression in endometriosis.

    Directory of Open Access Journals (Sweden)

    Matthew T Dyson

    2014-03-01

    identifies a novel role for the GATA family as key regulators of uterine physiology-aberrant DNA methylation in endometriotic cells correlates with a shift in GATA isoform expression that facilitates progesterone resistance and disease progression.

  11. DNA Methylation in Basal Metazoans: Insights from Ctenophores.

    Science.gov (United States)

    Dabe, Emily C; Sanford, Rachel S; Kohn, Andrea B; Bobkova, Yelena; Moroz, Leonid L

    2015-12-01

    Epigenetic modifications control gene expression without altering the primary DNA sequence. However, little is known about DNA methylation in invertebrates and its evolution. Here, we characterize two types of genomic DNA methylation in ctenophores, 5-methyl cytosine (5-mC) and the unconventional form of methylation 6-methyl adenine (6-mA). Using both bisulfite sequencing and an ELISA-based colorimetric assay, we experimentally confirmed the presence of 5-mC DNA methylation in ctenophores. In contrast to other invertebrates studied, Mnemiopsis leidyi has lower levels of genome-wide 5-mC methylation, but higher levels of 5-mC methylation in promoters when compared with gene bodies. Phylogenetic analysis showed that ctenophores have distinct forms of DNA methyltransferase 1 (DNMT1); the zf-CXXC domain type, which localized DNMT1 to CpG sites, and is a metazoan specific innovation. We also show that ctenophores encode the full repertoire of putative enzymes for 6-mA DNA methylation, and these genes are expressed in the aboral organ of Mnemiopsis. Using an ELISA-based colorimetric assay, we experimentally confirmed the presence of 6-mA methylation in the genomes of three different species of ctenophores, M. leidyi, Beroe abyssicola, and Pleurobrachia bachei. The functional role of this novel epigenomic mark is currently unknown. In summary, despite their compact genomes, there is a wide variety of epigenomic mechanisms employed by basal metazoans that provide novel insights into the evolutionary origins of biological novelties.

  12. Blood DNA methylation markers in prospectively identifiedhepatocellular carcinoma cases and controls from Taiwan

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    AIM To determine if gene-specific DNA methylation inprospectively collected blood samples is associated withlater development of hepatocellular carcinoma (HCC).METHODS: Comparing genome-wide DNA methylationprofiles using Illumina Human methylation 450Karrays, we previously identified a list of loci that weredifferentially methylated between tumor and adjacentnontumor tissues. To examine if dysregulation of DNA methylation patterns observed in tumor tissues can bedetected in white blood cell (WBC) DNA, we conducteda prospective case-control study nested within acommunity-based cancer screening cohort in Taiwanwith 16 years of follow up. We measured methylationlevels in ninety-six loci that were aberrant in DNAmethylation in HCC tumor tissues compared to adjacenttissues. Baseline WBC DNA from 159 HCC cases and 312matched controls were bisulfite treated and assayed byIllumina BeadArray. We used the χ 2 test for categoricalvariables and student's t -test for continuous variables toassess the difference in selected characteristics betweencases and controls. To estimate associations with HCCrisk, we used conditional logistic regression modelsstratified on the matching factors to calculate odds ratios(OR) and 95%CI.RESULTS: We found that high methylation level incg10272601 in WNK2 was associated with increasedrisk of HCC, with an OR of 1.91 (95%CI: 1.27-2.86).High methylation levels in both cg12680131 in TPO andcg22511877 in MYT1L , however, were associated withdecreased risk. The ORs (95%CI) were 0.59 (0.39-0.87)and 0.50 (0.33-0.77), respectively, for those with methylationlevels of cg12680131 and cg22511877 abovethe median compared with those with levels belowthe median. These associations were still statisticallysignificant in multivariable conditional logistic regressionmodels after adjusting for hepatitis B virus infection andalcohol consumption.CONCLUSION: These findings support the measurementof methylation markers in WBC DNA

  13. Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

    Directory of Open Access Journals (Sweden)

    Recillas-Targa Félix

    2011-06-01

    Full Text Available Abstract Background Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb gene promoter in different tumoral cell lines. Methods To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. Results We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. Conclusions This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing.

  14. DNA methylation and microRNAs in cancer

    Institute of Scientific and Technical Information of China (English)

    Xiang-Quan Li; Yuan-Yuan Guo; Wei De

    2012-01-01

    DNA methylation is a type of epigenetic modification in the human genome,which means that gene expression is regulated without altering the DNA sequence.Methylation and the relationship between methylation and cancer have been the focus of molecular biology researches.Methylation represses gene expression and can influence embryogenesis and tumorigenesis.In different tissues and at different stages of life,the level of methylation of DNA varies,implying a fundamental but distinct role for methylation.When genes are repressed by abnormal methylation,the resulting effects can include instability of that gene and inactivation of a tumor suppressor gene.MicroRNAs have some aspects in common with this regulation of gene expression.Here we reviewed the influence of gene methylation on cancer and analyzed the methods used to profile methylation.We also assessed the correlation between methylation and other epigenetic modifications and microRNAs.About 55 845 research papers have been published about methylation,and one-fifth of these are about the appearance of methylation in cancer.We conclude that methylation does play a role in some cancer types.

  15. DNA Methylation and Potential for Epigenetic Regulation in Pygospio elegans.

    Science.gov (United States)

    Kesäniemi, Jenni E; Heikkinen, Liisa; Knott, K Emily

    2016-01-01

    Transitions in developmental mode are common evolutionarily, but how and why they occur is not understood. Developmental mode describes larval phenotypes, including morphology, ecology and behavior of larvae, which typically are generalized across different species. The polychaete worm Pygospio elegans is one of few species polymorphic in developmental mode, with multiple larval phenotypes, providing a possibility to examine the potential mechanisms allowing transitions in developmental mode. We investigated the presence of DNA methylation in P. elegans, and, since maternal provisioning is a key factor determining eventual larval phenotype, we compared patterns of DNA methylation in females during oogenesis in this species. We demonstrate that intragenic CpG site DNA methylation and many relevant genes necessary for DNA methylation occur in P. elegans. Methylation-sensitive AFLP analysis showed that gravid females with offspring differing in larval developmental mode have significantly different methylation profiles and that the females with benthic larvae and non-reproductive females from the same location also differ in their epigenetic profiles. Analysis of CpG sites in transcriptome data supported our findings of DNA methylation in this species and showed that CpG observed/expected ratios differ among females gravid with embryos destined to different developmental modes. The differences in CpG site DNA methylation patterns seen among the samples suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. PMID:27008314

  16. [Profiles of DNA methylation in normal and cancer cells].

    Science.gov (United States)

    Weber, Michaël

    2008-01-01

    In eukaryotes, the epigenetic mark DNA methylation is found exclusively at cytosine residues in the CpG islands of genes, transposons and intergenic DNA. Among functional roles, DNA methylation is essential for mammalian embryonic development, and is classically thought to function by stably silencing promoter activity. However, until recently, understanding of the distribution of cytosine methylation in the whole genome - and hence, identification of its targets - was very limited. High-throughput methodologies, including methylated DNA immunoprecipitation, have recently revealed genome-wide mapping of DNA methylation, and provided new and unexpected data. Clearly DNA methylation is selectively associated with some key promoters- and is not a prerequisite for promoter inactivation, since strong CpG island promoters are mostly unmethylated, even when inactive. Most germline-specific genes are methylated and permanently silenced in somatic cells, suggesting a role of this mark in maintaining somatic cellular identity. These large scale studies will also help understanding the deregulation of DNA methylation associated with cancer, among which unmethylation of germinal cells genes, and recent observtion of large hypomethylated regions in tumoral specimens. The next challenge will be to understand if these methylation changes occur randomly, or more likely are specified by oncogenes or linked to environmental pressure. PMID:18789220

  17. Effects of LET, fluence and particle energy on inactivation, chromosomal aberrations and DNA strand breaks

    International Nuclear Information System (INIS)

    Experiments are described studying the inactivation and the induction of chromosomal aberrations in mammalian cells. In addition, experiments of the induction of single and double strand breaks of DNA in mammalian cells will be compared to the induction of single and double strand breaks of DNA in solution. (orig./MG)

  18. An atlas of DNA methylation in diverse bovine tissues

    Science.gov (United States)

    We launched an effort to produce a reference cattle DNA methylation resource to improve animal production. We will employ experimental pipelines built around next generation sequencing technologies to map DNA methylation in cultured cells and primary tissues systems frequently involved in animal pro...

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

    Science.gov (United States)

    Barrachina, Marta; Ferrer, Isidre

    2009-08-01

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

  20. The Silencing of CCND2 by Promoter Aberrant Methylation in Renal Cell Cancer and Analysis of the Correlation between CCND2 Methylation Status and Clinical Features.

    Science.gov (United States)

    Wang, Lu; Cui, Yun; Zhang, Lian; Sheng, Jindong; Yang, Yang; Kuang, Guanyu; Fan, Yu; Zhang, Qian; Jin, Jie

    2016-01-01

    Cyclin D2 (CCND2) is a member of the D-type cyclins, which plays a pivotal role in cell cycle regulation, differentiation and malignant transformation. However, its expression status and relative regulation mechanism remains unclear in renal cell cancer (RCC). In our study, the mRNA expression level of CCND2 is down-regulated in 22/23 paired RCC tissues (pTSA restored CCND2 expression in several methylated RCC cell lines. Among the 102 RCC tumors, methylation of CCND2 was detected in 29/102 (28%) cases. Only 2/23 (8.7%) adjacent non-malignant tissues showed methylation. We then analyzed the correlation of clinical features and its promoter methylation. Collectively, our data suggested that loss of CCND2 expression is closely associated with the promoter aberrant methylation. PMID:27583477

  1. Sequences sufficient for programming imprinted germline DNA methylation defined.

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    Yoon Jung Park

    Full Text Available Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC; the second carries only the DMD and repeats (DR from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice.

  2. Karyotype and DNA-Methylation Responses in Myelodysplastic Syndromes following Treatment with Traditional Chinese Formula Containing Arsenic

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    Sun Shuzhen

    2012-01-01

    Full Text Available We have previously shown that arsenic-containing Chinese herbal formula, Qing-Huang powder capsule (containing tetraarsenic tetrasulfide, As4S4, is effective in the treatment of myelodysplastic syndrome (MDS; yet the underlined mechanisms remain unclear. In this study, using standard cytogenetic analysis (G-banded and global DNA methylation method (ChIP-on-chip assays, we aimed to determine the effect of arsenic-containing Chinese herbal formula on karyotype status and the genomic methylation level in primarily diagnosed MDS patients. Correlation of aberrant DNA methylation and chromosome aberrations in MDS was also investigated. We found that the number of genes with aberrant DNA methylation was highest in MDS patients with normal karyotype, followed by trisomy 8 karyotype, and relatively low in patients with cytogenetic abnormalities other than trisomy 8. Treatment with arsenic-containing Chinese herbal formula had no effects on karyotype status, but resulted in a significant genome-wide demethylation. Our research uncovered a DNA demethylating activity of arsenic-containing Chinese herbal formula in the treatment of MDS.

  3. Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57

    DEFF Research Database (Denmark)

    Bak, Mads; Boonen, Susanne E; Dahl, Christina;

    2016-01-01

    involved in establishment and maintenance of methylation of imprinted loci. Our objective was to investigate whether additional regions are aberrantly methylated in ZFP57 mutation carriers. METHODS: Genome-wide DNA methylation analysis was performed on four individuals with homozygous or compound...... and HYMAI. A subset of patients with maternal hypomethylation at PLAGL1 have hypomethylation at additional imprinted loci throughout the genome, including GRB10, ZIM2 (PEG3), MEST (PEG1), KCNQ1OT1 and NESPAS (GNAS-AS1). About half of the TNDM1 patients carry mutations in ZFP57, a transcription factor...

  4. DNA methylation in serum of breast cancer patients: an independent prognostic marker.

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    Müller, Hannes M; Widschwendter, Andreas; Fiegl, Heidi; Ivarsson, Lennart; Goebel, Georg; Perkmann, Elisabeth; Marth, Christian; Widschwendter, Martin

    2003-11-15

    Changes in the status of DNA methylation are one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated whether aberrant DNA methylation in patient pretherapeutic sera is of prognostic significance in breast cancer. Using MethyLight, a high-throughput DNA methylation assay, we analyzed 39 genes in a gene evaluation set, consisting of 10 sera from metastasized patients, 26 patients with primary breast cancer, and 10 control patients. To determine the prognostic value of genes identified within the gene evaluation set, we finally analyzed pretreatment sera of 24 patients having had no adjuvant treatment (training set) to determine their prognostic value. An independent test set consisting of 62 patients was then used to test the validity of genes and combinations of genes, which in the training set were found to be good prognostic markers. In the gene evaluation set we identified five genes (ESR1, APC, HSD17B4, HIC1, and RASSF1A). In the training set, patients with methylated serum DNA for RASSF1A and/or APC had the worst prognosis (P < 0.001). This finding was confirmed by analyzing serum samples from the independent test set (P = 0.007). When analyzing all 86 of the investigated patients, multivariate analysis showed methylated RASSF1A and/or APC serum DNA to be independently associated with poor outcome, with a relative risk for death of 5.7. DNA methylation of particular genes in pretherapeutic sera of breast cancer patients, especially of RASSF1A/APC, is more powerful than standard prognostic parameters.

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

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    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.

  6. DNA Methylation Variation Trends during the Embryonic Development of Chicken.

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    Shizhao Li

    Full Text Available The embryogenesis period is critical for epigenetic reprogramming and is thus of great significance in the research field of poultry epigenetics for elucidation of the trends in DNA methylation variations during the embryonic development of birds, particularly due to differences in embryogenesis between birds and mammals. Here, we first examined the variations in genomic DNA methylation during chicken embryogenesis through high-performance liquid chromatography using broilers as the model organism. We then identified the degree of DNA methylation of the promoters and gene bodies involved in two specific genes (IGF2 and TNF-α using the bisulfite sequencing polymerase chain reaction method. In addition, we measured the expression levels of IGF2, TNF-α and DNA methyltransferase (DNMT 1, 3a and 3b. Our results showed that the genomic DNA methylation levels in the liver, heart and muscle increased during embryonic development and that the methylation level of the liver was significantly higher in mid-anaphase. In both the muscle and liver, the promoter methylation levels of TNF-α first increased and then decreased, whereas the gene body methylation levels remained lower at embryonic ages E8, 11 and 14 before increasing notably at E17. The promoter methylation level of IGF2 decreased persistently, whereas the methylation levels in the gene body showed a continuous increase. No differences in the expression of TNF-α were found among E8, 11 and 14, whereas a significant increase was observed at E17. IGF2 showed increasing expression level during the examined embryonic stages. In addition, the mRNA and protein levels of DNMTs increased with increasing embryonic ages. These results suggest that chicken shows increasing genomic DNA methylation patterns during the embryonic period. Furthermore, the genomic DNA methylation levels in tissues are closely related to the genes expression levels, and gene expression may be simultaneously regulated by promoter

  7. Methylation reaction for four DNA base molecules by methanediazonium ions

    Institute of Scientific and Technical Information of China (English)

    LI Lan; QU ZhiHao; WANG Hong; LI ZongHe

    2009-01-01

    The methylation reactions at ten nucleophilic sites in four DNA base molecules with methanediazonium ions (CH3N2+) have been theoretically investigated including solvent effects at the B3LYP/6-31G** and MP2/6-31G** levels. The results show that all the methylation reactions have relatively small activation energy (<33.5 kJ/mol), and the methylation process is exothermic reaction and easy to occur. This study shows that the ultimate carcinogen CH3N2+ by NDMA can easily methylate DNA base molecules and form carcinogenic products.

  8. Methylation reaction for four DNA base molecules by methanediazonium ions

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The methylation reactions at ten nucleophilic sites in four DNA base molecules with methanediazonium ions(CH3N2+) have been theoretically investigated including solvent effects at the B3LYP/6-31G and MP2/6-31G levels.The results show that all the methylation reactions have relatively small activation energy(<33.5 kJ/mol),and the methylation process is exothermic reaction and easy to occur.This study shows that the ultimate carcinogen CH3N2+ by NDMA can easily methylate DNA base molecules and form carcinogenic products.

  9. Hepatocellular carcinoma displays distinct DNA methylation signatures with potential as clinical predictors.

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    Hector Hernandez-Vargas

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions. METHODS AND FINDINGS: We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption. Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy. CONCLUSION: Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.

  10. Aberrant methylation frequency of TNFRSF10C promoter in pancreatic cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Hui-Hua Cai; Yue-Ming Sun; Yi Miao; Wen-Tao Gao; Quan Peng; JieYao; Han-Lin Zhao

    2011-01-01

    BACKGROUND: A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations, which promote tumor progression. Epigenetic abnormalities include changes in DNA methylation and in the modification of histones. This study aimed to assess the status of methylation in the CpG island (CGI) of the tumor necrosis factor receptor superfamily member 10c (TNFRSF10C) with combined bisulfite restriction analysis (COBRA) and to evaluate its role in the progression of pancreatic cancer (PC). METHODS: The methylation status of four PC cell lines was assessed using COBRA and/or bisulfite genomic sequencing (BGS). Changes in methylation and TNFRSF10C expression in PC cell lines before and after treatment with 5-aza-2'-deoxycytidine (5-aza-dC) and/or trichostatin A (TSA) were assessed by BGS and real-time RT-PCR. Apoptosis in the four cell lines was tested by flow cytometry (FCM) and TUNEL assay. RESULTS: The methylation status of the TNFRSF10C promoter was assessed in PC cells (BxPC-3: 68.84±8.71%; CFPAC-1: 0;PANC-1: 96.77±4.57%; SW1990: 54.97±7.33%) with the COBRA assay, which was confirmed by the results of BGS. After treatment with 5-aza-dC and/or TSA, apoptosis was induced in PC cells to different degrees, and the levels of TNFRSF10C transcriptional expression in the PC cell lines (except CFPAC-1) increased markedly after 5-aza-dC treatment. CONCLUSIONS: A high frequency of CGI methylation in the TNFRSF10C promoter results in inactivation of the gene and enhancement of tumor growth in most PC cell lines (except CFPAC-1). Inactivation of TNFRSF10C by CGI hypermethylation can play an important role in PC progression and be potentially useful as a diagnostic marker and a new therapeutic approach for PC.

  11. Genome-wide DNA methylation maps in follicular lymphoma cells determined by methylation-enriched bisulfite sequencing.

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    Jeong-Hyeon Choi

    Full Text Available BACKGROUND: Follicular lymphoma (FL is a form of non-Hodgkin's lymphoma (NHL that arises from germinal center (GC B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

  12. Genome-wide mapping of DNA methylation in chicken.

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    Qinghe Li

    Full Text Available Cytosine DNA methylation is an important epigenetic modification termed as the fifth base that functions in diverse processes. Till now, the genome-wide DNA methylation maps of many organisms has been reported, such as human, Arabidopsis, rice and silkworm, but the methylation pattern of bird remains rarely studied. Here we show the genome-wide DNA methylation map of bird, using the chicken as a model organism and an immunocapturing approach followed by high-throughput sequencing. In both of the red jungle fowl and the avian broiler, DNA methylation was described separately for the liver and muscle tissue. Generally, chicken displays analogous methylation pattern with that of animals and plants. DNA methylation is enriched in the gene body regions and the repetitive sequences, and depleted in the transcription start site (TSS and the transcription termination site (TTS. Most of the CpG islands in the chicken genome are kept in unmethylated state. Promoter methylation is negatively correlated with the gene expression level, indicating its suppressive role in regulating gene transcription. This work contributes to our understanding of epigenetics in birds.

  13. DNA methylation characteristics of primary melanomas with distinct biological behaviour.

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    Szilvia Ecsedi

    Full Text Available In melanoma, the presence of promoter related hypermethylation has previously been reported, however, no methylation-based distinction has been drawn among the diverse melanoma subtypes. Here, we investigated DNA methylation changes associated with melanoma progression and links between methylation patterns and other types of somatic alterations, including the most frequent mutations and DNA copy number changes. Our results revealed that the methylome, presenting in early stage samples and associated with the BRAF(V600E mutation, gradually decreased in the medium and late stages of the disease. An inverse relationship among the other predefined groups and promoter methylation was also revealed except for histologic subtype, whereas the more aggressive, nodular subtype melanomas exhibited hypermethylation as well. The Breslow thickness, which is a continuous variable, allowed for the most precise insight into how promoter methylation decreases from stage to stage. Integrating our methylation results with a high-throughput copy number alteration dataset, local correlations were detected in the MYB and EYA4 genes. With regard to the effects of DNA hypermethylation on melanoma patients' survival, correcting for clinical cofounders, only the KIT gene was associated with a lower overall survival rate. In this study, we demonstrate the strong influence of promoter localized DNA methylation changes on melanoma initiation and show how hypermethylation decreases in melanomas associated with less favourable clinical outcomes. Furthermore, we establish the methylation pattern as part of an integrated apparatus of somatic DNA alterations.

  14. The Application of Next Generation Sequencing in DNA Methylation Analysis

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    Yingying Zhang

    2010-06-01

    Full Text Available DNA methylation is a major form of epigenetic modification and plays essential roles in physiology and disease processes. In the human genome, about 80% of cytosines in the 56 million CpG sites are methylated to 5-methylcytosines. The methylation pattern of DNA is highly variable among cells types and developmental stages and influenced by disease processes and genetic factors, which brings considerable theoretical and technological challenges for its comprehensive mapping. Recently various high-throughput approaches based on bisulfite conversion combined with next generation sequencing have been developed and applied for the genome wide analysis of DNA methylation. These methods provide single base pair resolution, quantitative DNA methylation data with genome wide coverage. We review these methods here and discuss some technical points of special interest like the sequence depth necessary to reach conclusions, the identification of clonal DNA amplification after bisulfite conversion and the detection of non-CpG methylation. Future application of these methods will greatly facilitate the profiling of the DNA methylation in the genomes of different species, individuals and cell types under healthy and disease states.

  15. Array-based DNA methylation profiling for breast cancer subtype discrimination.

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    Ilse Van der Auwera

    poor patient prognosis. The results of the current study also suggest that aberrant DNA methylation is not the main force driving the molecular biology of IBC.

  16. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1

    Science.gov (United States)

    Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

    2016-01-01

    The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. DOI: http://dx.doi.org/10.7554/eLife.17101.001 PMID:27595565

  17. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines.

    Science.gov (United States)

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I (2) statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating "hub genes" - heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  18. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    Science.gov (United States)

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility.

  19. Simulation of the Formation of DNA Double Strand Breaks and Chromosome Aberrations in Irradiated Cells

    Science.gov (United States)

    Plante, Ianik; Ponomarev, Artem L.; Wu, Honglu; Blattnig, Steve; George, Kerry

    2014-01-01

    The formation of DNA double-strand breaks (DSBs) and chromosome aberrations is an important consequence of ionizing radiation. To simulate DNA double-strand breaks and the formation of chromosome aberrations, we have recently merged the codes RITRACKS (Relativistic Ion Tracks) and NASARTI (NASA Radiation Track Image). The program RITRACKS is a stochastic code developed to simulate detailed event-by-event radiation track structure: [1] This code is used to calculate the dose in voxels of 20 nm, in a volume containing simulated chromosomes, [2] The number of tracks in the volume is calculated for each simulation by sampling a Poisson distribution, with the distribution parameter obtained from the irradiation dose, ion type and energy. The program NASARTI generates the chromosomes present in a cell nucleus by random walks of 20 nm, corresponding to the size of the dose voxels, [3] The generated chromosomes are located within domains which may intertwine, and [4] Each segment of the random walks corresponds to approx. 2,000 DNA base pairs. NASARTI uses pre-calculated dose at each voxel to calculate the probability of DNA damage at each random walk segment. Using the location of double-strand breaks, possible rejoining between damaged segments is evaluated. This yields various types of chromosomes aberrations, including deletions, inversions, exchanges, etc. By performing the calculations using various types of radiations, it will be possible to obtain relative biological effectiveness (RBE) values for several types of chromosome aberrations.

  20. DNA methylation: conducting the orchestra from exposure to phenotype?

    Science.gov (United States)

    Leenen, Fleur A D; Muller, Claude P; Turner, Jonathan D

    2016-01-01

    DNA methylation, through 5-methyl- and 5-hydroxymethylcytosine (5mC and 5hmC), is considered to be one of the principal interfaces between the genome and our environment, and it helps explain phenotypic variations in human populations. Initial reports of large differences in methylation level in genomic regulatory regions, coupled with clear gene expression data in both imprinted genes and malignant diseases, provided easily dissected molecular mechanisms for switching genes on or off. However, a more subtle process is becoming evident, where small (exposure to a disease phenotype led to numerous observational/association studies being performed. Although this expectation remains unmet, there is now a growing body of literature on specific genes, suggesting wide ranging transcriptional and translational consequences of such subtle methylation changes. Data from the glucocorticoid receptor (NR3C1) has shown that a complex interplay between DNA methylation, extensive 5'UTR splicing, and microvariability gives rise to the overall level and relative distribution of total and N-terminal protein isoforms generated. Additionally, the presence of multiple AUG translation initiation codons throughout the complete, processed mRNA enables translation variability, hereby enhancing the translational isoforms and the resulting protein isoform diversity, providing a clear link between small changes in DNA methylation and significant changes in protein isoforms and cellular locations. Methylation changes in the NR3C1 CpG island alters the NR3C1 transcription and eventually protein isoforms in the tissues, resulting in subtle but visible physiological variability. This review addresses the current pathophysiological and clinical associations of such characteristically small DNA methylation changes, the ever-growing roles of DNA methylation and the evidence available, particularly from the glucocorticoid receptor of the cascade of events initiated by such subtle methylation changes

  1. DNA sequence explains seemingly disordered methylation levels in partially methylated domains of Mammalian genomes.

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    Dimos Gaidatzis

    2014-02-01

    Full Text Available For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs, recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40% of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS. Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2 of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.

  2. Role of TET enzymes in DNA methylation, development, and cancer

    Science.gov (United States)

    Rasmussen, Kasper Dindler

    2016-01-01

    The pattern of DNA methylation at cytosine bases in the genome is tightly linked to gene expression, and DNA methylation abnormalities are often observed in diseases. The ten eleven translocation (TET) enzymes oxidize 5-methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation. TET genes, and especially TET2, are frequently mutated in various cancers, but how the TET proteins contribute to prevent the onset and maintenance of these malignancies is largely unknown. Here, we highlight recent advances in understanding the physiological function of the TET proteins and their role in regulating DNA methylation and transcription. In addition, we discuss some of the key outstanding questions in the field. PMID:27036965

  3. Research Advances in Pituitary Adenoma and DNA Methylation.

    Science.gov (United States)

    Wei, Zhen-Qing; Li, Yang; Li, Wei-Hua; Lou, Jia-Cheng; Zhang, Bo

    2016-08-01

    DNA methylation is closely related to the genesis and development of pituitary adenoma. Studies have shown that high methylation in the promoter region of potassium voltage-gated chanel,shaker related subfamily,beta member 2,O-6-methylguanine-DNA methyltransferase,echinoderm microtubule associated protein like 2 ,ras homolog family member D ,homeobox B1 ,NNAT, and P16 inhibits the expression of these genes and regulates of the proliferation of pituitary adenoma. DNA methylation is also closely related to invasive pituitary adenoma. Therefore,further study on molecular mechanism of DNA methylation of pituitary adenoma will offer a new strategy for the diagnosis and treatment of pituitary adenoma. PMID:27594164

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

    Science.gov (United States)

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

    2016-08-01

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

  5. Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP assays in using in vitro methylated genomic DNA

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    Jia Jinsong

    2010-09-01

    Full Text Available Abstract Background DNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of in vitro methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples. Findings We performed MeDIP assays using in vitro methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using in vitro methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA, we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data. Conclusion We illustrate the use of in vitro methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.

  6. Lung fibroblasts from patients with idiopathic pulmonary fibrosis exhibit genome-wide differences in DNA methylation compared to fibroblasts from nonfibrotic lung.

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    Steven K Huang

    Full Text Available Excessive fibroproliferation is a central hallmark of idiopathic pulmonary fibrosis (IPF, a chronic, progressive disorder that results in impaired gas exchange and respiratory failure. Fibroblasts are the key effector cells in IPF, and aberrant expression of multiple genes contributes to their excessive fibroproliferative phenotype. DNA methylation changes are critical to the development of many diseases, but the DNA methylome of IPF fibroblasts has never been characterized. Here, we utilized the HumanMethylation 27 array, which assays the DNA methylation level of 27,568 CpG sites across the genome, to compare the DNA methylation patterns of IPF fibroblasts (n = 6 with those of nonfibrotic patient controls (n = 3 and commercially available normal lung fibroblast cell lines (n = 3. We found that multiple CpG sites across the genome are differentially methylated (as defined by P value less than 0.05 and fold change greater than 2 in IPF fibroblasts compared to fibroblasts from nonfibrotic controls. These methylation differences occurred both in genes recognized to be important in fibroproliferation and extracellular matrix generation, as well as in genes not previously recognized to participate in those processes (including organ morphogenesis and potassium ion channels. We used bisulfite sequencing to independently verify DNA methylation differences in 3 genes (CDKN2B, CARD10, and MGMT; these methylation changes corresponded with differences in gene expression at the mRNA and protein level. These differences in DNA methylation were stable throughout multiple cell passages. DNA methylation differences may thus help to explain a proportion of the differences in gene expression previously observed in studies of IPF fibroblasts. Moreover, significant variability in DNA methylation was observed among individual IPF cell lines, suggesting that differences in DNA methylation may contribute to fibroblast heterogeneity among patients with IPF

  7. Sensing DNA methylation in the protozoan parasite Entamoeba histolytica.

    Science.gov (United States)

    Lavi, Tal; Isakov, Elada; Harony, Hala; Fisher, Ohad; Siman-Tov, Rama; Ankri, Serge

    2006-12-01

    In the protozoan parasite Entamoeba histolytica, 5-methylcytosine (m5C) was found predominantly in repetitive elements. Its formation is catalysed by Ehmeth, a DNA methyltransferase that belongs to the Dnmt2 subfamily. Here we describe a 32 kDa nuclear protein that binds in vitro with higher affinity to the methylated form of a DNA encoding a reverse transcriptase of an autonomous non-long-terminal repeat retrotransposon (RT LINE) compared with the non-methylated RT LINE. This protein, named E. histolytica-methylated LINE binding protein (EhMLBP), was purified from E. histolytica nuclear lysate, identified by mass spectrometry, and its corresponding gene was cloned. EhMLBP corresponds to a gene of unknown function that shares strong homology with putative proteins present in Entamoeba dispar and Entamoeba invadens. In contrast, the homology dropped dramatically when non-Entamoebidae sequences were considered and only a weak sequence identity was found with Trypanosoma and several prokaryotic histone H1. Recombinant EhMLBP showed the same binding preference for methylated RT LINE as the endogenous EhMLBP. Deletion mapping analysis localized the DNA binding region at the C-terminal part of the protein. This region is sufficient to assure the binding to methylated RT LINE with high affinity. Western blot and immunofluorescence microscopy, using an antibody raised against EhMLBP, showed that it has a nuclear localization. Chromatin immunoprecipitation (ChIP) confirmed that EhMLBP interacts with RT LINE in vivo. Finally, we showed that EhMLBP can also bind rDNA episome, a DNA that is methylated in the parasite. This suggests that EhMLBP may serve as a sensor of methylated repetitive DNA. This is the first report of a DNA-methylated binding activity in protozoa.

  8. Epigenetic therapy of cancer stem and progenitor cells bytargeting DNA methylation machineries

    Institute of Scientific and Technical Information of China (English)

    Patompon Wongtrakoongate

    2015-01-01

    Recent advances in stem cell biology have shed light onhow normal stem and progenitor cells can evolve to acquiremalignant characteristics during tumorigenesis. The cancercounterparts of normal stem and progenitor cells might beoccurred through alterations of stem cell fates includingan increase in self-renewal capability and a decreasein differentiation and/or apoptosis. This oncogenicevolution of cancer stem and progenitor cells, which oftenassociates with aggressive phenotypes of the tumorigeniccells, is controlled in part by dysregulated epigeneticmechanisms including aberrant DNA methylation leadingto abnormal epigenetic memory. Epigenetic therapy bytargeting DNA methyltransferases (DNMT) 1, DNMT3Aand DNMT3B via 5-Azacytidine (Aza) and 5-Aza-2'-deoxycytidine (Aza-dC) has proved to be successfultoward treatment of hematologic neoplasms especially forpatients with myelodysplastic syndrome. In this review,I summarize the current knowledge of mechanismsunderlying the inhibition of DNA methylation by Aza andAza-dC, and of their apoptotic- and differentiation-inducingeffects on cancer stem and progenitor cells in leukemia,medulloblastoma, glioblastoma, neuroblastoma, prostatecancer, pancreatic cancer and testicular germ cell tumors.Since cancer stem and progenitor cells are implicatedin cancer aggressiveness such as tumor formation,progression, metastasis and recurrence, I proposethat effective therapeutic strategies might be achievedthrough eradication of cancer stem and progenitor cellsby targeting the DNA methylation machineries to interferetheir "malignant memory".

  9. Genome-Wide Analysis of DNA Methylation in Human Amnion

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    Jinsil Kim

    2013-01-01

    Full Text Available The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3 gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies.

  10. DNA Copy Number Aberrations in Breast Cancer by Array Comparative Genomic Hybridization

    Institute of Scientific and Technical Information of China (English)

    Jian Li; Kai Wang; Shengting Li; Vera Timmermans-Wielenga; Fritz Rank; Carsten Wiuf; Xiuqing Zhang; Huanming Yang; Lars Bolund

    2009-01-01

    Array comparative genomic hybridization (CGH) has been popularly used for an-alyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb reso-lution bacterial artificial chromosome CGH arrays. A number of highly frequent genomic aberrations were discovered, which may act as "drivers" of tumor pro-gression. Meanwhile, the genomic profiles of four "normal" breast tissue samples taken at least 2 cm away from the primary tumor sites were also found to have some genomic aberrations that recurred with high frequency in the primary tu-mors, which may have important implications for clinical therapy. Additionally, we performed class comparison and class prediction for various clinicopathological pa-rameters, and a list of characteristic genomic aberrations associated with different clinicopathological phenotypes was compiled. Our study provides clues for further investigations of the underlying mechanisms of breast carcinogenesis.

  11. Analysis of DNA methylation in various swine tissues.

    Directory of Open Access Journals (Sweden)

    Chun Yang

    Full Text Available DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively.In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome.

  12. The dynamics and prognostic potential of DNA methylation changes at stem cell gene loci in women's cancer.

    Directory of Open Access Journals (Sweden)

    Joanna Zhuang

    2012-02-01

    Full Text Available Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.

  13. Dynamics of nucleosome assembly and effects of DNA methylation.

    Science.gov (United States)

    Lee, Ju Yeon; Lee, Jaehyoun; Yue, Hongjun; Lee, Tae-Hee

    2015-02-13

    The nucleosome is the fundamental packing unit of the eukaryotic genome, and CpG methylation is an epigenetic modification associated with gene repression and silencing. We investigated nucleosome assembly mediated by histone chaperone Nap1 and the effects of CpG methylation based on three-color single molecule FRET measurements, which enabled direct monitoring of histone binding in the context of DNA wrapping. According to our observation, (H3-H4)2 tetramer incorporation must precede H2A-H2B dimer binding, which is independent of DNA termini wrapping. Upon CpG methylation, (H3-H4)2 tetramer incorporation and DNA termini wrapping are facilitated, whereas proper incorporation of H2A-H2B dimers is inhibited. We suggest that these changes are due to rigidified DNA and increased random binding of histones to DNA. According to the results, CpG methylation expedites nucleosome assembly in the presence of abundant DNA and histones, which may help facilitate gene packaging in chromatin. The results also indicate that the slowest steps in nucleosome assembly are DNA termini wrapping and tetramer positioning, both of which are affected heavily by changes in the physical properties of DNA.

  14. The prima donna of epigenetics: the regulation of gene expression by DNA methylation

    Directory of Open Access Journals (Sweden)

    K.F. Santos

    2005-10-01

    Full Text Available This review focuses on the mechanisms of DNA methylation, DNA methylation pattern formation and their involvement in gene regulation. Association of DNA methylation with imprinting, embryonic development and human diseases is discussed. Furthermore, besides considering changes in DNA methylation as mechanisms of disease, the role of epigenetics in general and DNA methylation in particular in transgenerational carcinogenesis, in memory formation and behavior establishment are brought about as mechanisms based on the cellular memory of gene expression patterns.

  15. Maternal Methyl-Group Donor Intake and Global DNA (HydroxyMethylation before and during Pregnancy

    Directory of Open Access Journals (Sweden)

    Sara Pauwels

    2016-08-01

    Full Text Available It is still unclear to which extent methyl-group intake during pregnancy can affect maternal global DNA (hydroxylmethylation. Pregnancy methylation profiling and its link with methyl-group intake in a healthy population could enhance our understanding of the development of pregnancy related disorders. One hundred forty-eight women were enrolled in the MANOE (MAternal Nutrition and Offspring’s Epigenome study. Thiry-four women were enrolled before pregnancy and 116 during the first trimester of pregnancy. Global DNA (hydroxymethylation in blood using LC-MS/MS and dietary methyl-group intake (methionine, folate, betaine, and choline using a food-frequency questionnaire were estimated pre-pregnancy, during each trimester, and at delivery. Global DNA (hydroxymethylation levels were highest pre-pregnancy and at weeks 18–22 of pregnancy. We observed a positive relation between folic acid and global DNA methylation (p = 0.04 and hydroxymethylation (p = 0.04. A high intake of methionine pre-pregnancy and in the first trimester showed lower (hydroxymethylation percentage in weeks 11–13 and weeks 18–22, respectively. Choline and betaine intake in the first weeks was negatively associated with hydroxymethylation. Women with a high intake of these three methyl groups in the second and third trimester showed higher hyrdoxymethylation/methylation levels in the third trimester. To conclude, a time trend in DNA (hydroxymethylation was found and women with higher methyl-group intake showed higher methylation in the third trimester, and not in earlier phases of pregnancy.

  16. Effects of cytosine methylation on DNA charge transport

    Science.gov (United States)

    Hihath, Joshua; Guo, Shaoyin; Zhang, Peiming; Tao, Nongjian

    2012-04-01

    The methylation of cytosine bases in DNA commonly takes place in the human genome and its abnormality can be used as a biomarker in the diagnosis of genetic diseases. In this paper we explore the effects of cytosine methylation on the conductance of DNA. Although the methyl group is a small chemical modification, and has a van der Waals radius of only 2 Å, its presence significantly changes the duplex stability, and as such may also affect the conductance properties of DNA. To determine if charge transport through the DNA stack is sensitive to this important biological modification we perform multiple conductance measurements on a methylated DNA molecule with an alternating G:C sequence and its non-methylated counterpart. From these studies we find a measurable difference in the conductance between the two types of molecules, and demonstrate that this difference is statistically significant. The conductance values of these molecules are also compared with a similar sequence that has been previously studied to help elucidate the charge transport mechanisms involved in direct DNA conductance measurements.

  17. DNA Methylation, Epigenetics, and Evolution in Vertebrates: Facts and Challenges

    Directory of Open Access Journals (Sweden)

    Annalisa Varriale

    2014-01-01

    Full Text Available DNA methylation is a key epigenetic modification in the vertebrate genomes known to be involved in biological processes such as regulation of gene expression, DNA structure and control of transposable elements. Despite increasing knowledge about DNA methylation, we still lack a complete understanding of its specific functions and correlation with environment and gene expression in diverse organisms. To understand how global DNA methylation levels changed under environmental influence during vertebrate evolution, we analyzed its distribution pattern along the whole genome in mammals, reptiles and fishes showing that it is correlated with temperature, independently on phylogenetic inheritance. Other studies in mammals and plants have evidenced that environmental stimuli can promote epigenetic changes that, in turn, might generate localized changes in DNA sequence resulting in phenotypic effects. All these observations suggest that environment can affect the epigenome of vertebrates by generating hugely different methylation patterns that could, possibly, reflect in phenotypic differences. We are at the first steps towards the understanding of mechanisms that underlie the role of environment in molding the entire genome over evolutionary times. The next challenge will be to map similarities and differences of DNA methylation in vertebrates and to associate them with environmental adaptation and evolution.

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

    Science.gov (United States)

    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...

  19. Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus

    Directory of Open Access Journals (Sweden)

    Rihong Zhai

    2012-01-01

    Full Text Available Aberrant DNA methylation (DNAm is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA and Barrett esophagus (BE, EA precursor. We performed genome-wide DNAm profiling in EA tissue DNA (n = 8 and matched serum DNA (n = 8, in serum DNA of BE (n = 10, and in healthy controls (n = 10 using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92 in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.

  20. MIR-9-1 ABERRANT METHYLATION IS A FREQUENT EVENT IN BREAST CANCER AND IS ASSOCIATED WITH BONE METASTASES

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    Anca Florescu

    2012-10-01

    Full Text Available Abstract: Background. Aberrant promoter methylation of classical tumor suppressor genes occurs frequently during carcinogenesis. Several lines of evidences suggest that this epigenetic change also regulates microRNAs expression and may represent a potential molecular marker for cancer. Methods. We examined the methylation status at the hsa-miR-9-1 gene promoter in a series of 66 breast cancer cases by methylation sensitive PCR (MSP analysis. For 43 of the 66 patients paired normal breast tissue and/or pre invasive (ADH, DCIS lesions were also available. As control methylation status was determined on 6 normal breast tissues obtained from reductive mammoplasty.   Results. Methylation at mir-9-1 gene was detected in 32 out of 66 breast tumours (49% and in none of the 6 normal breast tissues derived from reductive mammoplasty (P=0.02 χ2- Test. In all cases the same methylation status was demonstrated in tumour specimen, paired normal breast tissues and/or pre-invasive (ADH and DCIS lesions. An higher frequency of methylation was found in patients showing metastases at diagnosis as compared with non metastatic patients (P=0.03 χ2-Test. Moreover, methylation at mir-9-1 gene was more frequent in patients showing bone metastases as first metastatic sites (P=0.04 χ2-Test, and in the subgroup of patients developing only bone metastases as compared with patients developing metastases  to visceral organs (P=0.03 χ2-Test. Conclusions. This study give further evidence of epigenetic mechanisms as regulators of miR-9 expression in breast cancer. Moreover, our results suggest an association between hypermethylation  at the miR-9-1 gene and metastatic site.

  1. DNA methylation mediated silencing of microRNA-145 is a potential prognostic marker in patients with lung adenocarcinoma

    OpenAIRE

    Wenjie Xia; Qiang Chen; Jie Wang; Qixing Mao; Gaochao Dong; Run Shi; YanYan Zheng; Lin Xu; Feng Jiang

    2015-01-01

    The molecular mechanism of down-regulated microRNA-145 (miR-145) expression in lung adenocarcinoma (LAC) remains largely unknown. We hypothesized that aberrant hyper-methylation of the CpG sites silenced the expression of miR-145 in LAC. In consideration of its pivotal role in LAC development and progression, we also evaluated the clinical utility of miR-145 as a prognostic marker. We assessed the DNA methylation status of the miR-145 promoter region in 20 pairs of LAC and the matched non-tum...

  2. The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

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    Evan J. Ryer

    2015-05-01

    Full Text Available Abdominal aortic aneurysm (AAA is a complex disorder that has a significant impact on the aging population. While both genetic and environmental risk factors have been implicated in AAA formation, the precise genetic markers involved and the factors influencing their expression remain an area of ongoing investigation. DNA methylation has been previously used to study gene silencing in other inflammatory disorders and since AAA has an extensive inflammatory component, we sought to examine the genome-wide DNA methylation profiles in mononuclear blood cells of AAA cases and matched non-AAA controls. To this end, we collected blood samples and isolated mononuclear cells for DNA and RNA extraction from four all male groups: AAA smokers (n = 11, AAA non-smokers (n = 9, control smokers (n = 10 and control non-smokers (n = 11. Methylation data were obtained using the Illumina 450k Human Methylation Bead Chip and analyzed using the R language and multiple Bioconductor packages. Principal component analysis and linear analysis of CpG island subsets identified four regions with significant differences in methylation with respect to AAA: kelch-like family member 35 (KLHL35, calponin 2 (CNN2, serpin peptidase inhibitor clade B (ovalbumin member 9 (SERPINB9, and adenylate cyclase 10 pseudogene 1 (ADCY10P1. Follow-up studies included RT-PCR and immunostaining for CNN2 and SERPINB9. These findings are novel and suggest DNA methylation may play a role in AAA pathobiology.

  3. The potential role of DNA methylation in abdominal aortic aneurysms.

    Science.gov (United States)

    Ryer, Evan J; Ronning, Kaitryn E; Erdman, Robert; Schworer, Charles M; Elmore, James R; Peeler, Thomas C; Nevius, Christopher D; Lillvis, John H; Garvin, Robert P; Franklin, David P; Kuivaniemi, Helena; Tromp, Gerard

    2015-01-01

    Abdominal aortic aneurysm (AAA) is a complex disorder that has a significant impact on the aging population. While both genetic and environmental risk factors have been implicated in AAA formation, the precise genetic markers involved and the factors influencing their expression remain an area of ongoing investigation. DNA methylation has been previously used to study gene silencing in other inflammatory disorders and since AAA has an extensive inflammatory component, we sought to examine the genome-wide DNA methylation profiles in mononuclear blood cells of AAA cases and matched non-AAA controls. To this end, we collected blood samples and isolated mononuclear cells for DNA and RNA extraction from four all male groups: AAA smokers (n = 11), AAA non-smokers (n = 9), control smokers (n = 10) and control non-smokers (n = 11). Methylation data were obtained using the Illumina 450k Human Methylation Bead Chip and analyzed using the R language and multiple Bioconductor packages. Principal component analysis and linear analysis of CpG island subsets identified four regions with significant differences in methylation with respect to AAA: kelch-like family member 35 (KLHL35), calponin 2 (CNN2), serpin peptidase inhibitor clade B (ovalbumin) member 9 (SERPINB9), and adenylate cyclase 10 pseudogene 1 (ADCY10P1). Follow-up studies included RT-PCR and immunostaining for CNN2 and SERPINB9. These findings are novel and suggest DNA methylation may play a role in AAA pathobiology. PMID:25993294

  4. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    Science.gov (United States)

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  5. Accounting for population stratification in DNA methylation studies.

    Science.gov (United States)

    Barfield, Richard T; Almli, Lynn M; Kilaru, Varun; Smith, Alicia K; Mercer, Kristina B; Duncan, Richard; Klengel, Torsten; Mehta, Divya; Binder, Elisabeth B; Epstein, Michael P; Ressler, Kerry J; Conneely, Karen N

    2014-04-01

    DNA methylation is an important epigenetic mechanism that has been linked to complex diseases and is of great interest to researchers as a potential link between genome, environment, and disease. As the scale of DNA methylation association studies approaches that of genome-wide association studies, issues such as population stratification will need to be addressed. It is well-documented that failure to adjust for population stratification can lead to false positives in genetic association studies, but population stratification is often unaccounted for in DNA methylation studies. Here, we propose several approaches to correct for population stratification using principal components (PCs) from different subsets of genome-wide methylation data. We first illustrate the potential for confounding due to population stratification by demonstrating widespread associations between DNA methylation and race in 388 individuals (365 African American and 23 Caucasian). We subsequently evaluate the performance of our PC-based approaches and other methods in adjusting for confounding due to population stratification. Our simulations show that (1) all of the methods considered are effective at removing inflation due to population stratification, and (2) maximum power can be obtained with single-nucleotide polymorphism (SNP)-based PCs, followed by methylation-based PCs, which outperform both surrogate variable analysis and genomic control. Among our different approaches to computing methylation-based PCs, we find that PCs based on CpG sites chosen for their potential to proxy nearby SNPs can provide a powerful and computationally efficient approach to adjust for population stratification in DNA methylation studies when genome-wide SNP data are unavailable.

  6. Accounting for population stratification in DNA methylation studies.

    Science.gov (United States)

    Barfield, Richard T; Almli, Lynn M; Kilaru, Varun; Smith, Alicia K; Mercer, Kristina B; Duncan, Richard; Klengel, Torsten; Mehta, Divya; Binder, Elisabeth B; Epstein, Michael P; Ressler, Kerry J; Conneely, Karen N

    2014-04-01

    DNA methylation is an important epigenetic mechanism that has been linked to complex diseases and is of great interest to researchers as a potential link between genome, environment, and disease. As the scale of DNA methylation association studies approaches that of genome-wide association studies, issues such as population stratification will need to be addressed. It is well-documented that failure to adjust for population stratification can lead to false positives in genetic association studies, but population stratification is often unaccounted for in DNA methylation studies. Here, we propose several approaches to correct for population stratification using principal components (PCs) from different subsets of genome-wide methylation data. We first illustrate the potential for confounding due to population stratification by demonstrating widespread associations between DNA methylation and race in 388 individuals (365 African American and 23 Caucasian). We subsequently evaluate the performance of our PC-based approaches and other methods in adjusting for confounding due to population stratification. Our simulations show that (1) all of the methods considered are effective at removing inflation due to population stratification, and (2) maximum power can be obtained with single-nucleotide polymorphism (SNP)-based PCs, followed by methylation-based PCs, which outperform both surrogate variable analysis and genomic control. Among our different approaches to computing methylation-based PCs, we find that PCs based on CpG sites chosen for their potential to proxy nearby SNPs can provide a powerful and computationally efficient approach to adjust for population stratification in DNA methylation studies when genome-wide SNP data are unavailable. PMID:24478250

  7. DNA methylation profiling identifies two splenic marginal zone lymphoma subgroups with different clinical and genetic features.

    Science.gov (United States)

    Arribas, Alberto J; Rinaldi, Andrea; Mensah, Afua A; Kwee, Ivo; Cascione, Luciano; Robles, Eloy F; Martinez-Climent, Jose A; Oscier, David; Arcaini, Luca; Baldini, Luca; Marasca, Roberto; Thieblemont, Catherine; Briere, Josette; Forconi, Francesco; Zamò, Alberto; Bonifacio, Massimiliano; Mollejo, Manuela; Facchetti, Fabio; Dirnhofer, Stephan; Ponzoni, Maurilio; Bhagat, Govind; Piris, Miguel A; Gaidano, Gianluca; Zucca, Emanuele; Rossi, Davide; Bertoni, Francesco

    2015-03-19

    Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation. PMID:25612624

  8. DNA methylation in diploid inbred lines of potatoes and its possible role in the regulation of heterosis.

    Science.gov (United States)

    Nakamura, Sunao; Hosaka, Kazuyoshi

    2010-01-01

    Self-incompatible diploid potatoes were altered to self-compatible ones by a function of S-locus inhibitor gene and continued selfing generated highly homozygous inbreds. In this study, this process was investigated for the status of DNA methylation by a simple method using genomic DNA digested by methylation-sensitive restriction enzymes prior to RAPD analysis. We detected 31 methylation-sensitive RAPD bands, of which 11 were newly appeared in the selfed progenies, and 6 of them stably inherited to subsequent generations. Aberrant segregations and paternal- or atavism-like transmission were also found. Segregating methylation-sensitive bands in initial populations became fixed in the advanced selfed progenies by 75.0-93.8%, of which 41.7% were fixed to all present and 58.3% to all absent. Because DNA methylation is generally recognized to suppress gene expression as regulatory factors, homozygosity/heterozygosity of methylated DNA may be involved in inbreeding depression/heterosis. PMID:19455300

  9. Blood-derived DNA methylation markers of cancer risk.

    Science.gov (United States)

    Marsit, Carmen; Christensen, Brock

    2013-01-01

    The importance of somatic epigenetic alterations in tissues targeted for carcinogenesis is now well recognized and considered a key molecular step in the development of a tumor. Particularly, alteration of gene-specific and genomic DNA methylation has been extensively characterized in tumors, and has become an attractive biomarker of risk due to its specificity and stability in human samples. It also is clear that tumors do not develop as isolated phenomenon in their target tissue, but instead result from altered processes affecting not only the surrounding cells and tissues, but other organ systems, including the immune system. Thus, alterations to DNA methylation profiles detectable in peripheral blood may be useful not only in understanding the carcinogenic process and response to environmental insults, but can also provide critical insights in a systems biological view of tumorigenesis. Research to date has generally focused on how environmental exposures alter genomic DNA methylation content in peripheral blood. More recent work has begun to translate these findings to clinically useful endpoints, by defining the relationship between DNA methylation alterations and cancer risk. This chapter highlights the existing research linking the environment, blood-derived DNA methylation alterations, and cancer risk, and points out how these epigenetic alterations may be contributing fundamentally to carcinogenesis.

  10. Heritable Transmission of Diabetic Metabolic Memory in Zebrafish Correlates With DNA Hypomethylation and Aberrant Gene Expression

    OpenAIRE

    Olsen, Ansgar S.; Sarras, Michael P.; LEONTOVICH, ALEXEY; Intine, Robert V.

    2012-01-01

    Metabolic memory (MM) is the phenomenon whereby diabetes complications persist and progress after glycemic recovery is achieved. Here, we present data showing that MM is heritable and that the transmission correlates with hyperglycemia-induced DNA hypomethylation and aberrant gene expression. Streptozocin was used to induce hyperglycemia in adult zebrafish, and then, following streptozocin withdrawal, a recovery phase was allowed to reestablish a euglycemic state. Blood glucose and serum insu...

  11. DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter.

    Science.gov (United States)

    Han, Han; Cortez, Connie C; Yang, Xiaojing; Nichols, Peter W; Jones, Peter A; Liang, Gangning

    2011-11-15

    Despite the fact that 45% of all human gene promoters do not contain CpG islands, the role of DNA methylation in control of non-CpG island promoters is controversial and its relevance in normal and pathological processes is poorly understood. Among the few studies which investigate the correlation between DNA methylation and expression of genes with non-CpG island promoters, the majority do not support the view that DNA methylation directly leads to transcription silencing of these genes. Our reporter assays and gene reactivation by 5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly lead to transcriptional silencing in cells competent to express these genes in vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a single-molecule, high-resolution nucleosome positioning assay, we demonstrate that active, but not inactive, non-CpG island promoters display a nucleosome-depleted region (NDR) immediately upstream of the transcription start site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin configurations: one is unmethylated with an NDR upstream of the TSS; another is methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically methylated. Together, these results demonstrate that the epigenetic signatures comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis and should therefore be included in analyses of cancer epigenetics.

  12. Cytosine methylation of sperm DNA in horse semen after cryopreservation.

    Science.gov (United States)

    Aurich, Christine; Schreiner, Bettina; Ille, Natascha; Alvarenga, Marco; Scarlet, Dragos

    2016-09-15

    Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure. PMID:27242182

  13. DNA methylation analysis of the angiotensin converting enzyme (ACE gene in major depression.

    Directory of Open Access Journals (Sweden)

    Peter Zill

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

  14. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie;

    2010-01-01

    brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...... and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.......Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA...

  15. Global DNA methylation changes in Cucurbitaceae inter-species grafting

    Directory of Open Access Journals (Sweden)

    Evangelia Avramidou

    2015-04-01

    Full Text Available Grafting has been used to improve yield, fruit quality and disease resistance in a range of tree and vegetable species. The molecular mechanisms underpinning grafting responses have only recently started to be delineated. One of those mechanisms involves long distance transfer of genetic material from rootstock to scion alluding to an epigenetic component to the grafting process. In the research presented herein we extended published work on heritable changes in the DNA methylation pattern of Solanaceae scion genomes, in Cucurbitaceae inter-species grafting. Specifically, we examined global DNA methylation changes in scions of cucumber, melon and watermelon heterografted onto pumpkin rootstocks using MSAP analysis. We observed a significant increase of global DNA methylation in cucumber and melon scions pointing to an epigenetic effect in Cucurbitaceae heterografting. Exploitation of differential epigenetic marking in different rootstock-scion combinations could lead to development of epi-molecular markers for generation and selection of superior quality grafted vegetables.

  16. DNA methylation and gene expression of HIF3A

    DEFF Research Database (Denmark)

    Main, Ailsa Maria; Gillberg, Linn; Jacobsen, Anna Louisa;

    2016-01-01

    BACKGROUND: Associations between BMI and DNA methylation of hypoxia-inducible factor 3-alpha (HIF3A) in both blood cells and subcutaneous adipose tissue (SAT) have been reported. In this study, we investigated associations between BMI and HIF3A DNA methylation in the blood and SAT from the same...... individuals, and whether HIF3A gene expression in SAT and skeletal muscle biopsies showed associations with BMI and insulin resistance. Furthermore, we aimed to investigate gender specificity and heritability of these traits. METHODS: We studied 137 first-degree relatives of type 2 diabetes (T2D) patients...... from 48 families, from whom we had SAT and muscle biopsies. DNA methylation of four CpG sites in the HIF3A promoter was analyzed in the blood and SAT by pyrosequencing, and HIF3A gene expression was analyzed in SAT and muscle by qPCR. An index of whole-body insulin sensitivity was estimated from oral...

  17. The DNA methylation profile of activated human natural killer cells.

    Science.gov (United States)

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-01

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states. PMID:26967308

  18. RNA-directed DNA methylation and demethylation in plants

    Institute of Scientific and Technical Information of China (English)

    CHINNUSAMY; Viswanathan

    2009-01-01

    RNA-directed DNA methylation (RdDM) is a nuclear process in which small interfering RNAs (siRNAs) direct the cytosine methylation of DNA sequences that are complementary to the siRNAs. In plants, double stranded-RNAs (dsRNAs) generated by RNA-dependent RNA polymerase 2 (RDR2) serve as precursors for Dicer-like 3 dependent biogenesis of 24-nt siRNAs. Plant specific RNA polymerase IV (Pol IV) is presumed to generate the initial RNA transcripts that are substrates for RDR2. siRNAs are loaded onto an argonaute4-containing RISC (RNA-induced silencing complex) that targets the de novo DNA methyltransferase DRM2 to RdDM target loci. Nascent RNA transcripts from the target loci are generated by another plant-specific RNA polymerase, Pol V, and these transcripts help recruit com- plementary siRNAs and the associated RdDM effector complex to the target loci in a transcrip- tion-coupled DNA methylation process. Small RNA binding proteins such as ROS3 may direct tar- get-specific DNA demethylation by the ROS1 family of DNA demethylases. Chromatin remodeling en- zymes and histone modifying enzymes also participate in DNA methylation and possibly demethylation. One of the well studied functions of RdDM is transposon silencing and genome stability. In addition, RdDM is important for paramutation, imprinting, gene regulation, and plant development. Lo- cus-specific DNA methylation and demethylation, and transposon activation under abiotic stresses suggest that RdDM is also important in stress responses of plants. Further studies will help illuminate the functions of RdDM in the dynamic control of epigenomes during development and environmental stress responses.

  19. RNA-directed DNA methylation and demethylation in plants

    Institute of Scientific and Technical Information of China (English)

    CHINNUSAMY Viswanathan; ZHU Jian-Kang

    2009-01-01

    A-dlrected DNA methylation (RdDM) Is a nuclear process in which small Interfering RNAs (siRNAs)direct the cytosine methylation of DNA sequences that are complementary to the siRNAs. In plants,double stranded-RNAs (dsRNAs) generated by RNA-dependent RNA polymerase 2 (RDR2) serve as precursors for Dicer-like 3 dependent biogenesis of 24-nt siRNAs. Plant specific RNA polymerase IV (Pol IV) is presumed to generate the initial RNA transcripts that are substrates for RDR2. siRNAs are loaded onto an argonaute4-containlng RlSC (RNA-induced silencing complex) that targets the de novo DNA methyltransferase DRM2 to RdDM target locl. Nascent RNA transcripts from the target loci are generated by another plant-specific RNA polymerase, Pol V, and these transcripts help recruit com-plementary siRNAs and the associated RdDM effector complex to the target loci in a transcrip-tion-coupled DNA methylation process. Small RNA binding proteins such as ROS3 may direct tar-get-specific DNA demethyiation by the ROS1 family of DNA demethylases. Chromatin remodeling en-zymes and histone modifying enzymes also participate in DNA methylation and possibly demethylation.One of the well studied functions of RdOM is transposon silencing and genome stability. In addition,RdDM is important for paramutation, imprinting, gene regulation, and plant development. Locus-specific DNA methylation and demethylation, and transposon activation under abiotic stresses suggest that RdDM is also important in stress responses of plants. Further studies will help illuminate the functions of RdDM in the dynamic control of epigenomes during development and environmental stress responses.

  20. Dysfunction of endothelial NO system originated from homocysteine-induced aberrant methylation pattern in promoter region of DDAH2 gene

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jing-ge; LIU Jun-xu; LI Zhu-hua; WANG Li-zhen; JIANG Yi-deng; WANG Shu-ren

    2007-01-01

    Background Hyperhomocysteinemia (HHcy)-mediated dysfunction of endothelial NO system is an important mechanism for atherosclerotic pathogenesis.Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme for degrading asymmetric dimethylarginine (ADMA),which is an endogenous inhibitor of endothelial nitric oxide (NO) synthase (eNOS).This study was designed to investigate whether the dysfunction of endothelial NO system originates from HHcy-mediated aberrant methylation modification in promotor region of DDAH2 gene.Methods Human umbilical vein endothelial cells (HUVECs) were cultured to the third generation and treated with homocysteine (Hcy) at different concentrations (0,10,30,100,and 300 μmol/L) for 72 hours.The methylation pattern in promoter region CpG island of DDAH2 gene was analyzed by nested methylation-specific PCR (nMSP).The mRNA expression of eNOS gene and DDAH2 gene was detected by semi-quantitative RT-PCR.The activity of DDAH2 and eNOS in cells,and the concentrations of ADMA and NO in culture medium were assayed respectively.Results Mild increased concentration of Hcy (10 and 30 μmol/L) induced hypomethylation,while high concentration of Hcy (100 and 300 μmol/L) induced hypermethylation in the promoter CpG island of DDAH2 gene.The mRNA expression of DDAH2 increased in mild enhanced concentration of Hcy,and decreased in high concentration of Hcy correspondingly.The inhibition of DDAH2 activity,the increase of ADMA concentration,the reduction of eNOS activity and the decrease of NO production were all consistently relevant to the alteration of Hcy concentration Conclusion The increased concentration of Hcy induced aberrant methylation pattern in promotor region of DDAH2 gene and the successive alterations in DDAH/ADMA/NOS/NO pathway,which showed highly relevant and dose-effect relationship.The results suggested that the dysfunction of endothelial NO system induced by HHcy could be partially originated from Hcy-mediated aberrant methylation in

  1. Altered DNA methylation in leukocytes with trisomy 21.

    Directory of Open Access Journals (Sweden)

    Kristi Kerkel

    2010-11-01

    Full Text Available The primary abnormality in Down syndrome (DS, trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq confirmed strong differences in methylation (p<0.0001 for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2'deoxycytidine (5aza-dC plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.

  2. DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection

    International Nuclear Information System (INIS)

    Epigenetic alterations are a hallmark of human cancer. In this study, we aimed to investigate whether aberrant DNA methylation of cancer-associated genes is related to urinary bladder cancer recurrence. A set of 4 genes, including CDH1 (E-cadherin), SFN (stratifin), RARB (retinoic acid receptor, beta) and RASSF1A (Ras association (RalGDS/AF-6) domain family 1), had their methylation patterns evaluated by MSP (Methylation-Specific Polymerase Chain Reaction) analysis in 49 fresh urinary bladder carcinoma tissues (including 14 cases paired with adjacent normal bladder epithelium, 3 squamous cell carcinomas and 2 adenocarcinomas) and 24 cell sediment samples from bladder washings of patients classified as cancer-free by cytological analysis (control group). A third set of samples included 39 archived tumor fragments and 23 matched washouts from 20 urinary bladder cancer patients in post-surgical monitoring. After genomic DNA isolation and sodium bisulfite modification, methylation patterns were determined and correlated with standard clinic-histopathological parameters. CDH1 and SFN genes were methylated at high frequencies in bladder cancer as well as in paired normal adjacent tissue and exfoliated cells from cancer-free patients. Although no statistically significant differences were found between RARB and RASSF1A methylation and the clinical and histopathological parameters in bladder cancer, a sensitivity of 95% and a specificity of 71% were observed for RARB methylation (Fisher's Exact test (p < 0.0001; OR = 48.89) and, 58% and 17% (p < 0.05; OR = 0.29) for RASSF1A gene, respectively, in relation to the control group. Indistinct DNA hypermethylation of CDH1 and SFN genes between tumoral and normal urinary bladder samples suggests that these epigenetic features are not suitable biomarkers for urinary bladder cancer. However, RARB and RASSF1A gene methylation appears to be an initial event in urinary bladder carcinogenesis and should be considered as defining a

  3. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation.

    Science.gov (United States)

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei

    2016-12-01

    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met-p16). The probe, paired with Met-p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases. PMID:27325520

  4. Differential DNA Methylation Analysis without a Reference Genome

    Directory of Open Access Journals (Sweden)

    Johanna Klughammer

    2015-12-01

    Full Text Available Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS, which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish. Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org. The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

  5. DNA methylation alterations in grade II- and anaplastic pleomorphic xanthoastrocytoma

    International Nuclear Information System (INIS)

    Pleomorphic xanthoastrocytoma (PXA) is a rare WHO grade II tumor accounting for less than 1% of all astrocytomas. Malignant transformation into PXA with anaplastic features, is unusual and correlates with poorer outcome of the patients. Using a DNA methylation custom array, we have quantified the DNA methylation level on the promoter sequence of 807 cancer-related genes of WHO grade II (n = 11) and III PXA (n = 2) and compared to normal brain tissue (n = 10) and glioblastoma (n = 87) samples. DNA methylation levels were further confirmed on independent samples by pyrosequencing of the promoter sequences. Increasing DNA promoter hypermethylation events were observed in anaplastic PXA as compared with grade II samples. We further validated differential hypermethylation of CD81, HCK, HOXA5, ASCL2 and TES on anaplastic PXA and grade II tumors. Moreover, these epigenetic alterations overlap those described in glioblastoma patients, suggesting common mechanisms of tumorigenesis. Even taking into consideration the small size of our patient populations, our data strongly suggest that epigenome-wide profiling of PXA is a valuable tool to identify methylated genes, which may play a role in the malignant progression of PXA. These methylation alterations may provide useful biomarkers for decision-making in those patients with low-grade PXA displaying a high risk of malignant transformation

  6. Differential DNA Methylation Analysis without a Reference Genome.

    Science.gov (United States)

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-12-22

    Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

  7. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

    Science.gov (United States)

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei

    2016-06-01

    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met- p16). The probe, paired with Met- p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases.

  8. Platelet mitochondrial DNA methylation: a potential new marker of cardiovascular disease

    OpenAIRE

    Baccarelli, Andrea A.; Byun, Hyang-Min

    2015-01-01

    Background: Platelets are critical in the etiology of cardiovascular disease (CVD), and the mitochondria in these cells serve as an energy source for platelet function. Epigenetic factors, especially DNA methylation, have been employed as markers of CVD. Unlike nuclear DNA methylation, mitochondrial DNA (mtDNA) methylation has not been widely studied, in part, due to debate about its existence and role. In this study, we examined platelet mtDNA methylation in relation to CVD. Results: We meas...

  9. Aberrant methylation of LINE-1, SLIT2, MAL and IGFBP7 in non-small cell lung cancer.

    Science.gov (United States)

    Suzuki, Makoto; Shiraishi, Kenji; Eguchi, Ayami; Ikeda, Koei; Mori, Takeshi; Yoshimoto, Kentaro; Ohba, Yasuomi; Yamada, Tatsuya; Ito, Takaaki; Baba, Yoshifumi; Baba, Hideo

    2013-04-01

    Genome-wide DNA hypomethylation and gene hypermethylation play important roles in instability and carcino-genesis. Methylation in long interspersed nucleotide element 1 (LINE-1) is a good indicator of the global DNA methylation level within a cell. Slit homolog 2 (SLIT2), myelin and lymphocyte protein gene (MAL) and insulin-like growth factor binding protein 7 (IGFBP7) are known to be hypermethylated in various malignancies. The aim of the present study was to assess the precise methylation levels of LINE-1, SLIT2, MAL and IGFBP7 in non-small cell lung cancer (NSCLC) using a pyrosequencing assay. Methylation of all regions was examined in 56 primary NSCLCs using a pyrosequencing assay. Changes in mRNA expression levels of SLIT2, MAL and IGFBP7 were measured before and after treatment with a demethylating agent. Methylation of these genes was also examined in 9 lung cancer cell lines using RT-PCR and a pyrosequencing assay. Frequencies of hypomethylation of LINE-1 and hypermethylation of SLIT2, MAL and IGFBP7, defined by predetermined cut off values, were 55, 64, 46 and 54% in NSCLCs, respectively and exhibited tumor-specific features. The hypermethylation of all genes was well correlated with changes in expression. The methylation level and frequency of MAL were significantly higher in smokers and in patients without EGFR mutations. Through accurate measurement of methylation levels using pyrosequencing, hypomethylation of LINE-1 and hypermethylation of SLIT2, MAL and IGFBP7 were frequently detected in NSCLCs and associated with various clinical features. Analysis of the methylation profiles of these genes may, therefore, provide novel opportunities for the therapy of NSCLCs.

  10. The Role of DNA Methylation in the Development and Progression of Lung Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Keith M. Kerr

    2007-01-01

    Full Text Available Lung cancer, caused by smoking in ∼87% of cases, is the leading cause of cancer death in the United States and Western Europe. Adenocarcinoma is now the most common type of lung cancer in men and women in the United States, and the histological subtype most frequently seen in never-smokers and former smokers. The increasing frequency of adenocarcinoma, which occurs more peripherally in the lung, is thought to be at least partially related to modifications in cigarette manufacturing that have led to a change in the depth of smoke inhalation. The rising incidence of lung adenocarcinoma and its lethal nature underline the importance of understanding the development and progression of this disease. Alterations in DNA methylation are recognized as key epigenetic changes in cancer, contributing to chromosomal instability through global hypomethylation, and aberrant gene expression through alterations in the methylation levels at promoter CpG islands. The identification of sequential changes in DNA methylation during progression and metastasis of lung adenocarcinoma, and the elucidation of their interplay with genetic changes, will broaden our molecular understanding of this disease, providing insights that may be applicable to the development of targeted drugs, as well as powerful markers for early detection and patient classification.

  11. DNA methylation and leukemia susceptibility in China: Evidence from an updated meta-analysis

    Science.gov (United States)

    Jiang, Danjie; Li, Yirun; Hong, Qingxiao; Shen, Yusheng; Xu, Chunjing; Xu, Yan; Zhu, Huangkai; Dai, Dongjun; Ouyang, Guifang; Duan, Shiwei

    2016-01-01

    Mounting evidence supports a role for DNA methylation in the pathogenesis of leukemia; however, there no overview of these results in the Chinese population. The present study performed a comprehensive meta-analysis to establish candidate genes with an altered methylation status in Chinese leukemia patients. Eligible studies were identified through searching the National Center of Biotechnology Information PubMed and Wanfang databases. Studies were pooled and overall odds ratios with corresponding confidence intervals were calculated. A total of 4,325 leukemia patients and 2,010 controls from 94 studies on 53 genes were included in this meta-analysis, and 47 genes were found to be aberrantly methylated in leukemia patients. A further subgroup meta-analysis by leukemia subtype demonstrated that hypermethylation of 5 genes, namely cyclin-dependent kinase (CDKN)2A, DNA-binding protein inhibitor-4, CDKN2B, glioma pathogenesis-related protein 1 and p73, contributed to the risk of various subtypes of leukemia. In addition, a strong association between CDKN2A and leukemia was identified in Chinese (Pleukemia in Chinese patients.

  12. DNA甲基化与癌症%DNA Methylation and Cancer

    Institute of Scientific and Technical Information of China (English)

    韩竞男; 鲁昊骋; 梁静

    2012-01-01

    DNA甲基化是重要的表观遗传修饰,主要发生在DNA的CpG岛.DNA的甲基化通过DNA甲基转移酶( DNA methyltransferases,DNMTs)完成.DNA甲基化参与了细胞分化、基因组稳定性、X染色体失活、基因印记等多种细胞生物学过程.单基因水平及基因组范围内的DNA甲基化改变在肿瘤发生发展中亦发挥重要作用.抑癌基因的异常甲基化引起的表达抑制,可导致肿瘤细胞的增殖失控和侵袭转移,并参与肿瘤组织的血管生成过程.在许多肿瘤的研究中都发现了基因组整体DNA低甲基化所导致的染色体不稳定性.本文从DNA的异常高甲基化和低甲基化两方面论述了DNA甲基化在细胞恶变发生发展过程中的改变及其影响,并阐述了DNA甲基化改变在肿瘤诊断和治疗中的作用.%DNA methylation plays a crucial role in various cellular functions such as differentiation, genomic stability, X-inactivation and imprinting. DNA methylation is mediated by DNA methyltransferases (DNMTs), including DNMT1 , DNMT3a and DNMT3b. Aberrent DNA methylation has been observed in many types of cancer. Promoter hypermethylation causes silencing of key tumor supperssor genes, and consequently leads to cell proliferation, angiogenesis, invasion and metastasis. Genome-wide hypomethylation contributes to genomic instability, which is an important feature of various malignancies. This review examined alteration in DNA methylation and the consequent effect on gene expression in cancer, and discussed the use of DNA methylation inhibitors in cancer therapy.

  13. DNA methylation modifications associated with chronic fatigue syndrome.

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    Wilfred C de Vega

    Full Text Available Chronic Fatigue Syndrome (CFS, also known as myalgic encephalomyelitis, is a complex multifactorial disease that is characterized by the persistent presence of fatigue and other particular symptoms for a minimum of 6 months. Symptoms fail to dissipate after sufficient rest and have major effects on the daily functioning of CFS sufferers. CFS is a multi-system disease with a heterogeneous patient population showing a wide variety of functional disabilities and its biological basis remains poorly understood. Stable alterations in gene function in the immune system have been reported in several studies of CFS. Epigenetic modifications have been implicated in long-term effects on gene function, however, to our knowledge, genome-wide epigenetic modifications associated with CFS have not been explored. We examined the DNA methylome in peripheral blood mononuclear cells isolated from CFS patients and healthy controls using the Illumina HumanMethylation450 BeadChip array, controlling for invariant probes and probes overlapping polymorphic sequences. Gene ontology (GO and network analysis of differentially methylated genes was performed to determine potential biological pathways showing changes in DNA methylation in CFS. We found an increased abundance of differentially methylated genes related to the immune response, cellular metabolism, and kinase activity. Genes associated with immune cell regulation, the largest coordinated enrichment of differentially methylated pathways, showed hypomethylation within promoters and other gene regulatory elements in CFS. These data are consistent with evidence of multisystem dysregulation in CFS and implicate the involvement of DNA modifications in CFS pathology.

  14. The interplay between DNA methylation, folate and neurocognitive development.

    Science.gov (United States)

    Irwin, Rachelle E; Pentieva, Kristina; Cassidy, Tony; Lees-Murdock, Diane J; McLaughlin, Marian; Prasad, Girijesh; McNulty, Helene; Walsh, Colum P

    2016-06-01

    DNA methylation provides an attractive possible means for propagating the effects of environmental inputs during fetal life and impacting subsequent adult mental health, which is leading to increasing collaboration between molecular biologists, nutritionists and psychiatrists. An area of interest is the potential role of folate, not just in neural tube closure in early pregnancy, but in later major neurodevelopmental events, with consequences for later sociocognitive maturation. Here, we set the scene for recent discoveries by reviewing the major events of neural development during fetal life, with an emphasis on tissues and structures where dynamic methylation changes are known to occur. Following this, we give an indication of some of the major classes of genes targeted by methylation and important for neurological and behavioral development. Finally, we highlight some cognitive disorders where methylation changes are implicated as playing an important role. PMID:27319574

  15. Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family

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    Eriona Hysolli

    2016-07-01

    Full Text Available Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs have been shown to be highly similar to embryonic stem cells (ESCs. However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.

  16. Ancestry dependent DNA methylation and influence of maternal nutrition.

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    Khyobeni Mozhui

    Full Text Available There is extensive variation in DNA methylation between individuals and ethnic groups. These differences arise from a combination of genetic and non-genetic influences and potential modifiers include nutritional cues, early life experience, and social and physical environments. Here we compare genome-wide DNA methylation in neonatal cord blood from African American (AA; N = 112 and European American (EA; N = 91 participants of the CANDLE Study (Conditions Affecting Neurocognitive Development and Learning in Early Childhood. Our goal is to determine if there are replicable ancestry-specific methylation patterns that may implicate risk factors for diseases that have differential prevalence between populations. To identify the most robust ancestry-specific CpG sites, we replicate our results in lymphoblastoid cell lines from Yoruba African and CEPH European panels of HapMap. We also evaluate the influence of maternal nutrition--specifically, plasma levels of vitamin D and folate during pregnancy--on methylation in newborns. We define stable ancestry-dependent methylation of genes that include tumor suppressors and cell cycle regulators (e.g., APC, BRCA1, MCC. Overall, there is lower global methylation in African ancestral groups. Plasma levels of 25-hydroxy vitamin D are also considerably lower among AA mothers and about 60% of AA and 40% of EA mothers have concentrations below 20 ng/ml. Using a weighted correlation analysis, we define a network of CpG sites that is jointly modulated by ancestry and maternal vitamin D. Our results show that differences in DNA methylation patterns are remarkably stable and maternal micronutrients can exert an influence on the child epigenome.

  17. Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

    Science.gov (United States)

    George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further

  18. Advances in DNA methylation: 5-hydroxymethylcytosine revisited

    DEFF Research Database (Denmark)

    Dahl, Christina; Grønbæk, Kirsten; Guldberg, Per

    2011-01-01

    Mammalian DNA contains two modified cytosine bases; 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Both of these have been known for decades but have received very different levels of attention in the scientific literature. 5mC has been studied extensively, and its role as an epigenet...

  19. Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

    Science.gov (United States)

    Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

    2011-01-01

    Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

  20. Triplex-mediated analysis of cytosine methylation at CpA sites in DNA

    OpenAIRE

    Johannsen, Marie W.; Gerrard, Simon R.; Melvin, Tracy; Brown, Tom

    2014-01-01

    Modified triplex-forming oligonucleotides distinguish 5-methyl cytosine from unmethylated cytosine in DNA duplexes by differences in triplex melting temperatures. The discrimination is sequence-specific; dramatic differences in stabilisation are seen for CpA methylation, whereas CpG methylation is not detected. This direct detection of DNA methylation constitutes a new approach for epigenetic analysis.

  1. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

    OpenAIRE

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei

    2016-01-01

    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au–S bonds. The ssDNA contains a thiolated 5′-end, a regulatory domain of 12 adenine nucleotides, and a functional domain ...

  2. RNA-directed DNA methylation: Mechanisms and functions

    KAUST Repository

    Mahfouz, Magdy M.

    2010-07-01

    Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt- long siRNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental, and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives.

  3. DNA Methylation and Gene Expression Profiling of Ewing Sarcoma Primary Tumors Reveal Genes That Are Potential Targets of Epigenetic Inactivation

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    Nikul Patel

    2012-01-01

    Full Text Available The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1, and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

  4. Biomarkers measured in buccal and blood leukocyte DNA as proxies for colon tissue global methylation

    OpenAIRE

    Ashbury, Janet E.; Taylor, Sherryl A; Tse, M Yat; Stephen C Pang; Louw, Jacob A; Vanner, Stephen J.; King, Will D

    2014-01-01

    There is increasing interest in clarifying the role of global DNA methylation levels in colorectal cancer (CRC) etiology. Most commonly, in epidemiologic studies, methylation is measured in DNA derived from blood leukocytes as a proxy measure of methylation changes in colon tissue. However, little is known about the correlations between global methylation levels in DNA derived from colon tissue and more accessible tissues such as blood or buccal cells. This cross-sectional study utilized DNA ...

  5. Histone H1 Limits DNA Methylation in Neurospora crassa.

    Science.gov (United States)

    Seymour, Michael; Ji, Lexiang; Santos, Alex M; Kamei, Masayuki; Sasaki, Takahiko; Basenko, Evelina Y; Schmitz, Robert J; Zhang, Xiaoyu; Lewis, Zachary A

    2016-01-01

    Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the [Formula: see text]hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the [Formula: see text]hH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa. PMID:27172195

  6. Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules

    Science.gov (United States)

    Kelly, Theresa K.; Liu, Yaping; Lay, Fides D.; Liang, Gangning; Berman, Benjamin P.; Jones, Peter A.

    2012-01-01

    DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline, we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome-depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule, and thus, single cell level, that can be used to monitor disease progression and response to therapy. PMID:22960375

  7. Effect of phenylhexyl isothiocyanate on aberrant histone H3 methylation in primary human acute leukemia

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    Zou Yong

    2012-07-01

    Full Text Available Abstract Background We have previously studied the histone acetylation in primary human leukemia cells. However, histone H3 methylation in these cells has not been characterized. Methods This study examined the methylation status at histone H3 lysine 4 (H3K4 and histone H3 lysine 9 (H3K9 in primary acute leukemia cells obtained from patients and compared with those in the non-leukemia and healthy cells. We further characterized the effect of phenylhexyl isothiocyanate (PHI, Trichostatin A (TSA, and 5-aza-2’-deoxycytidine (5-Aza on the cells. Results We found that methylation of histone H3K4 was virtually undetectable, while methylation at H3K9 was significantly higher in primary human leukemia cells. The histone H3K9 hypermethylation and histone H3K4 hypomethylation were observed in both myeloid and lymphoid leukemia cells. PHI was found to be able to normalize the methylation level in the primary leukemia cells. We further showed that PHI was able to enhance the methyltransferase activity of H3K4 and decrease the activity of H3K9 methyltransferase. 5-Aza had similar effect on H3K4, but minimal effect on H3K9, whereas TSA had no effect on H3K4 and H3K9 methyltransferases. Conclusions This study revealed opposite methylation level of H3K4 and H3K9 in primary human leukemia cells and demonstrated for the first time that PHI has different effects on the methyltransferases for H3K4 and H3K9.

  8. Infant sex-specific placental cadmium and DNA methylation associations

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, April F., E-mail: april.mohanty@va.gov [Cardiovascular Health Research Unit, University of Washington, 1730 Minor Ave, Seattle, WA 98101 (United States); Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA (United States); Farin, Fred M., E-mail: freddy@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Bammler, Theo K., E-mail: tbammler@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); MacDonald, James W., E-mail: jmacdon@uw.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Afsharinejad, Zahra, E-mail: zafshari@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Burbacher, Thomas M., E-mail: tmb@uw.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Box: 357234, 1705 N.E. Pacific Street, Seattle, WA 98195 (United States); Siscovick, David S., E-mail: dsiscovick@nyam.org [Cardiovascular Health Research Unit, University of Washington, 1730 Minor Ave, Seattle, WA 98101 (United States); Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA (United States); Department of Medicine, University of Washington, Seattle, WA (United States); and others

    2015-04-15

    Background: Recent evidence suggests that maternal cadmium (Cd) burden and fetal growth associations may vary by fetal sex. However, mechanisms contributing to these differences are unknown. Objectives: Among 24 maternal-infant pairs, we investigated infant sex-specific associations between placental Cd and placental genome-wide DNA methylation. Methods: We used ANOVA models to examine sex-stratified associations of placental Cd (dichotomized into high/low Cd using sex-specific Cd median cutoffs) with DNA methylation at each cytosine-phosphate-guanine site or region. Statistical significance was defined using a false discovery rate cutoff (<0.10). Results: Medians of placental Cd among females and males were 5 and 2 ng/g, respectively. Among females, three sites (near ADP-ribosylation factor-like 9 (ARL9), siah E3 ubiquitin protein ligase family member 3 (SIAH3), and heparin sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3ST4) and one region on chromosome 7 (including carnitine O-octanoyltransferase (CROT) and TP5S target 1 (TP53TG1)) were hypomethylated in high Cd placentas. Among males, high placental Cd was associated with methylation of three sites, two (hypomethylated) near MDS1 and EVI1 complex locus (MECOM) and one (hypermethylated) near spalt-like transcription factor 1 (SALL1), and two regions (both hypomethylated, one on chromosome 3 including MECOM and another on chromosome 8 including rho guanine nucleotide exchange factor (GEF) 10 (ARHGEF10). Differentially methylated sites were at or close to transcription start sites of genes involved in cell damage response (SIAH3, HS3ST4, TP53TG1) in females and cell differentiation, angiogenesis and organ development (MECOM, SALL1) in males. Conclusions: Our preliminary study supports infant sex-specific placental Cd-DNA methylation associations, possibly accounting for previously reported differences in Cd-fetal growth associations across fetal sex. Larger studies are needed to replicate and extend these

  9. Infant sex-specific placental cadmium and DNA methylation associations

    International Nuclear Information System (INIS)

    Background: Recent evidence suggests that maternal cadmium (Cd) burden and fetal growth associations may vary by fetal sex. However, mechanisms contributing to these differences are unknown. Objectives: Among 24 maternal-infant pairs, we investigated infant sex-specific associations between placental Cd and placental genome-wide DNA methylation. Methods: We used ANOVA models to examine sex-stratified associations of placental Cd (dichotomized into high/low Cd using sex-specific Cd median cutoffs) with DNA methylation at each cytosine-phosphate-guanine site or region. Statistical significance was defined using a false discovery rate cutoff (<0.10). Results: Medians of placental Cd among females and males were 5 and 2 ng/g, respectively. Among females, three sites (near ADP-ribosylation factor-like 9 (ARL9), siah E3 ubiquitin protein ligase family member 3 (SIAH3), and heparin sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3ST4) and one region on chromosome 7 (including carnitine O-octanoyltransferase (CROT) and TP5S target 1 (TP53TG1)) were hypomethylated in high Cd placentas. Among males, high placental Cd was associated with methylation of three sites, two (hypomethylated) near MDS1 and EVI1 complex locus (MECOM) and one (hypermethylated) near spalt-like transcription factor 1 (SALL1), and two regions (both hypomethylated, one on chromosome 3 including MECOM and another on chromosome 8 including rho guanine nucleotide exchange factor (GEF) 10 (ARHGEF10). Differentially methylated sites were at or close to transcription start sites of genes involved in cell damage response (SIAH3, HS3ST4, TP53TG1) in females and cell differentiation, angiogenesis and organ development (MECOM, SALL1) in males. Conclusions: Our preliminary study supports infant sex-specific placental Cd-DNA methylation associations, possibly accounting for previously reported differences in Cd-fetal growth associations across fetal sex. Larger studies are needed to replicate and extend these

  10. Analysis of the association between CIMP and BRAF in colorectal cancer by DNA methylation profiling.

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    Toshinori Hinoue

    Full Text Available A CpG island methylator phenotype (CIMP is displayed by a distinct subset of colorectal cancers with a high frequency of DNA hypermethylation in a specific group of CpG islands. Recent studies have shown that an activating mutation of BRAF (BRAF(V600E is tightly associated with CIMP, raising the question of whether BRAF(V600E plays a causal role in the development of CIMP or whether CIMP provides a favorable environment for the acquisition of BRAF(V600E. We employed Illumina GoldenGate DNA methylation technology, which interrogates 1,505 CpG sites in 807 different genes, to further study this association. We first examined whether expression of BRAF(V600E causes DNA hypermethylation by stably expressing BRAF(V600E in the CIMP-negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We determined 100 CIMP-associated CpG sites and examined changes in DNA methylation in eight stably transfected clones over multiple passages. We found that BRAF(V600E is not sufficient to induce CIMP in our system. Secondly, considering the alternative possibility, we identified genes whose DNA hypermethylation was closely linked to BRAF(V600E and CIMP in 235 primary colorectal tumors. Interestingly, genes that showed the most significant link include those that mediate various signaling pathways implicated in colorectal tumorigenesis, such as BMP3 and BMP6 (BMP signaling, EPHA3, KIT, and FLT1 (receptor tyrosine kinases and SMO (Hedgehog signaling. Furthermore, we identified CIMP-dependent DNA hypermethylation of IGFBP7, which has been shown to mediate BRAF(V600E-induced cellular senescence and apoptosis. Promoter DNA hypermethylation of IGFBP7 was associated with silencing of the gene. CIMP-specific inactivation of BRAF(V600E-induced senescence and apoptosis pathways by IGFBP7 DNA hypermethylation might create a favorable context for the acquisition of BRAF(V600E in CIMP+ colorectal cancer. Our data will be useful for future investigations toward

  11. Minimal evidence for consistent changes in maize DNA methylation patterns following environmental stress.

    Directory of Open Access Journals (Sweden)

    Steven R Eichten

    2015-05-01

    Full Text Available DNA methylation is a chromatin modification that is sometimes associated with epigenetic regulation of gene expression. As DNA methylation can be reversible at some loci, it is possible that methylation patterns may change within an organism that is subjected to environmental stress. In order to assess the effects of abiotic stress on DNA methylation patterns in maize (Zea mays, seeding plants were subjected to heat, cold, and UV stress treatments. Tissue was later collected from individual adult plants that had been subjected to stress or control treatments and used to perform DNA methylation profiling to determine whether there were consistent changes in DNA methylation triggered by specific stress treatments. DNA methylation profiling was performed by immunoprecipitation of methylated DNA followed by microarray hybridization to allow for quantitative estimates of DNA methylation abundance throughout the low-copy portion of the maize genome. By comparing the DNA methylation profiles of each individual plant to the average of the control plants it was possible to identify regions of the genome with variable DNA methylation. However, we did not find evidence of consistent DNA methylation changes resulting from the stress treatments used in this study. Instead, the data suggest that there is a low-rate of stochastic variation that is present in both control and stressed plants.

  12. New strategy to address DNA-methyl transferase activity in ovarian cancer cell cultures by monitoring the formation of 5-methylcytosine using HPLC-UV.

    Science.gov (United States)

    Iglesias González, T; Blanco-González, E; Montes-Bayón, M

    2016-08-15

    Methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs). Aberrant expression and activity of these enzymes has been reported to play an important role in the initiation and progression of tumors and its response to chemotherapy. Therefore, there is a great interest in developing strategies to detect human DNMTs activity. We propose a simple, antibody-free, label-free and non-radioactive analytical strategy in which methyltransferase activity is measured trough the determination of the 5-methylcytosine (5mC) content in DNA by a chromatographic method (HPLC-UV) previously developed. For this aim, a correlation between the enzyme activity and the concentration of 5mC obtained by HPLC-UV is previously obtained under optimized conditions using both, un-methylated and hemi-methylated DNA substrates and the prokaryotic methyltransferase M.SssI as model enzyme. The evaluation of the methylation yield in un-methylated known sequences (a 623bp PCR-amplicon) turned to be quantitative (110%) in experiments conducted in-vitro. Methylation of hemi-methylated and low-methylated sequences could be also detected with the proposed approach. The application of the methodology to the determination of the DNMTs activity in nuclear extracts from human ovarian cancer cells has revealed the presence of matrix effects (also confirmed by standard additions) that hampered quantitative enzyme recovery. The obtained results showed the high importance of adequate sample clean-up steps. PMID:27318640

  13. DNA methylation patterns in cord blood DNA and body size in childhood.

    Directory of Open Access Journals (Sweden)

    Caroline L Relton

    Full Text Available Epigenetic markings acquired in early life may have phenotypic consequences later in development through their role in transcriptional regulation with relevance to the developmental origins of diseases including obesity. The goal of this study was to investigate whether DNA methylation levels at birth are associated with body size later in childhood.A study design involving two birth cohorts was used to conduct transcription profiling followed by DNA methylation analysis in peripheral blood. Gene expression analysis was undertaken in 24 individuals whose biological samples and clinical data were collected at a mean ± standard deviation (SD age of 12.35 (0.95 years, the upper and lower tertiles of body mass index (BMI were compared with a mean (SD BMI difference of 9.86 (2.37 kg/m(2. This generated a panel of differentially expressed genes for DNA methylation analysis which was then undertaken in cord blood DNA in 178 individuals with body composition data prospectively collected at a mean (SD age of 9.83 (0.23 years. Twenty-nine differentially expressed genes (>1.2-fold and p<10(-4 were analysed to determine DNA methylation levels at 1-3 sites per gene. Five genes were unmethylated and DNA methylation in the remaining 24 genes was analysed using linear regression with bootstrapping. Methylation in 9 of the 24 (37.5% genes studied was associated with at least one index of body composition (BMI, fat mass, lean mass, height at age 9 years, although only one of these associations remained after correction for multiple testing (ALPL with height, p(Corrected = 0.017.DNA methylation patterns in cord blood show some association with altered gene expression, body size and composition in childhood. The observed relationship is correlative and despite suggestion of a mechanistic epigenetic link between in utero life and later phenotype, further investigation is required to establish causality.

  14. Genome aberrations in canine mammary carcinomas and their detection in cell-free plasma DNA.

    Directory of Open Access Journals (Sweden)

    Julia Beck

    Full Text Available Mammary tumors are the most frequent cancers in female dogs exhibiting a variety of histopathological differences. There is lack of knowledge about the genomes of these common dog tumors. Five tumors of three different histological subtypes were evaluated. Massive parallel sequencing (MPS was performed in comparison to the respective somatic genome of each animal. Copy number and structural aberrations were validated using droplet digital PCR (ddPCR. Using mate-pair sequencing chromosomal aneuploidies were found in two tumors, frequent smaller deletions were found in one, inter-chromosomal fusions in one other, whereas one tumor was almost normal. These aberrations affect several known cancer associated genes such as cMYC, and KIT. One common deletion of the proximal end of CFA27, harboring the tumor suppressor gene PFDN5 was detected in four tumors. Using ddPCR, this deletion was validated and detected in 50% of tumors (N = 20. Breakpoint specific dPCRs were established for four tumors and tumor specific cell-free DNA (cfDNA was detected in the plasma. In one animal tumor-specific cfDNA was found >1 year after surgery, attributable to a lung metastasis. Paired-end sequencing proved that copy-number imbalances of the tumor are reflected by the cfDNA. This report on chromosomal instability of canine mammary cancers reveals similarities to human breast cancers as well as special canine alterations. This animal model provides a framework for using MPS for screening for individual cancer biomarkers with cost effective confirmation and monitoring using ddPCR. The possibility exists that ddPCR can be expanded to screening for common cancer related variants.

  15. DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development.

    Directory of Open Access Journals (Sweden)

    Yosuke Ichijima

    Full Text Available During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

  16. Inheritance and Variation of Genomic DNA Methylation in Diploid and Triploid Pacific Oyster (Crassostrea gigas).

    Science.gov (United States)

    Jiang, Qun; Li, Qi; Yu, Hong; Kong, Lingfeng

    2016-02-01

    DNA methylation is an important epigenetic mechanism that could be responsive to environmental changes indicating a potential role in natural selection and adaption. In order to evaluate an evolutionary role of DNA methylation, it is essential to first gain a better insight into inheritability. To address this question, this study investigated DNA methylation variation from parents to offspring in the Pacific oyster Crassostrea gigas using fluorescent-labeled methylation-sensitive amplified polymorphism (F-MSAP) analysis. Most of parental methylated loci were stably transmitted to offspring segregating following Medelian expectation. However, methylated loci deviated more often than non-methylated loci and offspring showed a few de novo methylated loci indicating DNA methylation changes from parents to offspring. Interestingly, some male-specific methylated loci were found in this study which might help to explore sex determination in oyster. Despite environmental stimuli, genomic stresses such as polyploidization also can induce methylation changes. This study also compared global DNA methylation level and individual methylated loci between diploid and triploid oysters. Results showed no difference in global methylation state but a few ploidy-specific loci were detected. DNA methylation variation during polyploidization was less than autonomous methylation variation from parents to offspring.

  17. Structure of DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Song, Jikui; Rechkoblit, Olga; Bestor, Timothy H.; Patel, Dinshaw J. (MSKCC); (Columbia)

    2011-09-06

    Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.

  18. Structure of DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation

    Energy Technology Data Exchange (ETDEWEB)

    J Song; O Rechkoblit; T Bestor; D Patel

    2011-12-31

    Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.

  19. Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer’s disease model cell line

    International Nuclear Information System (INIS)

    Highlights: ► Genome-wide DNA methylation pattern in Alzheimer’s disease model cell line. ► Integrated analysis of CpG methylation and mRNA expression profiles. ► Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. ► 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′-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 contribute to the pathogenesis of AD.

  20. Biological implications and therapeutic significance of DNA methylation regulated genes in cervical cancer.

    Science.gov (United States)

    Bhat, Samatha; Kabekkodu, Shama Prasada; Noronha, Ashish; Satyamoorthy, Kapaettu

    2016-02-01

    Cervical cancer is the second most common cancer among women worldwide. About 528,000 women are diagnosed with cervical cancer contributing to around 266,000 deaths, across the globe every year. Out of these, the burden of 226,000 (85%) deaths occurs in the developing countries, who are less resource intensive to manage the disease. This is despite the fact that cervical cancer is amenable for early detection due to its long and relatively well-known natural history prior to its culmination as invasive disease. Infection with high risk human papillomavirus (hrHPVs) is essential but not sufficient to cause cervical cancer. Although it was thought that genetic mutations alone was sufficient to cause cervical cancer, the current epidemiological and molecular studies have shown that HPV infection along with genetic and epigenetic changes are frequently associated and essential for initiation, development and progression of the disease. Moreover, aberrant DNA methylation in host and HPV genome can be utilized not only as biomarkers for early detection, disease progression, diagnosis and prognosis of cervical cancer but also to design effective therapeutic strategies. In this review, we focus on recent studies on DNA methylation changes in cervical cancer and their potential role as biomarkers for early diagnosis, prognosis and targeted therapy. PMID:26743075

  1. Naturally occurring endo-siRNA silences LINE-1 retrotransposons in human cells through DNA methylation.

    Science.gov (United States)

    Chen, Long; Dahlstrom, Jane E; Lee, Sung-Hun; Rangasamy, Danny

    2012-07-01

    Long interspersed nuclear element 1 (LINE-1) retrotransposons are mutagens that are capable of generating deleterious mutations by inserting themselves into genes and affecting gene function in the human genome. In normal cells, the activity of LINE-1 retrotransposon is mostly repressed, maintaining a stable genome structure. In contrast, cancer cells are characterized by aberrant expression of LINE-1 retrotransposons, which, in principle, have the potential to contribute to genomic instability. The mechanistic pathways that regulate LINE-1 expression remain unclear. Using deep-sequencing small RNA analysis, we identified a subset of differentially expressed endo-siRNAs that directly regulate LINE-1 expression. Detailed analyses suggest that these endo-siRNAs are significantly depleted in human breast cancer cells compared with normal breast cells. The overexpression of these endo-siRNAs in cancer cells markedly silences endogenous LINE-1 expression through increased DNA methylation of the LINE-1 5'-UTR promoter. The finding that endo-siRNAs can silence LINE-1 activity through DNA methylation suggests that a functional link exists between the expression of endo-siRNAs and LINE-1 retrotransposons in human cells.

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

    Science.gov (United States)

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

    2010-08-01

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

  3. Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism.

    Science.gov (United States)

    Vaes, Bart L T; Lute, Carolien; van der Woning, Sebastian P; Piek, Ester; Vermeer, Jenny; Blom, Henk J; Mathers, John C; Müller, Michael; de Groot, Lisette C P G M; Steegenga, Wilma T

    2010-02-01

    S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2'-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.

  4. Quantitative analysis of methylation of genomic loci in early-stage rectal cancer predicts distant recurrence.

    NARCIS (Netherlands)

    Maat, M.F. de; Velde, C.J. van de; Werff, M.P. van der; Putter, H.; Umetani, N.; Klein-Kranenbarg, E.M.; Turner, R.R.; Krieken, J.H.J.M. van; Bilchik, A.; Tollenaar, R.A.; Hoon, D.S.

    2008-01-01

    PURPOSE: There are no accurate prognostic biomarkers specific for rectal cancer. Epigenetic aberrations, in the form of DNA methylation, accumulate early during rectal tumor formation. In a preliminary study, we investigated absolute quantitative methylation changes associated with tumor progression

  5. Aberrant 5'-CpG Methylation of Cord Blood TNFα Associated with Maternal Exposure to Polybrominated Diphenyl Ethers.

    Directory of Open Access Journals (Sweden)

    Tyna Dao

    Full Text Available Growing evidence suggests that maternal exposures to endocrine disrupting chemicals during pregnancy may lead to poor pregnancy outcomes and increased fetal susceptibility to adult diseases. Polybrominated diphenyl ethers (PBDEs, which are ubiquitously used flame-retardants, could leach into the environment; and become persistent organic pollutants via bioaccumulation. In the United States, blood PBDE levels in adults range from 30-100 ng/g- lipid but the alarming health concern revolves around children who have reported blood PBDE levels 3 to 9-fold higher than adults. PBDEs disrupt endocrine, immune, reproductive and nervous systems. However, the mechanism underlying its adverse health effect is not fully understood. Epigenetics is a possible biological mechanism underlying maternal exposure-child health outcomes by regulating gene expression without changes in the DNA sequence. We sought to examine the relationship between maternal exposure to environmental PBDEs and promoter methylation of a proinflammatory gene, tumor necrosis factor alpha (TNFα. We measured the maternal blood PBDE levels and cord blood TNFα promoter methylation levels on 46 paired samples of maternal and cord blood from the Boston Birth Cohort (BBC. We showed that decreased cord blood TNFα methylation associated with high maternal PBDE47 exposure. CpG site-specific methylation showed significantly hypomethylation in the girl whose mother has a high blood PBDE47 level. Consistently, decreased TNFα methylation associated with an increase in TNFα protein level in cord blood. In conclusion, our finding provided evidence that in utero exposure to PBDEs may epigenetically reprogram the offspring's immunological response through promoter methylation of a proinflammatory gene.

  6. Aberrant 5'-CpG Methylation of Cord Blood TNFα Associated with Maternal Exposure to Polybrominated Diphenyl Ethers.

    Science.gov (United States)

    Dao, Tyna; Hong, Xiumei; Wang, Xiaobin; Tang, Wan-Yee

    2015-01-01

    Growing evidence suggests that maternal exposures to endocrine disrupting chemicals during pregnancy may lead to poor pregnancy outcomes and increased fetal susceptibility to adult diseases. Polybrominated diphenyl ethers (PBDEs), which are ubiquitously used flame-retardants, could leach into the environment; and become persistent organic pollutants via bioaccumulation. In the United States, blood PBDE levels in adults range from 30-100 ng/g- lipid but the alarming health concern revolves around children who have reported blood PBDE levels 3 to 9-fold higher than adults. PBDEs disrupt endocrine, immune, reproductive and nervous systems. However, the mechanism underlying its adverse health effect is not fully understood. Epigenetics is a possible biological mechanism underlying maternal exposure-child health outcomes by regulating gene expression without changes in the DNA sequence. We sought to examine the relationship between maternal exposure to environmental PBDEs and promoter methylation of a proinflammatory gene, tumor necrosis factor alpha (TNFα). We measured the maternal blood PBDE levels and cord blood TNFα promoter methylation levels on 46 paired samples of maternal and cord blood from the Boston Birth Cohort (BBC). We showed that decreased cord blood TNFα methylation associated with high maternal PBDE47 exposure. CpG site-specific methylation showed significantly hypomethylation in the girl whose mother has a high blood PBDE47 level. Consistently, decreased TNFα methylation associated with an increase in TNFα protein level in cord blood. In conclusion, our finding provided evidence that in utero exposure to PBDEs may epigenetically reprogram the offspring's immunological response through promoter methylation of a proinflammatory gene. PMID:26406892

  7. DNA methylation and folate metabolism in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Shun Shi Zhu; Shu Dong Xiao; Zhi Ping Chen; Yao Shi; Jing Yuan Fang; Rong Rong Li; Joel B Masor

    2000-01-01

    AIM To investigate DNA methylation status in gastric cancer and its relationship with folate metabolism.METHODS Serum before operation, the gastric mucosa from the lesion, and the surrounding area inpatients with gastric cancer and the remote normal-appearing mucosa of the resected stomach were collectedrespectively. The serum folate, mucosal tissue folate, S-adenosylmethionine ( SAM ), S-adenosylhomocysteine (SAH), and the DNA methylation levels were determined.RESULTS The tissue folate was significantly lower than that in ulcers, especially in the surrounding andnormal mucosa (0.38±0.13, 0.50±0.17 vs 0.53±0.50, 0.79±0.82ng/mg protein, P < 0.01), and itdecreased gradually in the lesion areas. The DNA methylation status showed similar decreasing trend incancers compared with the methylation increasing trend in ulcers. The SAM level ascended in the lesion areaswith a higher. concentration in cancer mueosa (63.5±43.0 vs 25.9±11.9nmol/g tissue, P < 0.01 ). Theaccumulation of SAH in the surrounding and normal mucosa of cancers was observed (17.3±24.6, 15.5±8.6vs 14.6±4.2, 10.0±1.9nmol/g tissue, P < 0.05 - 0.01). There were significantly negative correlationsbetween tissue folate and the SAM and SAH levels in the three areas.CONCLUSION Patients with gastric cancer have the regional folate deficiency in the stomach mucosa,although the serum folate level remains normal. This disturbs the local SAM and SAH metabolism withaccumulation of SAH and DNA hypomethylation which has been known as an important molecularmechanism for carcinogenesis. Folic acid can modulate DNA methylation status by its effect in one-carbongroup metabolism and thus affect the process of the carcinogenesis. Therefore, this may be an access for theprevention of gastric cancer.

  8. SETDB1 is involved in postembryonic DNA methylation and gene silencing in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dawei Gou

    Full Text Available DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9 by members of the Su(var3-9 family of histone methyltransferases (HMTs triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1 can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2 and Su(var205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1. By enlisting Dnmt2 and Su(var205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.

  9. Aberrant methylation of NPY, PENK, and WIF1 as a promising marker for blood-based diagnosis of colorectal cancer

    KAUST Repository

    Roperch, J.-P.

    2013-12-01

    Background: DNA methylation is a well-known epigenetic mechanism involved in epigenetic gene regulation. Several genes were reported hypermethylated in CRC, althought no gene marker was proven to be individually of sufficient sensitivity or specificity in routine clinical practice. Here, we identified novel epigenetic markers and assessed their combined use for diagnostic accuracy.Methods: We used methylation arrays on samples from several effluents to characterize methylation profiles in CRC samples and controls, as established by colonoscopy and pathology findings, and selected two differentially methylated candidate epigenetic genes (NPY, PENK). To this gene panel we added WIF, on the basis of being reported in literature as silenced by promoter hypermethylation in several cancers, including CRC. We measured their methylation degrees by quantitative multiplex-methylation specific PCR (QM-MSP) on 15 paired carcinomas and adjacent non-cancerous colorectal tissues and we subsequently performed a clinical validation on two different series of 266 serums, subdivided in 32 CRC, 26 polyps, 47 other cancers and 161 with normal colonoscopy. We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.Results: We obtained CRC detection on tissues with both sensitivity and specificity of 100%. On serum CRC samples, we obtained sensitivity/specificity values of, e.g., 87%/80%, 78%/90% and 59%/95%, and negative predictive value/positive predictive value figures of 97%/47%, 95%/61% and 92%/70%. On serum samples from other cancers we obtained sensitivity/specificity of, e.g, 89%/25%, 43%/80% and 28%/91%.Conclusions: We showed the potential of NPY, PENK, and WIF1 as combined epigenetic markers for CRC diagnosis, both in tissue and serum and tested their use as serum biomarkers in other cancers. We optimized a QM-MSP for simultaneously quantifying their methylation levels. Our assay can be an effective

  10. Genomic patterns of DNA methylation: targets and function of an epigenetic mark.

    Science.gov (United States)

    Weber, Michael; Schübeler, Dirk

    2007-06-01

    Methylation of cytosines can mediate epigenetic gene silencing and is the only known DNA modification in eukaryotes. Recent efforts to map DNA methylation across mammalian genomes revealed limited DNA methylation at regulatory regions but widespread methylation in intergenic regions and repeats. This is consistent with the idea that hypermethylation is the default epigenetic state and serves in maintaining genome integrity. DNA methylation patterns at regulatory regions are generally stable, but a minor subset of regulatory regions show variable DNA methylation between cell types, suggesting an additional dynamic component. Such promoter de novo methylation might be involved in the maintenance rather than the initiation of silencing of defined genes during development. How frequently such dynamic methylation occurs, its biological relevance and the pathways involved deserve investigation. PMID:17466503

  11. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Science.gov (United States)

    Schrader, Anna; Gross, Thomas; Thalhammer, Verena; Längst, Gernot

    2015-01-01

    The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  12. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Directory of Open Access Journals (Sweden)

    Anna Schrader

    Full Text Available The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  13. Functions of DNA methylation and hydroxymethylation in mammalian development.

    Science.gov (United States)

    Guibert, Sylvain; Weber, Michael

    2013-01-01

    DNA methylation occurs at cytosines, predominantly in the CpG dinucleotide context and is a key epigenetic regulator of embryogenesis and stem-cell differentiation in mammals. The genomic patterns of 5-methylcytosine are extensively reprogrammed during early embryonic development as well as in the germ-cell lineage. Thanks to improvements in high-throughput mapping technologies, it is now possible to characterize the dynamics of this epigenetic mark at the genome scale. DNA methylation plays multiple roles during development and serves to establish long-term gene silencing. In 2009, it was revealed that 5-hydroxymethylcytosine (5hmC) is another prominent cytosine modification catalyzed by the enzymes of the TET family and abundant in certain cell types. 5hmC has been thought to serve as an intermediate in the reaction of DNA demethylation or act as a signal for chromatin factors. Here, we review the current knowledge on the roles of these DNA epigenetic marks in development, epigenetic reprogramming, and pluripotency. PMID:23587238

  14. Effect of aspirin on chromosome aberration and DNA damage induced by X-rays in mice

    Science.gov (United States)

    Niikawa, M.; Chuuriki, K.; Shibuya, K.; Seo, M.; Nagase, H.

    In order to reveal the anticlastogenic potency of aspirin, we evaluated the suppressive ability of aspirin on chromosome aberrations induced by X-ray. Aspirin at doses of 0.5, 5 and 50 mg/kg was administrated intraperitoneally or orally at 0.5 h after or before the X-ray irradiation. The anticlastogenic activity of aspirin on chromosome aberrations induced by X-ray was determined in the mouse micronucleus test and alkaline single cell gel electrophoresis (SCG) assay in vivo. The frequency by polychromatic erythrocytes with micronuclei (MNPCEs) was decreased by about 19-61% at 0.5 h after and about 23-62% at 0.5 h before the X-ray irradiation. DNA damage by X-ray was significantly decreased by oral administration of aspirin at 0.5 h after or before the X-ray irradiation for the SCG assay. We consider aspirin can be used as preventive agents against exposure of X-ray.

  15. Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids.

    NARCIS (Netherlands)

    Vaissiere, T.; Cuenin, C.; Paliwal, A.; Vineis, P.; Hoek, G.; Krzyzanowski, M.; Airoldi, L.; Dunning, A.; Garte, S.; Malaveille, C.; Overvad, K.; Clavel-Chapelon, F.; Linseisen, J.; Boeing, H.; Trichopoulou, A.; Trichopoulous, D.; Kaladidi, A.; Palli, D.; Krogh, V.; Tumino, R.; Panico, S.; Bueno de Mesquita, H.B.; Peeters, P.H.M.; Kumle, M.; Gonzalez, C.A.; Martinez, C.; Dorronsoro, M.; Barricarte, A.; Navarro, C.; Quiros, J.R.; Berglund, B.; Janzon, L.; Jarvholm, B.; Day, N.E.; Key, T.J.; Saracci, R.; Kaaks, R.; Riboli, E.; Hainaut, P.; Herceg, Z.

    2009-01-01

    Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has prov

  16. Trichloroethylene-induced gene expression and DNA methylation changes in B6C3F1 mouse liver.

    Directory of Open Access Journals (Sweden)

    Yan Jiang

    Full Text Available Trichloroethylene (TCE, widely used as an organic solvent in the industry, is a common contaminant in air, soil, and water. Chronic TCE exposure induced hepatocellular carcinoma in mice, and occupational exposure in humans was suggested to be associated with liver cancer. To understand the role of non-genotoxic mechanism(s for TCE action, we examined the gene expression and DNA methylation changes in the liver of B6C3F1 mice orally administered with TCE (0, 100, 500 and 1000 mg/kg b.w. per day for 5 days. After 5 days TCE treatment at a dose level of 1000 mg/kg b.w., a total of 431 differentially expressed genes were identified in mouse liver by microarray, of which 291 were up-regulated and 140 down-regulated. The expression changed genes were involved in key signal pathways including PPAR, proliferation, apoptosis and homologous recombination. Notably, the expression level of a number of vital genes involved in the regulation of DNA methylation, such as Utrf1, Tet2, DNMT1, DNMT3a and DNMT3b, were dysregulated. Although global DNA methylation change was not detected in the liver of mice exposed to TCE, the promoter regions of Cdkn1a and Ihh were found to be hypo- and hypermethylated respectively, which correlated negatively with their mRNA expression changes. Furthermore, the gene expression and DNA methylation changes induced by TCE were dose dependent. The overall data indicate that TCE exposure leads to aberrant DNA methylation changes, which might alter the expression of genes involved in the TCE-induced liver tumorgenesis.

  17. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation.

    Directory of Open Access Journals (Sweden)

    Si-Yang Liu

    Full Text Available Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase of species with validated DNA methylation. The lack of repeat contents of the A. flavus' genome and the high RIP-index of the small amount of remanent repeat potentially support our speculation that DNA methylation may be absent in A. flavus or that it may possess de novo DNA methylation which occurs very transiently during the obscure sexual stage of this fungal species. This work contributes to our understanding on the DNA methylation status of A. flavus, as well as reinforces our views on the DNA methylation in fungal species. In addition, our strategy of applying bisulfite sequencing to DNA methylation detection in species with low DNA methylation may serve as a reference for later scientific investigations in other hypomethylated species.

  18. A systematic analysis on DNA methylation and the expression of both mRNA and microRNA in bladder cancer.

    Directory of Open Access Journals (Sweden)

    Jialou Zhu

    Full Text Available BACKGROUND: DNA methylation aberration and microRNA (miRNA deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported. METHODOLOGY/PRINCIPAL FINDINGS: The DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to "neurogenesis" and "cell differentiation" by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes (HIC1, SLIT2, RASAL1, and KRT17 by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples. CONCLUSIONS/SIGNIFICANCE: We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research.

  19. Indices of methylation in sperm DNA from fertile men differ between distinct geographical regions

    NARCIS (Netherlands)

    Consales, C.; Leter, G.; Bonde, J. P E; Toft, G.; Eleuteri, P.; Moccia, T.; Budillon, A.; Jönsson, B. A G; Giwercman, A.; Pedersen, H. S.; Ludwicki, J. K.; Zviezdai, V.; Heederik, D.; Spanò, M.

    2014-01-01

    STUDY QUESTION Which are the main determinants, if any, of sperm DNA methylation levels? SUMMARY ANSWER Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences. WHAT IS KNOWN ALREADY DNA methylation level, assessed on repetitive sequence

  20. DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress

    Institute of Scientific and Technical Information of China (English)

    Wensheng Wang; Xiuqin Zhao; Yajiao Pan; Linghua Zhu; Binying Fu; Zhikang Li

    2011-01-01

    DNA methylation,one of the most important epigenetic phenomena,plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli.In the present study,a rnethylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress.Consistent with visibly different phenotypes in response to salt stress,epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salttolerant FL478 and salt-sensitive IR29.In addition,most tissue-specific DNA methylation loci were conserved,while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes.Strikingly,salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478.This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage,and helps to further elucidate the implications of DNA methylation in crop growth and development.

  1. Radioprotective properties of DNA methylation-disrupting agents

    International Nuclear Information System (INIS)

    5-Azacytidine and sodium butyrate, two DNA methylation-disrupting agents, were tested for radioprotective properties on V79A03 cells. Both compounds can activate genes not previously expressed (e.g. metallothionein). 5-Azecytidine treatment (3 μM, 24h) caused a 50% decrease in the 5-methylcytosine content of V79A03 DNA whereas sodium butyrate treatment (1 mM, 24h) resulted in a 700% increase in 5-methylcytosine content. Additionally, 5-azacytidine treatment resulted in the increased survival of V79A03 cells, with treatment 24 h prior to exposure to gamma radiation providing a dose reduction factor of 1.8. Sodium butyrate treatment did not result in a significant increase in survival. These results indicate that the hypomethylation of genomic DNA prior to exposure to gamma radiation correlates with an increase in survival of V79A03 cells, possibly due to the activation of the enzymes involved in repair. (author)

  2. Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

    Science.gov (United States)

    Kraiczy, J; Nayak, K; Ross, A; Raine, T; Mak, T N; Gasparetto, M; Cario, E; Rakyan, V; Heuschkel, R; Zilbauer, M

    2016-05-01

    DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.

  3. Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

    Science.gov (United States)

    Kraiczy, J; Nayak, K; Ross, A; Raine, T; Mak, T N; Gasparetto, M; Cario, E; Rakyan, V; Heuschkel, R; Zilbauer, M

    2016-05-01

    DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD. PMID:26376367

  4. Identification of DNA methylation biomarkers from Infinium arrays

    Directory of Open Access Journals (Sweden)

    Richard D Emes

    2012-08-01

    Full Text Available Epigenetic modifications of DNA, such as cytosine methylation are differentially abundant in diseases such as cancer. A goal for clinical research is finding sites that are differentially methylated between groups of samples to act as potential biomarkers for disease outcome. However, clinical samples are often limited in availability, represent a heterogeneous collection of cells or are of uncertain clinical class. Array based methods for identification of methylation provide a cost effective method to survey a proportion of the methylome at single base resolution. The Illumina Infinium array has become a popular and reliable high throughput method in this field and are proving useful in the identification of biomarkers for disease. Here, we compare a commonly used statistical test with a new intuitive and flexible computational approach to quickly detect differentially methylated sites. The method rapidly identifies and ranks candidate lists with greatest inter-group variability whilst controlling for intra-group variability. Intuitive and biologically relevant filters can be imposed to quickly identify sites and genes of interest.

  5. Unmasking risk loci: DNA methylation illuminates the biology of cancer predisposition: analyzing DNA methylation of transcriptional enhancers reveals missed regulatory links between cancer risk loci and genes.

    Science.gov (United States)

    Aran, Dvir; Hellman, Asaf

    2014-02-01

    Paradoxically, DNA sequence polymorphisms in cancer risk loci rarely correlate with the expression of cancer genes. Therefore, the molecular mechanism underlying an individual's susceptibility to cancer has remained largely unknown. However, recent evaluations of the correlations between DNA methylation and gene expression levels across healthy and cancerous genomes have revealed enrichment of disease-related DNA methylation variations within disease-associated risk loci. Moreover, it appears that transcriptional enhancers embedded in cancer risk loci often contain DNA methylation sites that closely define the expression of prominent cancer genes, despite the lack of significant correlations between gene expression levels and the surrounding disease-associated polymorphic sequences. We suggest that DNA methylation variations may obscure the effect of co-residing risk sequence alleles. Analysis of enhancer methylation data may help to reveal the regulatory circuits underlying predisposition to cancers and other common diseases.

  6. Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Richards Eric J

    2008-09-01

    Full Text Available Abstract Background DNA methylation is an important biochemical mark that silences repetitive sequences, such as transposons, and reinforces epigenetic gene expression states. An important class of repetitive genes under epigenetic control in eukaryotic genomes encodes ribosomal RNA (rRNA transcripts. The ribosomal genes coding for the 45S rRNA precursor of the three largest eukaryotic ribosomal RNAs (18S, 5.8S, and 25–28S are found in nucleolus organizer regions (NORs, comprised of hundreds to thousands of repeats, only some of which are expressed in any given cell. An epigenetic switch, mediated by DNA methylation and histone modification, turns rRNA genes on and off. However, little is known about the mechanisms that specify and maintain the patterns of NOR DNA methylation. Results Here, we explored the extent of naturally-occurring variation in NOR DNA methylation among accessions of the flowering plant Arabidopsis thaliana. DNA methylation in coding regions of rRNA genes was positively correlated with copy number of 45S rRNA gene and DNA methylation in the intergenic spacer regions. We investigated the inheritance of NOR DNA methylation patterns in natural accessions with hypomethylated NORs in inter-strain crosses and defined three different categories of inheritance in F1 hybrids. In addition, subsequent analysis of F2 segregation for NOR DNA methylation patterns uncovered different patterns of inheritance. We also revealed that NOR DNA methylation in the Arabidopsis accession Bor-4 is influenced by the vim1-1 (variant in methylation 1-1 mutation, but the primary effect is specified by the NORs themselves. Conclusion Our results indicate that the NORs themselves are the most significant determinants of natural variation in NOR DNA methylation. However, the inheritance of NOR DNA methylation suggests the operation of a diverse set of mechanisms, including inheritance of parental methylation patterns, reconfiguration of parental NOR DNA

  7. Effect of DNA methylation on protein-DNA interaction of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    何忠效; 白坚石; 张昱

    1999-01-01

    HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.

  8. Relationship between tumor DNA methylation status and patient characteristics in African-American and European-American women with breast cancer.

    Directory of Open Access Journals (Sweden)

    Songping Wang

    Full Text Available Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA and European-American (EA breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women. Five of the genes, all known tumor suppressor genes (RASSF1A, RARβ2, CDH13, HIN1 and SFRP1, were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50. In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy naïve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARβ2 and CDH13 and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients.

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

    Directory of Open Access Journals (Sweden)

    Guillaume eRiviere

    2014-04-01

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

  10. DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide

    International Nuclear Information System (INIS)

    The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide

  11. Chromosomal aberrations and oxidative DNA adduct 8-hydroxy-2-deoxyguanosine as biomarkers of radiotoxicity in radiation workers

    Directory of Open Access Journals (Sweden)

    Sanaa A. El-Benhawy

    2016-07-01

    Conclusions: Scoring of chromosome aberrations such as breaks, fragments and dicentrics is a reliable method to detect previous exposure to ionizing radiation. This type of monitoring may be used as a biological dosimeter instead of physical dosimetry.8-OHdG is a useful oxidative DNA marker among radiation workers and those exposed to environmental carcinogens.

  12. Human age estimation from blood using mRNA, DNA methylation, DNA rearrangement, and telomere length.

    Science.gov (United States)

    Zubakov, Dmitry; Liu, Fan; Kokmeijer, Iris; Choi, Ying; van Meurs, Joyce B J; van IJcken, Wilfred F J; Uitterlinden, André G; Hofman, Albert; Broer, Linda; van Duijn, Cornelia M; Lewin, Jörn; Kayser, Manfred

    2016-09-01

    Establishing the age of unknown persons, or persons with unknown age, can provide important leads in police investigations, disaster victim identification, fraud cases, and in other legal affairs. Previous methods mostly relied on morphological features available from teeth or skeletal parts. The development of molecular methods for age estimation allowing to use human specimens that possess no morphological age information, such as bloodstains, is extremely valuable as this type of samples is commonly found at crime scenes. Recently, we introduced a DNA-based approach for human age estimation from blood based on the quantification of T-cell specific DNA rearrangements (sjTRECs), which achieves accurate assignment of blood DNA samples to one of four 20-year-interval age categories. Aiming at improving the accuracy of molecular age estimation from blood, we investigated different types of biomarkers. We started out by systematic genome-wide surveys for new age-informative mRNA and DNA methylation markers in blood from the same young and old individuals using microarray technologies. The obtained candidate markers were validated in independent samples covering a wide age range using alternative technologies together with previously proposed DNA methylation, sjTREC, and telomere length markers. Cross-validated multiple regression analysis was applied for estimating and validating the age predictive power of various sets of biomarkers within and across different marker types. We found that DNA methylation markers outperformed mRNA, sjTREC, and telomere length in age predictive power. The best performing model included 8 DNA methylation markers derived from 3 CpG islands reaching a high level of accuracy (cross-validated R(2)=0.88, SE±6.97 years, mean absolute deviation 5.07 years). However, our data also suggest that mRNA markers can provide independent age information: a model using a combined set of 5 DNA methylation markers and one mRNA marker could provide

  13. Elevations in Circulating Methylated and Unmethylated Preproinsulin DNA in New-Onset Type 1 Diabetes.

    Science.gov (United States)

    Fisher, Marisa M; Watkins, Renecia A; Blum, Janice; Evans-Molina, Carmella; Chalasani, Naga; DiMeglio, Linda A; Mather, Kieren J; Tersey, Sarah A; Mirmira, Raghavendra G

    2015-11-01

    Elevated ratios of circulating unmethylated to methylated preproinsulin (INS) DNA have been suggested to reflect β-cell death in type 1 diabetes (T1D). We tested the hypothesis that absolute levels (rather than ratios) of unmethylated and methylated INS DNA differ between subjects with new-onset T1D and control subjects and assessed longitudinal changes in these parameters. We used droplet digital PCR to measure levels of unmethylated and methylated INS DNA in serum from subjects at T1D onset and at 8 weeks and 1 year post-onset. Compared with control subjects, levels of both unmethylated and methylated INS DNA were elevated at T1D onset. At 8 weeks post-onset, methylated INS DNA remained elevated, but unmethylated INS DNA fell. At 1 year postonset, both unmethylated and methylated INS DNA returned to control levels. Subjects with obesity, type 2 diabetes, and autoimmune hepatitis exhibited lower levels of unmethylated and methylated INS compared with subjects with T1D at onset and no differences compared with control subjects. Our study shows that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. Our work emphasizes the need to consider absolute levels of differentially methylated DNA species as potential biomarkers of disease. PMID:26216854

  14. Conservation of DNA Methylation Programming Between Mouse and Human Gametes and Preimplantation Embryos.

    Science.gov (United States)

    White, Carlee R; MacDonald, William A; Mann, Mellissa R W

    2016-09-01

    In mice, assisted reproductive technologies (ARTs) applied during gametogenesis and preimplantation development can result in disruption of genomic imprinting. In humans, these technologies and/or subfertility have been linked to perturbations in genomic imprinting. To understand how ARTs and infertility affect DNA methylation, it is important to understand DNA methylation dynamics and the role of regulatory factors at these critical stages. Recent genome studies performed using mouse and human gametes and preimplantation embryos have shed light onto these processes. Here, we comprehensively review the current state of knowledge regarding global and imprinted DNA methylation programming in the mouse and human. Available data highlight striking similarities in mouse and human DNA methylation dynamics during gamete and preimplantation development. Just as fascinating, these studies have revealed sex-, gene-, and allele-specific differences in DNA methylation programming, warranting future investigation to untangle the complex regulation of DNA methylation dynamics during gamete and preimplantation development.

  15. Linkage of DNA Methylation Quantitative Trait Loci to Human Cancer Risk

    Directory of Open Access Journals (Sweden)

    Holger Heyn

    2014-04-01

    Full Text Available Epigenetic regulation and, in particular, DNA methylation have been linked to the underlying genetic sequence. DNA methylation quantitative trait loci (meQTL have been identified through significant associations between the genetic and epigenetic codes in physiological and pathological contexts. We propose that interrogating the interplay between polymorphic alleles and DNA methylation is a powerful method for improving our interpretation of risk alleles identified in genome-wide association studies that otherwise lack mechanistic explanation. We integrated patient cancer risk genotype data and genome-scale DNA methylation profiles of 3,649 primary human tumors, representing 13 solid cancer types. We provide a comprehensive meQTL catalog containing DNA methylation associations for 21% of interrogated cancer risk polymorphisms. Differentially methylated loci harbor previously reported and as-yet-unidentified cancer genes. We suggest that such regulation at the DNA level can provide a considerable amount of new information about the biology of cancer-risk alleles.

  16. Analysis of the DNA methylome and transcriptome in granulopoiesis reveal timed changes and dynamic enhancer methylation

    DEFF Research Database (Denmark)

    Rönnerblad, Michelle; Andersson, Robin; Olofsson, Tor;

    2014-01-01

    In development, epigenetic mechanisms such as DNA methylation have been suggested to provide a cellular memory to maintain multipotency but also stabilize cell fate decisions and direct lineage restriction. In this study, we set out to characterize changes in DNA methylation and gene expression...... active during differentiation. Overall, this study depicts in detail the epigenetic and transcriptional changes that occur during granulopoiesis and supports the role of DNA methylation as a regulatory mechanism in blood cell differentiation....

  17. Pancreatic cancer patient survival correlates with DNA methylation of pancreas development genes.

    Science.gov (United States)

    Thompson, Michael J; Rubbi, Liudmilla; Dawson, David W; Donahue, Timothy R; Pellegrini, Matteo

    2015-01-01

    DNA methylation is an epigenetic mark associated with regulation of transcription and genome structure. These markers have been investigated in a variety of cancer settings for their utility in differentiating normal tissue from tumor tissue. Here, we examine the direct correlation between DNA methylation and patient survival. We find that changes in the DNA methylation of key pancreatic developmental genes are strongly associated with patient survival.

  18. Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study.

    Science.gov (United States)

    Wu, Hui-Chen; Wang, Qiao; Chung, Wendy K; Andrulis, Irene L; Daly, Mary B; John, Esther M; Keegan, Theresa H M; Knight, Julia; Bradbury, Angela R; Kappil, Maya A; Gurvich, Irina; Santella, Regina M; Terry, Mary Beth

    2014-07-01

    Many epidemiologic studies of environmental exposures and disease susceptibility measure DNA methylation in white blood cells (WBC). Some studies are also starting to use saliva DNA as it is usually more readily available in large epidemiologic studies. However, little is known about the correlation of methylation between WBC and saliva DNA. We examined DNA methylation in three repetitive elements, Sat2, Alu, and LINE-1, and in four CpG sites, including AHRR (cg23576855, cg05575921), cg05951221 at 2q37.1, and cg11924019 at CYP1A1, in 57 girls aged 6-15 years with blood and saliva collected on the same day. We measured all DNA methylation markers by bisulfite-pyrosequencing, except for Sat2 and Alu, which were measured by the MethyLight assay. Methylation levels measured in saliva DNA were lower than those in WBC DNA, with differences ranging from 2.8% for Alu to 14.1% for cg05575921. Methylation levels for the three repetitive elements measured in saliva DNA were all positively correlated with those in WBC DNA. However, there was a wide range in the Spearman correlations, with the smallest correlation found for Alu (0.24) and the strongest correlation found for LINE-1 (0.73). Spearman correlations for cg05575921, cg05951221, and cg11924019 were 0.33, 0.42, and 0.79, respectively. If these findings are replicated in larger studies, they suggest that, for selected methylation markers (e.g., LINE-1), methylation levels may be highly correlated between blood and saliva, while for others methylation markers, the levels may be more tissue specific. Thus, in studies that differ by DNA source, each interrogated site should be separately examined in order to evaluate the correlation in DNA methylation levels across DNA sources.

  19. DNA methylation mediates genetic variation for adaptive transgenerational plasticity.

    Science.gov (United States)

    Herman, Jacob J; Sultan, Sonia E

    2016-09-14

    Environmental stresses experienced by individual parents can influence offspring phenotypes in ways that enhance survival under similar conditions. Although such adaptive transgenerational plasticity is well documented, its transmission mechanisms are generally unknown. One possible mechanism is environmentally induced DNA methylation changes. We tested this hypothesis in the annual plant Polygonum persicaria, a species known to express adaptive transgenerational plasticity in response to parental drought stress. Replicate plants of 12 genetic lines (sampled from natural populations) were grown in dry versus moist soil. Their offspring were exposed to the demethylating agent zebularine or to control conditions during germination and then grown in dry soil. Under control germination conditions, the offspring of drought-stressed parents grew longer root systems and attained greater biomass compared with offspring of well-watered parents of the same genetic lines. Demethylation removed these adaptive developmental effects of parental drought, but did not significantly alter phenotypic expression in offspring of well-watered parents. The effect of demethylation on the expression of the parental drought effect varied among genetic lines. Differential seed provisioning did not contribute to the effect of parental drought on offspring phenotypes. These results demonstrate that DNA methylation can mediate adaptive, genotype-specific effects of parental stress on offspring phenotypes. PMID:27629032

  20. Genome-Wide Discriminatory Information Patterns of Cytosine DNA Methylation.

    Science.gov (United States)

    Sanchez, Robersy; Mackenzie, Sally A

    2016-01-01

    Cytosine DNA methylation (CDM) is a highly abundant, heritable but reversible chemical modification to the genome. Herein, a machine learning approach was applied to analyze the accumulation of epigenetic marks in methylomes of 152 ecotypes and 85 silencing mutants of Arabidopsis thaliana. In an information-thermodynamics framework, two measurements were used: (1) the amount of information gained/lost with the CDM changes I R and (2) the uncertainty of not observing a SNP L C R . We hypothesize that epigenetic marks are chromosomal footprints accounting for different ontogenetic and phylogenetic histories of individual populations. A machine learning approach is proposed to verify this hypothesis. Results support the hypothesis by the existence of discriminatory information (DI) patterns of CDM able to discriminate between individuals and between individual subpopulations. The statistical analyses revealed a strong association between the topologies of the structured population of Arabidopsis ecotypes based on I R and on LCR, respectively. A statistical-physical relationship between I R and L C R was also found. Results to date imply that the genome-wide distribution of CDM changes is not only part of the biological signal created by the methylation regulatory machinery, but ensures the stability of the DNA molecule, preserving the integrity of the genetic message under continuous stress from thermal fluctuations in the cell environment. PMID:27322251

  1. DNA Methylation and the HOXC6 Paradox in Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Vinarskaja, Anna; Yamanaka, Masanori; Ingenwerth, Marc; Schulz, Wolfgang A., E-mail: wolfgang.schulz@uni-duesseldorf.de [Department of Urology, Heinrich Heine University, Moorenstr. 5, 40225 Düsseldorf (Germany)

    2011-09-27

    Overexpression of the classical homeobox transcription factor HOXC6 is frequent in prostate cancers and correlates with adverse clinical parameters. Since surprisingly many HOXC6 target genes are downregulated in prostate cancer, it has been posited that oncogenic effects of HOXC6 in prostate cancer may be unmasked by concurrent epigenetic downregulation of target genes exerting tumor suppressive effects. To test this hypothesis, we have studied the expression of three HOXC6 target genes, CNTN1 (encoding a cell adhesion protein), DKK3 and WIF1 (encoding WNT growth factor antagonists) as well as DNA methylation of DKK3 and WIF1. HOXC6 upregulation and association with poor prognosis were confirmed in our tissue series. The three target genes were each significantly downregulated in cancer tissues and expression of each one correlated inversely with that of HOXC6. Cases with lower WIF1 expression showed significantly earlier recurrence (p = 0.021), whereas no statistical significance was reached for CNTN1 and DKK3. Hypermethylation of DKK3 or WIF1 gene promoters was observed in a subset of cancers with downregulated expression, but was often weak. Our data support the hypothesis that HOXC6 target genes exerting tumor-suppressive effects are epigenetically downregulated in prostate cancer, but DNA methylation appears to follow or bolster rather than to cause their transcriptional inactivation.

  2. Genome-Wide Discriminatory Information Patterns of Cytosine DNA Methylation

    Science.gov (United States)

    Sanchez, Robersy; Mackenzie, Sally A.

    2016-01-01

    Cytosine DNA methylation (CDM) is a highly abundant, heritable but reversible chemical modification to the genome. Herein, a machine learning approach was applied to analyze the accumulation of epigenetic marks in methylomes of 152 ecotypes and 85 silencing mutants of Arabidopsis thaliana. In an information-thermodynamics framework, two measurements were used: (1) the amount of information gained/lost with the CDM changes IR and (2) the uncertainty of not observing a SNP LCR. We hypothesize that epigenetic marks are chromosomal footprints accounting for different ontogenetic and phylogenetic histories of individual populations. A machine learning approach is proposed to verify this hypothesis. Results support the hypothesis by the existence of discriminatory information (DI) patterns of CDM able to discriminate between individuals and between individual subpopulations. The statistical analyses revealed a strong association between the topologies of the structured population of Arabidopsis ecotypes based on IR and on LCR, respectively. A statistical-physical relationship between IR and LCR was also found. Results to date imply that the genome-wide distribution of CDM changes is not only part of the biological signal created by the methylation regulatory machinery, but ensures the stability of the DNA molecule, preserving the integrity of the genetic message under continuous stress from thermal fluctuations in the cell environment. PMID:27322251

  3. Antagonism between DNA and H3K27 methylation at the imprinted Rasgrf1 locus

    DEFF Research Database (Denmark)

    Lindroth, Anders M; Park, Yoon Jung; McLean, Chelsea M;

    2008-01-01

    At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited...... DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated...

  4. PPARGC1A DNA methylation in subcutaneous adipose tissue in low birth weight subjects

    DEFF Research Database (Denmark)

    Gillberg, Linn; Jacobsen, Stine; Rönn, Tina;

    2014-01-01

    -fat overfeeding increases PPARGC1A DNA methylation in muscle in a birth weight dependent manner. However, PPARGC1A DNA methylation in subcutaneous adipose tissue (SAT) in LBW subjects has not previously been investigated. Our objective was to determine PPARGC1A DNA methylation and mRNA expression in basal......OBJECTIVE: Increased DNA methylation of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in skeletal muscle from type 2 diabetes (T2D) subjects and from low birth weight (LBW) subjects with an increased risk of T2D. High...

  5. Environmental pollution and DNA methylation: carcinogenesis, clinical significance, and practical applications.

    Science.gov (United States)

    Cao, Yi

    2015-09-01

    Environmental pollution is one of the main causes of human cancer. Exposures to environmental carcinogens result in genetic and epigenetic alterations which induce cell transformation. Epigenetic changes caused by environmental pollution play important roles in the development and progression of environmental pollution-related cancers. Studies on DNA methylation are among the earliest and most conducted epigenetic research linked to cancer. In this review, the roles of DNA methylation in carcinogenesis and their significance in clinical medicine were summarized, and the effects of environmental pollutants, particularly air pollutants, on DNA methylation were introduced. Furthermore, prospective applications of DNA methylation to environmental pollution detection and cancer prevention were discussed.

  6. Chordoma characterization of significant changes of the DNA methylation pattern.

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    Beate Rinner

    Full Text Available Chordomas are rare mesenchymal tumors occurring exclusively in the midline from clivus to sacrum. Early tumor detection is extremely important as these tumors are resistant to chemotherapy and irradiation. Despite continuous research efforts surgical excision remains the main treatment option. Because of the often challenging anatomic location early detection is important to enable complete tumor resection and to reduce the high incidence of local recurrences. The aim of this study was to explore whether DNA methylation, a well known epigenetic marker, may play a role in chordoma development and if hypermethylation of specific CpG islands may serve as potential biomarkers correlated with SNP analyses in chordoma. The study was performed on tumor samples from ten chordoma patients. We found significant genomic instability by Affymetrix 6.0. It was interesting to see that all chordomas showed a loss of 3q26.32 (PIK 3CA and 3q27.3 (BCL6 thus underlining the potential importance of the PI3K pathway in chordoma development. By using the AITCpG360 methylation assay we elucidated 20 genes which were hyper/hypomethylated compared to normal blood. The most promising candidates were nine hyper/hypomethylated genes C3, XIST, TACSTD2, FMR1, HIC1, RARB, DLEC1, KL, and RASSF1. In summary, we have shown that chordomas are characterized by a significant genomic instability and furthermore we demonstrated a characteristic DNA methylation pattern. These findings add new insights into chordoma development, diagnosis and potential new treatment options.

  7. Germline DNA copy number aberrations identified as potential prognostic factors for breast cancer recurrence.

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    Yadav Sapkota

    Full Text Available Breast cancer recurrence (BCR is a common treatment outcome despite curative-intent primary treatment of non-metastatic breast cancer. Currently used prognostic and predictive factors utilize tumor-based markers, and are not optimal determinants of risk of BCR. Germline-based copy number aberrations (CNAs have not been evaluated as determinants of predisposition to experience BCR. In this study, we accessed germline DNA from 369 female breast cancer subjects who received curative-intent primary treatment following diagnosis. Of these, 155 experienced BCR and 214 did not, after a median duration of follow up after breast cancer diagnosis of 6.35 years (range = 0.60-21.78 and 8.60 years (range = 3.08-13.57, respectively. Whole genome CNA genotyping was performed on the Affymetrix SNP array 6.0 platform. CNAs were identified using the SNP-Fast Adaptive States Segmentation Technique 2 algorithm implemented in Nexus Copy Number 6.0. Six samples were removed due to poor quality scores, leaving 363 samples for further analysis. We identified 18,561 CNAs with ≥1 kb as a predefined cut-off for observed aberrations. Univariate survival analyses (log-rank tests identified seven CNAs (two copy number gains and five copy neutral-loss of heterozygosities, CN-LOHs showing significant differences (P<2.01×10(-5 in recurrence-free survival (RFS probabilities with and without CNAs.We also observed three additional but distinct CN-LOHs showing significant differences in RFS probabilities (P<2.86×10(-5 when analyses were restricted to stratified cases (luminal A, n = 208 only. After adjusting for tumor stage and grade in multivariate analyses (Cox proportional hazards models, all the CNAs remained strongly associated with the phenotype of BCR. Of these, we confirmed three CNAs at 17q11.2, 11q13.1 and 6q24.1 in representative samples using independent genotyping platforms. Our results suggest further investigations on the potential use of germline DNA

  8. Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer

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    Chung Jae

    2009-06-01

    Full Text Available Abstract Background Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition. Methods To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis. Results Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99 between the total number of hypermethylated CGIs and GI50 values (i.e., increased drug resistance following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells. Conclusion Selective epigenetic disruption of distinct biological

  9. A novel method for sensitive and specific detection of DNA methylation biomarkers based on DNA restriction during PCR cycling.

    Science.gov (United States)

    Kneip, Christoph; Schmidt, Bernd; Fleischhacker, Michael; Seegebarth, Anke; Lewin, Jörn; Flemming, Nadja; Seemann, Stefanie; Schlegel, Thomas; Witt, Christian; Liebenberg, Volker; Dietrich, Dimo

    2009-09-01

    DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcino-genesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of background DNA during amplification using the heat-stable enzyme Tsp509I. An assay for the lung cancer methylation biomarker BARHL2 was used to show clinical and analytical performance of the method in comparison with methylation-specific PCR technology. Both technologies showed comparable performance when analyzing technical DNA mixtures and bronchial lavage samples from 75 patients suspected of having lung cancer. The results demonstrate that the approach is useful for sensitive and specific detection of a few copies of methylated DNA in samples with a high background of unmethylated DNA, such as in clinical samples from body fluids.

  10. Temporal stability of epigenetic markers: sequence characteristics and predictors of short-term DNA methylation variations.

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    Hyang-Min Byun

    Full Text Available BACKGROUND: DNA methylation is an epigenetic mechanism that has been increasingly investigated in observational human studies, particularly on blood leukocyte DNA. Characterizing the degree and determinants of DNA methylation stability can provide critical information for the design and conduction of human epigenetic studies. METHODS: We measured DNA methylation in 12 gene-promoter regions (APC, p16, p53, RASSF1A, CDH13, eNOS, ET-1, IFNγ, IL-6, TNFα, iNOS, and hTERT and 2 of non-long terminal repeat elements, i.e., L1 and Alu in blood samples obtained from 63 healthy individuals at baseline (Day 1 and after three days (Day 4. DNA methylation was measured by bisulfite-PCR-Pyrosequencing. We calculated intraclass correlation coefficients (ICCs to measure the within-individual stability of DNA methylation between Day 1 and 4, subtracted of pyrosequencing error and adjusted for multiple covariates. RESULTS: Methylation markers showed different temporal behaviors ranging from high (IL-6, ICC = 0.89 to low stability (APC, ICC = 0.08 between Day 1 and 4. Multiple sequence and marker characteristics were associated with the degree of variation. Density of CpG dinucleotides nearby the sequence analyzed (measured as CpG(o/e or G+C content within ±200 bp was positively associated with DNA methylation stability. The 3' proximity to repeat elements and range of DNA methylation on Day 1 were also positively associated with methylation stability. An inverted U-shaped correlation was observed between mean DNA methylation on Day 1 and stability. CONCLUSIONS: The degree of short-term DNA methylation stability is marker-dependent and associated with sequence characteristics and methylation levels.

  11. Aberrant methylation of Glutathione S-transferase P1 and E-cadherin in invasive ductal breast carcinoma and fibroadenoma

    Institute of Scientific and Technical Information of China (English)

    Wings Tjing Yung Loo; Mary Ngan Bing Cheung; Louis Wing Cheong Chow

    2010-01-01

    Objective To investigate the hypermethylation status of glutathione transferase P1(GSTP1) and E cadherin (ECAD), TSGs (tumor suppressor genes) in our breast cancer samples and explore their correlation with clinicopathological features of corresponding cancer patients. Methods One hundred and thirty six IDC (invasive ductal carcinoma) patients were recruited for analysis and 16 fibroadenoma patients acted as control. DNA extraction and methylation specific PCR (MSP) were subsequently performed preceded by pathological examination. Results The percentage of hypermethylated GSTP1 in carcinoma and fibroadenoma groups was 34.92% and 15.79% respectively and the percentage of hypermethylated ECAD in carcinomas and fibroadenomas was 18.00% and 0.00% respectively. Carcinoma had the highest percentage of c erbB2 overexpression being 54.55% among the clinicopathological parameters. Conclusion Hypermethylation patterns are frequent in IDC and seem to relate to c erbB2 overexpression, and such epigenetic change should not be neglected in fibroadenoma. Tumor methylation status in cancer patients can be determined at early stage and it may be a reference for better treatment planning.

  12. New advances of microRNAs in glioma stem cells, with special emphasis on aberrant methylation of microRNAs.

    Science.gov (United States)

    Zhao, Bing; Bian, Er-Bao; Li, Jia; Li, Jun

    2014-09-01

    Malignant brain tumors are thought to be originate from a small population of cells that display stem cell properties, including the capacity of self-renewal, multipotent differentiation, initiation of tumor tissues. Cancer stem cells (CSCs) have been identified in gliomas in which they are named as glioma stem cells (GSCs). GSCs, sharing some characteristics with normal neural stem cells (NSCs), contribute to the cellular origin for primary gliomas and the recurrence of malignant gliomas after current conventional therapy. Recently, increasing evidences have showed that miRNAs play a central role in GSCs. In this review we focus on the role of GSCs in gliomas and in the abnomal expression of miRNAs in GSCs. Furthermore, we also discuss epigenetic dysregulation of tumor-suppressor miRNAs by promoter DNA methylation is involved in the regulation of GSCs biology. Recent advances in understanding dysregulated expression of miRNAs and methylation of tumor-suppressor miRNAs in GSCs and their possible use as new therapeutic targets of gliomas.

  13. The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma.

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    Fumiharu Ohka

    Full Text Available Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4 have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ, and even low grade gliomas (LGGs, WHO grade 2 eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6-methylguanine-DNA methyltransferase (MGMT that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1 IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2 LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3 LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4 higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.

  14. Characterization and directed evolution of a methyl-binding domain protein for high-sensitivity DNA methylation analysis.

    Science.gov (United States)

    Heimer, Brandon W; Tam, Brooke E; Sikes, Hadley D

    2015-12-01

    Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application. PMID:26384511

  15. DNA methylation analysis in the intestinal epithelium-effect of cell separation on gene expression and methylation profile.

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    Andreas C Jenke

    Full Text Available BACKGROUND: Epigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore desirable for all epigenetic studies. However, to date little information is available on whether separation protocols might influence epigenetic and/or gene expression signatures and hence might be less beneficial. We investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (IECs from 6 healthy individuals. MATERIALS AND METHODS: Epithelial cells were isolated from small bowel (i.e. terminal ileum biopsies using EDTA/DTT and enzymatic release followed by magnetic bead sorting via EPCAM labeled microbeads. Effects on gene/mRNA expression were analyzed using a real time PCR based expression array. DNA methylation was assessed by pyrosequencing of bisulfite converted DNA and methylated DNA immunoprecipitation (MeDIP. RESULTS: While cell purity was >95% using both cell separation approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable and only revealed subtle differences. Comparison of DNA methylation of the epithelial cell marker EPCAM in unseparated whole biopsy samples with separated epithelium (i.e. EPCAM positive and negative fraction demonstrated significant differences in DNA methylation between all three tissue fractions indicating cell type specific methylation patterns can be masked in unseparated tissue samples. CONCLUSIONS: Taken together, our data highlight the importance of considering the potential effect of cell separation on gene expression as well as DNA methylation signatures. The decision to separate tissue samples will therefore depend on study design and specific separation protocols.

  16. Position-dependent correlations between DNA methylation and the evolutionary rates of mammalian coding exons

    Science.gov (United States)

    Chuang, Trees-Juen; Chen, Feng-Chi; Chen, Yen-Zho

    2012-01-01

    DNA cytosine methylation is a central epigenetic marker that is usually mutagenic and may increase the level of sequence divergence. However, methylated genes have been reported to evolve more slowly than unmethylated genes. Hence, there is a controversy on whether DNA methylation is correlated with increased or decreased protein evolutionary rates. We hypothesize that this controversy has resulted from the differential correlations between DNA methylation and the evolutionary rates of coding exons in different genic positions. To test this hypothesis, we compare human–mouse and human–macaque exonic evolutionary rates against experimentally determined single-base resolution DNA methylation data derived from multiple human cell types. We show that DNA methylation is significantly related to within-gene variations in evolutionary rates. First, DNA methylation level is more strongly correlated with C-to-T mutations at CpG dinucleotides in the first coding exons than in the internal and last exons, although it is positively correlated with the synonymous substitution rate in all exon positions. Second, for the first exons, DNA methylation level is negatively correlated with exonic expression level, but positively correlated with both nonsynonymous substitution rate and the sample specificity of DNA methylation level. For the internal and last exons, however, we observe the opposite correlations. Our results imply that DNA methylation level is differentially correlated with the biological (and evolutionary) features of coding exons in different genic positions. The first exons appear more prone to the mutagenic effects, whereas the other exons are more influenced by the regulatory effects of DNA methylation. PMID:23019368

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

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    Tomoyuki Nagata

    2015-03-01

    Full Text Available 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 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.

  18. Human leukocyte telomere length is associated with DNA methylation levels in multiple subtelomeric and imprinted loci.

    Science.gov (United States)

    Buxton, Jessica L; Suderman, Matthew; Pappas, Jane J; Borghol, Nada; McArdle, Wendy; Blakemore, Alexandra I F; Hertzman, Clyde; Power, Christine; Szyf, Moshe; Pembrey, Marcus

    2014-05-14

    In humans, leukocyte telomere length (LTL) is positively correlated with lifespan, and shorter LTL is associated with increased risk of age-related disease. In this study we tested for association between telomere length and methylated cytosine levels. Measurements of mean telomere length and DNA methylation at >450,000 CpG sites were obtained for both blood (N = 24) and EBV-transformed cell-line (N = 36) DNA samples from men aged 44-45 years. We identified 65 gene promoters enriched for CpG sites at which methylation levels are associated with leukocyte telomere length, and 36 gene promoters enriched for CpG sites at which methylation levels are associated with telomere length in DNA from EBV-transformed cell-lines. We observed significant enrichment of positively associated methylated CpG sites in subtelomeric loci (within 4 Mb of the telomere) (P telomere length, DNA methylation and gene expression in health and disease.

  19. Dynamic heterogeneity and DNA methylation in embryonic stem cells.

    KAUST Repository

    Singer, Zakary S

    2014-07-01

    Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the "2i" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.

  20. DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

    Science.gov (United States)

    Rathore, Mangal S; Jha, Bhavanath

    2016-03-01

    The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93-32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53 % and 13.33-25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions. PMID:26588922

  1. Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis).

    Science.gov (United States)

    Nilsen, Frances M; Parrott, Benjamin B; Bowden, John A; Kassim, Brittany L; Somerville, Stephen E; Bryan, Teresa A; Bryan, Colleen E; Lange, Ted R; Delaney, J Patrick; Brunell, Arnold M; Long, Stephen E; Guillette, Louis J

    2016-03-01

    Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida's north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different "treatments" of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics.

  2. Genome-wide DNA methylation patterns and transcription analysis in sheep muscle.

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    Christine Couldrey

    Full Text Available DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS. While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.

  3. Base-resolution DNA methylation landscape of zebrafish brain and liver

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    Aniruddha Chatterjee

    2014-12-01

    To our knowledge, these datasets are the only RRBS datasets and base-resolution DNA methylation data available at this time for zebrafish brain and liver. These datasets could serve as a resource for future studies to document the functional role of DNA methylation in zebrafish. In addition, these datasets could be used as controls while performing analysis on treated samples.

  4. Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis).

    Science.gov (United States)

    Nilsen, Frances M; Parrott, Benjamin B; Bowden, John A; Kassim, Brittany L; Somerville, Stephen E; Bryan, Teresa A; Bryan, Colleen E; Lange, Ted R; Delaney, J Patrick; Brunell, Arnold M; Long, Stephen E; Guillette, Louis J

    2016-03-01

    Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida's north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different "treatments" of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics. PMID:26748003

  5. Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis)

    Science.gov (United States)

    Nilsen, Frances M.; Parrott, Benjamin B.; Bowden, John A.; Kassim, Brittany L.; Somerville, Stephen E.; Bryan, Teresa A.; Bryan, Colleen E.; Lange, Ted R.; Delaney, J. Patrick; Brunell, Arnold M.; Long, Stephen E.; Guillette, Louis J.

    2016-01-01

    Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida’s north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different “treatments” of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics. PMID:26748003

  6. Adult global DNA methylation in relation to pre-natal nutrition

    OpenAIRE

    Lumey, LH; Terry, Mary Beth; Delgado-Cruzata, Lissette; Liao, Yuyan; Wang, Qiao; Susser, Ezra; McKeague, Ian; Santella, Regina M.

    2011-01-01

    Background Exposure to a pre-natal famine environment has been associated with a persistent decrease in DNA methylation of the IGF2 gene, although study findings on other loci have been highly variable. There have been no studies to date of the relation between pre-natal famine and overall global DNA methylation in adulthood.

  7. Effect of chronic heroin and cocaine administration on global DNA methylation in brain and liver.

    Science.gov (United States)

    Fragou, Domniki; Zanos, Panos; Kouidou, Sofia; Njau, Samuel; Kitchen, Ian; Bailey, Alexis; Kovatsi, Leda

    2013-04-26

    Drug abuse is associated with epigenetic changes, such as histone modifications and DNA methylation. The purpose of the present study was to examine the effect of chronic cocaine and heroin administration on global DNA methylation in brain and liver. Male, 8 week old, C57BL/6J mice received heroin in a chronic 'intermittent' escalating dose paradigm, or cocaine in a chronic escalating dose 'binge' paradigm, which mimic the human pattern of opioid or cocaine abuse respectively. Following sacrifice, livers and brains were removed and DNA was extracted from them. The extracted DNA was hydrolyzed and 2'-deoxycytidine and 5-methyl-2'-deoxycytidine were determined by HPLC-UV. The % 5-methyl-2'-deoxycytidine content of DNA was significantly higher in the brain compared to the liver. There were no differences between the control animals and the cocaine or heroin treated animals in neither of the tissues examined, which is surprising since cocaine administration induced gross morphological changes in the liver. Moreover, there was no difference in the % 5-methyl-2'-deoxycytidine content of DNA between the cocaine and the heroin treated animals. The global DNA methylation status in the brain and liver of mice chronically treated with cocaine or heroin remains unaffected, but this finding cannot exclude the existence of anatomical region or gene-specific methylation differences. This is the first time that global DNA methylation in the liver and whole brain has been studied following chronic cocaine or heroin treatment. PMID:23454526

  8. Influence of IL17A polymorphisms on the aberrant methylation of DAPK and CDH1 in non-cancerous gastric mucosa

    Directory of Open Access Journals (Sweden)

    Arisawa Tomiyasu

    2012-07-01

    Full Text Available Abstract Background CpG island aberrant methylation is shown to be an important mechanism in gene silencing. The important role of IL-17 in inflammatory response to H. pylori colonization has been indicated. We investigated the influence of IL17A polymorphisms, -197 G > A (rs2275913 and *1249 C > T (rs3748067, on the methylation of DAPK and CDH1. Methods Gastric mucosal samples were obtained from 401 subjects without malignancies. Methylation status of gene was determined by MSP. The genotyping of IL17A was performed by PCR-SSCP. Results Methylations of DAPK and CDH1 were seen in 196 and 149 of all 401 subjects, respectively. Overall, *1249 T carrier was associated with a decreased risk for DAPK methylation, whereas -197 G > A was not. In the subjects older than 60 years old, *1249 T carrier was more strongly associated with gene methylation and -197 A carrier tended to be associated with an increased risk for CDH1 methylation. When evaluating by inflammation promoting haplotype (-197 mutant carrier with *1249 homozygote, this haplotype had a more strongly increased risk for both DAPK and CDH1 methylations in comparatively older subjects. Both atrophy and metaplasia scores were significantly increased with age in -197 A carrier or *1249 CC homozygote, whereas were not in -197 GG homozygote or *1249 T carrier. PG I/II ratio was more significantly decreased in -197 A carrier than in GG homozygote under influence of H. pylori infection. Conclusions In -197 A allele carrier with *1249 CC homozygote, the methylations of both DAPK and CDH1 may be increased gradually, but more rapidly than the other genotypes, with age and altered gastric mucosal structure induced by H. pylori infection.

  9. Dynamics of DNA methylation in recent human and great ape evolution

    OpenAIRE

    Irene Hernando-Herraez; Javier Prado-Martinez; Paras Garg; Marcos Fernandez-Callejo; Holger Heyn; Christina Hvilsom; Arcadi Navarro; Manel Esteller; Sharp, Andrew J.; Tomas Marques-Bonet

    2013-01-01

    DNA methylation is an epigenetic modification involved in regulatory processes such as cell differentiation during development, X-chromosome inactivation, genomic imprinting and susceptibility to complex disease. However, the dynamics of DNA methylation changes between humans and their closest relatives are still poorly understood. We performed a comparative analysis of CpG methylation patterns between 9 humans and 23 primate samples including all species of great apes (chimpanzee, bonobo, go...

  10. EXTRACELLULAR DNA AND THE LEVEL OF ITS METHYLATION IN DIFFERENT RHEUMATIC DISEASES

    Directory of Open Access Journals (Sweden)

    N O Shubayeva

    2012-01-01

    Conclusion. RDs are characterized by the higher concentration of apoptotic and necrotic DNA, impaired exDNA methylation, varying complexification of exDNA with monometinic proteins, which is associated with the hyperproduction of autoantibodies (including anti-exDNA antibodies and inflammatory markers.

  11. Pros and cons of methylation-based enrichment methods for ancient DNA

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine; Gamba, Cristina; Der Sarkissian, Clio;

    2015-01-01

    The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules...... containing methylated CpGs. Using remains of a Palaeo-Eskimo Saqqaq individual, woolly mammoths, polar bears and two equine species, we confirm that DNA methylation survives in a variety of tissues, environmental contexts and over a large temporal range (4,000 to over 45,000 years before present). MBD...... enrichment, however, appears principally biased towards the recovery of CpG-rich and long DNA templates and is limited by the fast post-mortem cytosine deamination rates of methylated epialleles. This method, thus, appears only appropriate for the analysis of ancient methylomes from very well preserved...

  12. Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Induction of DNA repair by the carcinogens methyl and ethyl nitrosourea and methyl methanesulfonate.

    Science.gov (United States)

    Thielmann, H W; Vosberg, H P; Reygers, U

    1975-08-15

    Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RFI) of phage phiX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5' leads to 3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5' leads 3' exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds. PMID:170107

  13. Recent progress towards understanding the role of DNA methylation in human placental development

    Science.gov (United States)

    Mayne, Benjamin T; Buckberry, Sam; Breen, James; Rodriguez Lopez, Carlos M; Roberts, Claire T

    2016-01-01

    Epigenetic modifications, and particularly DNA methylation, have been studied in many tissues, both healthy and diseased, and across numerous developmental stages. The placenta is the only organ that has a transient life of 9 months and undergoes rapid growth and dynamic structural and functional changes across gestation. Additionally, the placenta is unique because although developing within the mother, its genome is identical to that of the foetus. Given these distinctive characteristics, it is not surprising that the epigenetic landscape affecting placental gene expression may be different to that in other healthy tissues. However, the role of epigenetic modifications, and particularly DNA methylation, in placental development remains largely unknown. Of particular interest is the fact that the placenta is the most hypomethylated human tissue and is characterized by the presence of large partially methylated domains (PMDs) containing silenced genes. Moreover, how and why the placenta is hypomethylated and what role DNA methylation plays in regulating placental gene expression across gestation are poorly understood. We review genome-wide DNA methylation studies in the human placenta and highlight that the different cell types that make up the placenta have very different DNA methylation profiles. Summarizing studies on DNA methylation in the placenta and its relationship with pregnancy complications are difficult due to the limited number of studies available for comparison. To understand the key steps in placental development and hence what may be perturbed in pregnancy complications requires large-scale genome-wide DNA methylation studies coupled with transcriptome analyses. PMID:27026712

  14. Epigenetic Variation in Monozygotic Twins: A Genome-Wide Analysis of DNA Methylation in Buccal Cells

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    Jenny van Dongen

    2014-05-01

    Full Text Available DNA methylation is one of the most extensively studied epigenetic marks in humans. Yet, it is largely unknown what causes variation in DNA methylation between individuals. The comparison of DNA methylation profiles of monozygotic (MZ twins offers a unique experimental design to examine the extent to which such variation is related to individual-specific environmental influences and stochastic events or to familial factors (DNA sequence and shared environment. We measured genome-wide DNA methylation in buccal samples from ten MZ pairs (age 8–19 using the Illumina 450k array and examined twin correlations for methylation level at 420,921 CpGs after QC. After selecting CpGs showing the most variation in the methylation level between subjects, the mean genome-wide correlation (rho was 0.54. The correlation was higher, on average, for CpGs within CpG islands (CGIs, compared to CGI shores, shelves and non-CGI regions, particularly at hypomethylated CpGs. This finding suggests that individual-specific environmental and stochastic influences account for more variation in DNA methylation in CpG-poor regions. Our findings also indicate that it is worthwhile to examine heritable and shared environmental influences on buccal DNA methylation in larger studies that also include dizygotic twins.

  15. Extensive sequence-influenced DNA methylation polymorphism in the human genome

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    Hellman Asaf

    2010-05-01

    Full Text Available Abstract Background Epigenetic polymorphisms are a potential source of human diversity, but their frequency and relationship to genetic polymorphisms are unclear. DNA methylation, an epigenetic mark that is a covalent modification of the DNA itself, plays an important role in the regulation of gene expression. Most studies of DNA methylation in mammalian cells have focused on CpG methylation present in CpG islands (areas of concentrated CpGs often found near promoters, but there are also interesting patterns of CpG methylation found outside of CpG islands. Results We compared DNA methylation patterns on both alleles between many pairs (and larger groups of related and unrelated individuals. Direct observation and simulation experiments revealed that around 10% of common single nucleotide polymorphisms (SNPs reside in regions with differences in the propensity for local DNA methylation between the two alleles. We further showed that for the most common form of SNP, a polymorphism at a CpG dinucleotide, the presence of the CpG at the SNP positively affected local DNA methylation in cis. Conclusions Taken together with the known effect of DNA methylation on mutation rate, our results suggest an interesting interdependence between genetics and epigenetics underlying diversity in the human genome.

  16. Determining methylation status of methylguanine DNA methyl transferase (MGMT) from formalin-fixed, paraffin embedded tumor tissue

    OpenAIRE

    ABREU, FRANCINE B.; Torrey L. Gallagher; Liu, Emmeline Z.; Tsongalis, Gregory J.

    2014-01-01

    O-6-methylguanine-DNA methyltransferase (MGMT) has been associated with resistance to alkylating agent cancer therapy in Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. Lower expression or silencing of the MGMT protein by promoter methylation has been reported to improve survival in patients with GBM [1]. This protocol describes bisulfite conversion, methylation sensitive PCR amplification and data analysis/interpretation. This protocol differs from publis...

  17. Determining methylation status of methylguanine DNA methyl transferase (MGMT) from formalin-fixed, paraffin embedded tumor tissue

    Science.gov (United States)

    de Abreu, Francine B.; Gallagher, Torrey L.; Liu, Emmeline Z.; Tsongalis, Gregory J.

    2014-01-01

    O-6-methylguanine-DNA methyltransferase (MGMT) has been associated with resistance to alkylating agent cancer therapy in Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. Lower expression or silencing of the MGMT protein by promoter methylation has been reported to improve survival in patients with GBM [1]. This protocol describes bisulfite conversion, methylation sensitive PCR amplification and data analysis/interpretation. This protocol differs from published protocols in that it:•Describes a detailed method to measure MGMT using DNA extracted from solid tumor tissue. We have optimized the DNA extraction by using FFPE tissue blocks that contain greater than 50% tumor tissue, when non-tumor tissue was also present. Performance of this assay is compromised when lower quantities of tumor cells are used as the methylation status of tumor cells is diluted out by methylation status of normal cells.•The measurement of MGMT could be further (enhanced) optimized using a percentage of methylation ration cutoff of 2 as methylated.•The machine specifications detailed here are specific to measuring MGMT from PPFE tumor tissue. PMID:26150933

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

    OpenAIRE

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

    2012-01-01

    DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N[superscript 6]-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss o...

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

    OpenAIRE

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

    2013-01-01

    DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N6-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces...

  20. Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1

    Energy Technology Data Exchange (ETDEWEB)

    Bendall, Matthew L. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Luong, Khai [Pacific Biosciences, Menlo Park, CA (United States); Wetmore, Kelly M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Blow, Matthew [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Korlach, Jonas [Pacific Biosciences, Menlo Park, CA (United States); Deutschbauer, Adam [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Malmstrom, Rex [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2013-08-30

    We performed whole genome analyses of DNA methylation in Shewanella 17 oneidensis MR-1 to examine its possible role in regulating gene expression and 18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 19 revealed extensive methylation of adenine (N6mA) throughout the 20 genome. These methylated bases were located in five sequence motifs, 21 including three novel targets for Type I restriction/modification enzymes. The 22 sequence motifs targeted by putative methyltranferases were determined via 23 SMRT sequencing of gene knockout mutants. In addition, we found S. 24 oneidensis MR-1 cultures grown under various culture conditions displayed 25 different DNA methylation patterns. However, the small number of differentially 26 methylated sites could not be directly linked to the much larger number of 27 differentially expressed genes in these conditions, suggesting DNA methylation is 28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 29 of methylated GATC motifs in the origin of replication indicate DNA methylation 30 may regulate genome replication in a manner similar to that seen in Escherichia 31 coli. Furthermore, comparative analyses suggest that many 32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.

  1. Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

    Institute of Scientific and Technical Information of China (English)

    Bin-Zhong Li; Guo-Liang Xu; Zheng Huang; Qing-Yan Cui; Xue-Hui Song; Lin Du; Albert Jeltsch; Ping Chen; Guohong Li; En Li

    2011-01-01

    Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyltransferase is largely unknown. Here, we show that histone H3 tails lacking lysine 4 (K4) methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain (PHD). In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation - first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails - the latter might serve as a checkpoint for the methylation activity.

  2. Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols.

    Directory of Open Access Journals (Sweden)

    Yuh Shiwa

    Full Text Available Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03 when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50 when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14 by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45 and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17. These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

  3. Understanding the connection between epigenetic DNA methylation and nucleosome positioning from computer simulations.

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    Guillem Portella

    Full Text Available Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling.

  4. The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach.

    Science.gov (United States)

    Gonzalez, Diego; Kozdon, Jennifer B; McAdams, Harley H; Shapiro, Lucy; Collier, Justine

    2014-04-01

    DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria.

  5. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene® fixed samples compared to restored FFPE DNA

    DEFF Research Database (Denmark)

    Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte;

    2014-01-01

    , precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap......Chip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better...... compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective....

  6. Methylation effect on the ohmic resistance of a poly-GC DNA-like chain

    Science.gov (United States)

    de Moura, F. A. B. F.; Lyra, M. L.; de Almeida, M. L.; Ourique, G. S.; Fulco, U. L.; Albuquerque, E. L.

    2016-10-01

    We determine, by using a tight-binding model Hamiltonian, the characteristic current-voltage (IxV) curves of a 5-methylated cytosine single strand poly-GC DNA-like finite segment, considering the methyl groups attached laterally to a random fraction of the cytosine basis. Striking, we found that the methylation significantly impacts the ohmic resistance (R) of the DNA-like segments, indicating that measurements of R can be used as a biosensor tool to probe the presence of anomalous methylation.

  7. Determination and Difference Analysis of DNA Methylation Content Both in Blood and Muscle Tissue of Pigs

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this experiment, DNA samples from parental lines Large White, Landrace, and Meishan pigs, and their hybrids Large White×Landrace, Landrace×Large White, Large White×Meishan, and Meishan×Large White pigs were used for the determination of DNA methylation content in both blood and muscle tissue. The differences about DNA methylation content between parental lines and their hybrids were analyzed. These will offer theoretical support from molecular level for heterosis. High performance liquid chromatography (HPLC) was firstly used to detect DNA methylation content. The average DNA methylation content in 163 DNA samples of muscle tissue was 16.92%, whereas, the average DNA methylation content in 182 samples of blood was 6.49%, the difference between which was especially prominent (P 0.05); and the differences between reciprocal cross hybrids in both hybrid systems were not significant (P > 0.05), but between different hybrid systems, the hybrids had a significant difference (P<0.05). The average methylation content in muscle samples was higher than that in blood samples, and the methylation in different tissues was different.

  8. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

    Directory of Open Access Journals (Sweden)

    James I. McDonald

    2016-06-01

    Full Text Available Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.

  9. Identification of body fluid-specific DNA methylation markers for use in forensic science.

    Science.gov (United States)

    Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

    2014-11-01

    DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science.

  10. Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression.

    Directory of Open Access Journals (Sweden)

    Francesca Bonvicini

    Full Text Available CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.

  11. Identification of body fluid-specific DNA methylation markers for use in forensic science.

    Science.gov (United States)

    Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

    2014-11-01

    DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. PMID:25128690

  12. A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation

    Directory of Open Access Journals (Sweden)

    Tierling Sascha

    2010-06-01

    Full Text Available Abstract Background DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. Results This combined method allows detection of 14 pg (that is, four to five genomic copies of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2 and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. Conclusion The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.

  13. The prognostic significance of whole blood global and specific DNA methylation levels in gastric adenocarcinoma.

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    Mansour S Al-Moundhri

    Full Text Available BACKGROUND: Epigenetics, particularly DNA methylation, has recently been elucidated as important in gastric cancer (GC initiation and progression. We investigated the clinical and prognostic importance of whole blood global and site-specific DNA methylation in GC. METHODS: Genomic DNA was extracted from the peripheral blood of 105 Omani GC patients at diagnosis. DNA methylation was quantified by pyrosequencing of global DNA and specific gene promoter regions at 5 CpG sites for CDH1, 7 CpG sites for p16, 4 CpG sites for p53, and 3 CpG sites for RUNX3. DNA methylation levels in patients were categorized into low, medium, and high tertiles. Associations between methylation level category and clinicopathological features were evaluated using χ(2 tests. Survival analyses were carried out using the Kaplan-Meier method and log rank test. A backward conditional Cox proportional hazards regression model was used to identify independent predictors of survival. RESULTS: Older GC patients had increased methylation levels at specific CpG sites within the CDH1, p53, and RUNX-3 promoters. Male gender was significantly associated with reduced global and increased site-specific DNA methylation levels in CDH1, p16, and p53 promoters. Global DNA low methylation level was associated with better survival on univariate analysis. Patients with high and medium methylation vs. low methylation levels across p16 promoter CpG sites, site 2 in particular, had better survival. Multivariate analysis showed that global DNA hypermethylation was a significant independent predictor of worse survival (hazard ratio (HR = 2.0, 95% CI: 1.1-3.8; p = 0.02 and high methylation mean values across p16 promoter sites 1-7 were associated with better survival with HR of 0.3 (95% CI, 0.1-0.8; p = 0.02 respectively. CONCLUSIONS: Analysis of global and site-specific DNA methylation in peripheral blood by pyrosequencing provides quantitative DNA methylation values that may serve as important

  14. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

    Directory of Open Access Journals (Sweden)

    Sharma Shikhar

    2012-01-01

    Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

  15. Hydrophobicity of methylated DNA as a possible mechanism for gene silencing

    International Nuclear Information System (INIS)

    AFM images show that chromatin reconstituted on methylated DNA (meDNA) is compacted when imaged under water. Chromatin reconstituted on unmethylated DNA is less compacted and less sensitive to hydration. These differences must reflect changes in the physical properties of DNA on methylation, but prior studies have not revealed large differences between methylated and unmethylated DNA. Quasi-elastic light scattering studies of solutions of methylated and unmethylated DNA support this view. In contrast, AFM images of molecules at a water/solid interface yield a persistence length that nearly doubles (to 92.5 ± 4 nm) when 9% of the total DNA is methylated. This increase in persistence length is accompanied by a decrease in contour length, suggesting that a significant fraction of the meDNA changes into the stiffer A form as the more hydrophobic meDNA is dehydrated at the interface. This suggests a simple mechanism for gene silencing as the stiffer meDNA is more difficult to remove from nucleosomes. (paper)

  16. DNA methylation results depend on DNA integrity – role of post mortem interval

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    Mathias eRhein

    2015-05-01

    Full Text Available Major questions of neurological and psychiatric mechanisms involve the brain functions on a molecular level and cannot be easily addressed due to limitations in access to tissue samples. Post mortem studies are able to partly bridge the gap between brain tissue research retrieved from animal trials and the information derived from peripheral analysis (e.g. measurements in blood cells in patients. Here, we wanted to know how fast DNA degradation is progressing under controlled conditions in order to define thresholds for tissue quality to be used in respective trials. Our focus was on the applicability of partly degraded samples for bisulfite sequencing and the determination of simple means to define cut-off values.After opening the brain cavity, we kept two consecutive pig skulls at ambient temperature (19-21°C and removed cortex tissue up to a post mortem interval (PMI of 120h. We calculated the percentage of degradation on DNA gel electrophoresis of brain DNA to estimate quality and relate this estimation spectrum to the quality of human post-mortem control samples. Functional DNA quality was investigated by bisulfite sequencing of two functionally relevant genes for either the serotonin receptor 5 (SLC6A4 or aldehyde dehydrogenase 2 (ALDH2.Testing our approach in a heterogeneous collective of human blood and brain samples, we demonstrate integrity of measurement quality below the threshold of 72h PMI.While sequencing technically worked for all timepoints irrespective of conceivable DNA degradation, there is a good correlation between variance of methylation to degradation levels documented in the gel (R2=0.4311, p=0.0392 for advancing post mortem intervals (PMI. This otherwise elusive phenomenon is an important prerequisite for the interpretation and evaluation of samples prior to in-depth processing via an affordable and easy assay to estimate identical sample quality and thereby comparable methylation measurements.

  17. Global DNA methylation in gonads of adult zebrafish Danio rerio under bisphenol A exposure.

    Science.gov (United States)

    Liu, Yan; Zhang, Yingying; Tao, Shiyu; Guan, Yongjing; Zhang, Ting; Wang, Zaizhao

    2016-08-01

    Altered DNA methylation is pervasively associated with changes in gene expression and signal transduction after exposure to a wide range of endocrine disrupting chemicals. As a weak estrogenic chemical, bisphenol A (BPA) has been extensively studied for reproductive toxicity. In order to explore the effects of BPA on epigenetic modification in gonads of zebrafish Danio rerio, we measured the global DNA methylation together with the gene expression of DNA methyltransferase (dnmts), glycine N-methyltransferase (gnmt), and ten-eleven translocation (tets) in gonads of D. rerio under BPA exposure by ELISA and quantitative real-time PCR method, respectively. The global level of DNA methylation was significantly decreased in ovaries after exposed to BPA for 7 days, and testes following 35-day exposure. Moreover, the global level of DNA methylation was also significantly reduced in testes after exposed to 15μg/L BPA for 7 days. Besides the alteration of the global level of DNA methylation, varying degrees of transcriptional changes of dnmts, gnmt and tets were detected in gonads of D. rerio under BPA exposure. The present study suggested that BPA might cause the global DNA demethylation in gonads of zebrafish by regulating the transcriptional changes of the DNA methylation/demethylation-associated genes (dnmts, gnmt, and tets). PMID:27101439

  18. Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.

    Science.gov (United States)

    Zhang, Baohua; Denomme, Michelle M; White, Carlee R; Leung, Kit-Yi; Lee, Martin B; Greene, Nicholas D E; Mann, Mellissa R W; Trasler, Jacquetta M; Baltz, Jay M

    2015-03-01

    The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst. The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the folate cycle, but only a few cell types generate SAM from betaine (N,N,N-trimethylglycine) via betaine-homocysteine methyltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the folate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos. Inhibiting both pathways, however, much more severely affected ICM development (7-8 cells). Total SAM levels in mouse blastocysts decreased significantly only when both pathways were inhibited (from 3.1 to 1.6 pmol/100 blastocysts). DNA methylation, detected as 5-methylcytosine (5-MeC) immunofluorescence in isolated ICMs, was minimally affected by inhibition of either pathway alone but decreased by at least 45-55% when both BHMT and the folate cycle were inhibited simultaneously. Effects on cell numbers and 5-MeC levels in the ICM were completely rescued by methionine (immediate SAM precursor) or SAM. Both the folate cycle and betaine/BHMT appear to contribute to a methyl pool required for normal ICM development and establishing initial embryonic DNA methylation. PMID:25466894

  19. Differential DNA methylation patterns of polycystic ovarian syndrome in whole blood of Chinese women

    DEFF Research Database (Denmark)

    Li, Shuxia; Zhu, Dongyi; Duan, Hongmei;

    2016-01-01

    interference of the genome with the environment through integrative biological mechanisms including epigenetics. We have performed a genome-wide DNA methylation analysis on polycystic ovarian syndrome and identified a substantial number of genomic sites differentially methylated in the whole blood of PCOS...... patients and healthy controls (52 sites, false discovery rate discovery rate ... in the DNA methylome from ovarian tissue under PCOS condition. Most importantly, our genome-wide profiling focusing on PCOS patients revealed a large number of DNA methylation sites and their enriched functional pathways significantly associated with diverse clinical features (levels of prolactin, estradiol...

  20. Genetic analysis of DNA methylation and gene expression levels in whole blood of healthy human subjects

    Directory of Open Access Journals (Sweden)

    van Eijk Kristel R

    2012-11-01

    Full Text Available Abstract Background The predominant model for regulation of gene expression through DNA methylation is an inverse association in which increased methylation results in decreased gene expression levels. However, recent studies suggest that the relationship between genetic variation, DNA methylation and expression is more complex. Results Systems genetic approaches for examining relationships between gene expression and methylation array data were used to find both negative and positive associations between these levels. A weighted correlation network analysis revealed that i both transcriptome and methylome are organized in modules, ii co-expression modules are generally not preserved in the methylation data and vice-versa, and iii highly significant correlations exist between co-expression and co-methylation modules, suggesting the existence of factors that affect expression and methylation of different modules (i.e., trans effects at the level of modules. We observed that methylation probes associated with expression in cis were more likely to be located outside CpG islands, whereas specificity for CpG island shores was present when methylation, associated with expression, was under local genetic control. A structural equation model based analysis found strong support in particular for a traditional causal model in which gene expression is regulated by genetic variation via DNA methylation instead of gene expression affecting DNA methylation levels. Conclusions Our results provide new insights into the complex mechanisms between genetic markers, epigenetic mechanisms and gene expression. We find strong support for the classical model of genetic variants regulating methylation, which in turn regulates gene expression. Moreover we show that, although the methylation and expression modules differ, they are highly correlated.

  1. 线粒体DNA与DNA甲基化的关系%The relationship between mitochondrial DNA and DNA methylation

    Institute of Scientific and Technical Information of China (English)

    王萍; 房静远

    2009-01-01

    线粒体DNA(mitochondrial DNA,mtDNA)遗传信息量虽小,却控制着线粒体一些最基本的性质,对细胞及其功能有着重要影响.mtDNA的损伤与衰老、肿瘤等疾病的发生有关.DNA甲基化是调节基因表达的重要方式之一.mtDNA基因的表达受核DNA (nuclear DNA,nDNA)的调控,mtDNA和nDNA协同作用参与机体代谢调节和发病.本文就近年来mtDNA与DNA甲基化的关系作一综述.%Mitochondrial DNA(mtDNA) determines the primary nature of mitochondrial and plays an important role in cell function.The damage of mtDNA is associated with aging, tumor and other diseases. DNA methylation is a major way to regulate gene expression. mtDNA expression is regulated by nuclear DNA. mtDNA and nDNA participating in metabolic regulation and pathogenesy synergisticly. The relationship between mitochondrial DNA and DNA methylation were reviewed here.

  2. Comparison of methods for quantification of global DNA methylation in human cells and tissues.

    Directory of Open Access Journals (Sweden)

    Sofia Lisanti

    Full Text Available DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

  3. BEclear: Batch Effect Detection and Adjustment in DNA Methylation Data.

    Science.gov (United States)

    Akulenko, Ruslan; Merl, Markus; Helms, Volkhard

    2016-01-01

    Batch effects describe non-natural variations of, for example, large-scale genomic data sets. If not corrected by suitable numerical algorithms, batch effects may seriously affect the analysis of these datasets. The novel array platform independent software tool BEclear enables researchers to identify those portions of the data that deviate statistically significant from the remaining data and to replace these portions by typical values reconstructed from neighboring data entries based on latent factor models. In contrast to other comparable methods that often use some sort of global normalization of the data, BEclear avoids changing the apparently unaffected parts of the data. We tested the performance of this approach on DNA methylation data for various tumor data sets taken from The Cancer Genome Atlas and compared the results to those obtained with the existing algorithms ComBat, Surrogate Variable Analysis, RUVm and Functional normalization. BEclear constantly performed at par with or better than these methods. BEclear is available as an R package at the Bioconductor project http://bioconductor.org/packages/release/bioc/html/BEclear.html. PMID:27559732

  4. BEclear: Batch Effect Detection and Adjustment in DNA Methylation Data

    Science.gov (United States)

    Akulenko, Ruslan; Merl, Markus; Helms, Volkhard

    2016-01-01

    Batch effects describe non-natural variations of, for example, large-scale genomic data sets. If not corrected by suitable numerical algorithms, batch effects may seriously affect the analysis of these datasets. The novel array platform independent software tool BEclear enables researchers to identify those portions of the data that deviate statistically significant from the remaining data and to replace these portions by typical values reconstructed from neighboring data entries based on latent factor models. In contrast to other comparable methods that often use some sort of global normalization of the data, BEclear avoids changing the apparently unaffected parts of the data. We tested the performance of this approach on DNA methylation data for various tumor data sets taken from The Cancer Genome Atlas and compared the results to those obtained with the existing algorithms ComBat, Surrogate Variable Analysis, RUVm and Functional normalization. BEclear constantly performed at par with or better than these methods. BEclear is available as an R package at the Bioconductor project http://bioconductor.org/packages/release/bioc/html/BEclear.html. PMID:27559732

  5. DNA Methylation and Chromatin Remodeling: The Blueprint of Cancer Epigenetics

    Directory of Open Access Journals (Sweden)

    Dipanjan Bhattacharjee

    2016-01-01

    Full Text Available Epigenetics deals with the interactions between genes and the immediate cellular environment. These interactions go a long way in shaping up each and every person’s individuality. Further, reversibility of epigenetic interactions may offer a dynamic control over the expression of various critical genes. Thus, tweaking the epigenetic machinery may help cause or cure diseases, especially cancer. Therefore, cancer epigenetics, especially at a molecular level, needs to be scrutinised closely, as it could potentially serve as the future pharmaceutical goldmine against neoplastic diseases. However, in view of its rapidly enlarging scope of application, it has become difficult to keep abreast of scientific information coming out of various epigenetic studies directed against cancer. Using this review, we have attempted to shed light on two of the most important mechanisms implicated in cancer, that is, DNA (deoxyribonucleic acid methylation and histone modifications, and their place in cancer pathogenesis. Further, we have attempted to take stock of the new epigenetic drugs that have emerged onto the market as well as those in the pipeline that offer hope in mankind’s fight against cancer.

  6. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing

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    Rajeevan Mangalathu S

    2009-06-01

    Full Text Available Abstract Background Differential expression of perforin (PRF1, a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction. Findings We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA or DNA extracted from peripheral blood mononuclear cells (PBMC DNA from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18% to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92% in the distal enhancer region (CpG sites 1–10, a variable methylation (range 49%–83% in the methylation sensitive region (CpG sites 11–17, and a progressively declining methylation level (range 12%–80% in the proximal promoter region (CpG sites 18–32 of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA. Conclusion This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune

  7. Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer

    Directory of Open Access Journals (Sweden)

    Laird Peter W

    2008-07-01

    Full Text Available Abstract Background Lung cancer is the leading cause of cancer death in men and women in the United States and Western Europe. Over 160,000 Americans die of this disease every year. The five-year survival rate is 15% – significantly lower than that of other major cancers. Early detection is a key factor in increasing lung cancer patient survival. DNA hypermethylation is recognized as an important mechanism for tumor suppressor gene inactivation in cancer and could yield powerful biomarkers for early detection of lung cancer. Here we focused on developing DNA methylation markers for squamous cell carcinoma of the lung. Using the sensitive, high-throughput DNA methylation analysis technique MethyLight, we examined the methylation profile of 42 loci in a collection of 45 squamous cell lung cancer samples and adjacent non-tumor lung tissues from the same patients. Results We identified 22 loci showing significantly higher DNA methylation levels in tumor tissue than adjacent non-tumor lung. Of these, eight showed highly significant hypermethylation in tumor tissue (p Conclusion We have identified 22 DNA methylation markers for squamous cell lung cancer, several of which have not previously been reported to be methylated in any type of human cancer. The top eight markers show great promise as a sensitive and specific DNA methylation marker panel for squamous cell lung cancer.

  8. Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing

    Science.gov (United States)

    Grasso, Chiara; Trevisan, Morena; Fiano, Valentina; Tarallo, Valentina; De Marco, Laura; Sacerdote, Carlotta; Richiardi, Lorenzo; Merletti, Franco; Gillio-Tos, Anna

    2016-01-01

    Background Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Objective Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. Methods We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. Results We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. Conclusion The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing. PMID

  9. Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Chiara Grasso

    Full Text Available Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis.Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results.We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1 by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36 and DNA from blood fractions of healthy people (DD study, N = 28, respectively.We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites.The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

  10. A review of dietary factors and its influence on DNA methylation in colorectal carcinogenesis.

    Science.gov (United States)

    Arasaradnam, R P; Commane, D M; Bradburn, D; Mathers, J C

    2008-01-01

    Colorectal cancer (CRC) is the most common cancer in non-smokers posing a significant health burden in the UK. Observational studies lend support to the impact of environmental factors especially diet on colorectal carcinogenesis. Significant advances have been made in understanding the biology of CRC carcinogenesis in particular epigenetic modifications such as DNA methylation. DNA methylation is thought to occur at least as commonly as inactivation of tumor suppressor genes. In fact compared with other human cancers, promoter gene methylation occurs most commonly within the gastrointestinal tract. Emerging data suggest the direct influence of certain micronutrients for example folic acid, selenium as well as interaction with toxins such as alcohol on DNA methylation. Such interactions are likely to have a mechanistic impact on CRC carcinogenesis through the methylation pathway but also, may offer possible therapeutic potential as nutraceuticals.

  11. The damage and repair of DNA in teleosts after administration of N-methyl-N-nitrosourea

    International Nuclear Information System (INIS)

    14C-MNU, dissolved in a DMSO-citratebuffer solution, was given intraperitoneally to Black Mollies (B.M.) and Poecilia formosa (P.f.). Gills, liver, tailfinmuscles, intestine, gonads and brain were removed from each fish and DNA was isolated by phenol extraction. The DNA was hydrolysed and then the purines were separated using HPLC. Methylation of purines was determined by a liquid scintillation counter. Maximum methylation was formed in the N-7 position of guanine in the DNA from intestine of B.M. The highest content of O6-methyl guanine was found in the DNA of tailfinmuscles of B.M. whereas DNA from brain of B.M. showed the maximum methylation in N-3 position of adenine. The methylation of the purines from B.M. showed the similar pattern as in P.f. but was quantitatively double the amount as that found in P.f. The methylation of O6-position of guanine and N-3 position of adenine occured earlier in P.f. than in B.M. Maximum methylation of purines from each of the organs investigated was found to occur after 1/2 to 8 hours. The amount of methylation as low as 10% of the maximum was observed in a period from 1/2 to 16 hours after the application of 14C-MNU. Excision repair seems to be responsible for removal of N-3 methyl adenine and N-7 methyl guanine whereas O6-methyl guanine seems to be repaired by methyltransferases. Removal of methylgroups from O6-position of guanine and the excision repair known to exist in mammals and bacteria probably play a role in these two species of teleosts as well. (Author)

  12. The role of cytosine methylation on charge transport through a DNA strand

    Science.gov (United States)

    Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

    2015-09-01

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  13. The role of cytosine methylation on charge transport through a DNA strand

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu [Department of Electrical Engineering, University of Washington, Seattle, Washington 98195-2500 (United States); Govind, Niranjan, E-mail: niri.govind@pnnl.gov [William R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 (United States)

    2015-09-07

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  14. Divergence of gene body DNA methylation and evolution of plant duplicate genes.

    Directory of Open Access Journals (Sweden)

    Jun Wang

    Full Text Available 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.

  15. Genome-wide DNA methylation and gene expression patterns provide insight into polycystic ovary syndrome development

    OpenAIRE

    Wang, Xiu-Xia; Wei, Jing-Zan; Jiao, Jiao; Jiang, Shu-Yi; Yu, Da-hai; Li, Da

    2014-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. However, the epigenetic mechanism involved in PCOS progression remains largely unknown. Here, combining the DNA methylation profiling together with transcriptome analysis, we showed that (i) there were 7929 differentially methylated CpG sites (β > 0.1, P 1.5, P < 0.005) in PCOS compared to normal ovaries; (ii) 54 genes were identified with methylated...

  16. High Quality Assessment of DNA Methylation in Archival Tissues from Colorectal Cancer Patients Using Quantitative High-Resolution Melting Analysis

    OpenAIRE

    Balic, Marija; Pichler, Martin; Strutz, Jasmin; Heitzer, Ellen; Ausch, Christoph; Samonigg, Hellmut; Cote, Richard J.; Dandachi, Nadia

    2009-01-01

    High-resolution melting (HRM) analysis is a novel tool for analysis of promoter methylation. The aim of the present study was to establish and validate HRM analysis for detection of promoter methylation on archival formalin-fixed paraffin-embedded tissues from colorectal cancer patients. We first evaluated HRM assays for O6-methylguanine-DNA methyltransferase (MGMT) and adenomatous polyposis coli (APC) promoter methylation on a methylated DNA dilution matrix and DNA extracted from eight fresh...

  17. Critical period of nonpromoter DNA methylation acquisition during prenatal male germ cell development.

    Directory of Open Access Journals (Sweden)

    Kirsten M Niles

    Full Text Available The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility.

  18. Chronic opioid use is associated with increased DNA methylation correlating with increased clinical pain.

    Science.gov (United States)

    Doehring, Alexandra; Oertel, Bruno Georg; Sittl, Reinhard; Lötsch, Jörn

    2013-01-01

    Environmentally caused changes in chromosomes that do not alter the DNA sequence but cause phenotypic changes by altering gene transcription are summarized as epigenetics. A major epigenetic mechanism is methylation or demethylation at CpG-rich DNA islands. DNA methylation triggered by drugs has largely unexplored therapeutic consequences. Here we report increased methylation at a CpG rich island in the OPRM1 gene coding for μ-opioid receptors and at a global methylation site (LINE-1) in leukocytes of methadone-substituted former opiate addicts compared with matched healthy controls. Higher DNA methylation associated with chronic opioid exposure was reproduced in an independent cohort of opioid-treated as compared to non-opioid-treated pain patients. This suggests that opioids may stimulate DNA methylation. The OPRM1 methylation had no immediate effect on μ-opioid receptor transcription and was not associated with opioid dosing requirements. However, the global DNA methylation at LINE-1 was significantly correlated with increased chronic pain. This suggests inhibitory effects on the transcription of still unspecified nocifensive gene products. It further implies that opioids may be causally associated with increased genome-wide DNA methylation, although currently there is no direct evidence of this. This has phenotypic consequences for pain and may provide a new, epigenetics-associated mechanism of opioid-induced hyperalgesia. The results indicate a potential influence of opioid analgesics on the patients' epigenome. They emphasize the need for reliable and cost-effective screening tools and may imply that high-throughput screening for lead compounds in artificial expression systems may not provide the best tools for identifying new pain medications.

  19. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    Science.gov (United States)

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITINABSTRACT Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  20. Proximity ligation in situ assay for monitoring the global DNA methylation in cells

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    Vallette François M

    2011-04-01

    Full Text Available Abstract Background DNA methylation has a central role in the epigenetic control of mammalian gene expression, and is required for X inactivation, genomics imprinting and silencing of retrotransposons and repetitive sequences. Thus, several technologies have been developed to measure the degree of DNA methylation. Results We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since the quantification of Dnmt1/PCNA interaction in cells reflects the degree of DNA methylation. Conclusion This method being directly realizable on cells, it appears that it could suggest a wide range of applications in basic research and drug development. More particularly, this method is specially adapted for the investigations realized from a weak quantity of biologic materiel such as stem cells or primary cultured tumor cells for examples.

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

    NARCIS (Netherlands)

    Weber, M.; Hellmann, I.; Stadler, M.B.; Ramos, L.; Paabo, S.; Rebhan, M.; Schubeler, D.

    2007-01-01

    To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in

  2. Genome-Wide Analysis of DNA Methylation and Cigarette Smoking in a Chinese Population

    Science.gov (United States)

    Zhu, Xiaoyan; Li, Jun; Deng, Siyun; Yu, Kuai; Liu, Xuezhen; Deng, Qifei; Sun, Huizhen; Zhang, Xiaomin; He, Meian; Guo, Huan; Chen, Weihong; Yuan, Jing; Zhang, Bing; Kuang, Dan; He, Xiaosheng; Bai, Yansen; Han, Xu; Liu, Bing; Li, Xiaoliang; Yang, Liangle; Jiang, Haijing; Zhang, Yizhi; Hu, Jie; Cheng, Longxian; Luo, Xiaoting; Mei, Wenhua; Zhou, Zhiming; Sun, Shunchang; Zhang, Liyun; Liu, Chuanyao; Guo, Yanjun; Zhang, Zhihong; Hu, Frank B.; Liang, Liming; Wu, Tangchun

    2016-01-01

    Background: Smoking is a risk factor for many human diseases. DNA methylation has been related to smoking, but genome-wide methylation data for smoking in Chinese populations is limited. Objectives: We aimed to investigate epigenome-wide methylation in relation to smoking in a Chinese population. Methods: We measured the methylation levels at > 485,000 CpG sites (CpGs) in DNA from leukocytes using a methylation array and conducted a genome-wide meta-analysis of DNA methylation and smoking in a total of 596 Chinese participants. We further evaluated the associations of smoking-related CpGs with internal polycyclic aromatic hydrocarbon (PAH) biomarkers and their correlations with the expression of corresponding genes. Results: We identified 318 CpGs whose methylation levels were associated with smoking at a genome-wide significance level (false discovery rate Zhang X, He M, Guo H, Chen W, Yuan J, Zhang B, Kuang D, He X, Bai Y, Han X, Liu B, Li X, Yang L, Jiang H, Zhang Y, Hu J, Cheng L, Luo X, Mei W, Zhou Z, Sun S, Zhang L, Liu C, Guo Y, Zhang Z, Hu FB, Liang L, Wu T. 2016. Genome-wide analysis of DNA methylation and cigarette smoking in Chinese. Environ Health Perspect 124:966–973; http://dx.doi.org/10.1289/ehp.1509834 PMID:26756918

  3. Changes in liver cell DNA methylation status in diabetic mice affect its FT-IR characteristics.

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    Benedicto de Campos Vidal

    Full Text Available Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile.The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO and adequate software.Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1 and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

  4. Control of Glycosylation-Related Genes by DNA Methylation: the Intriguing Case of the B3GALT5 Gene and Its Distinct Promoters.

    Science.gov (United States)

    Trinchera, Marco; Zulueta, Aida; Caretti, Anna; Dall'Olio, Fabio

    2014-08-04

    Glycosylation is a metabolic pathway consisting of the enzymatic modification of proteins and lipids through the stepwise addition of sugars that gives rise to glycoconjugates. To determine the full complement of glycoconjugates that cells produce (the glycome), a variety of genes are involved, many of which are regulated by DNA methylation. The aim of the present review is to briefly describe some relevant examples of glycosylation-related genes whose DNA methylation has been implicated in their regulation and to focus on the intriguing case of a glycosyltransferase gene (B3GALT5). Aberrant promoter methylation is frequently at the basis of their modulation in cancer, but in the case of B3GALT5, at least two promoters are involved in regulation, and a complex interplay is reported to occur between transcription factors, chromatin remodelling and DNA methylation of typical CpG islands or even of other CpG dinucleotides. Transcription of the B3GALT5 gene underwent a particular evolutionary fate, so that promoter hypermethylation, acting on one transcript, and hypomethylation of other sequences, acting on the other, cooperate on one gene to obtain full cancer-associated silencing. The findings may also help in unravelling the complex origin of serum CA19.9 antigen circulating in some patients.

  5. The dynamic DNA methylation cycle from egg to sperm in the honey bee Apis mellifera.

    Science.gov (United States)

    Drewell, Robert A; Bush, Eliot C; Remnant, Emily J; Wong, Garrett T; Beeler, Suzannah M; Stringham, Jessica L; Lim, Julianne; Oldroyd, Benjamin P

    2014-07-01

    In honey bees (Apis mellifera), the epigenetic mark of DNA methylation is central to the developmental regulation of caste differentiation, but may also be involved in additional biological functions. In this study, we examine the whole genome methylation profiles of three stages of the haploid honey bee genome: unfertilised eggs, the adult drones that develop from these eggs and the sperm produced by these drones. These methylomes reveal distinct patterns of methylation. Eggs and sperm show 381 genes with significantly different CpG methylation patterns, with the vast majority being more methylated in eggs. Adult drones show greatly reduced levels of methylation across the genome when compared with both gamete samples. This suggests a dynamic cycle of methylation loss and gain through the development of the drone and during spermatogenesis. Although fluxes in methylation during embryogenesis may account for some of the differentially methylated sites, the distinct methylation patterns at some genes suggest parent-specific epigenetic marking in the gametes. Extensive germ line methylation of some genes possibly explains the lower-than-expected frequency of CpG sites in these genes. We discuss the potential developmental and evolutionary implications of methylation in eggs and sperm in this eusocial insect species. PMID:24924193

  6. Oxidative Stress Modulates DNA Methylation during Melanocyte Anchorage Blockade Associated with Malignant Transformation

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    Ana C.E. Campos

    2007-12-01

    Full Text Available Both oxidative/nitrosative stress and alterations in DNA methylation are observed during carcinogenesis of different tumor types, but no clear correlation between these events has been demonstrated until now. Melanoma cell lines were previously established after submitting the nontumorigenic melanocyte lineage, melan-a, to cycles of anchorage blockade. In this work, increased intracellular oxidative species and nitric oxide levels, as well as alterations in the DNA methylation, were observed after melan-a detachment, which were also associated with a decrease in intracellular homocysteine (Hey, an element in the methionine (universal methyl donor cycle. This alteration was accompanied by increase in glutathione (GSH levels and methylated DNA content. Furthermore, a significant increase in dnmti and 3b expression was identified along melan-a anchorage blockade. LG-Nitro-L-arginine methyl esther (L-NAME, known as a nitric oxide synthase (NOS inhibitor, and N-acetyl-L-cysteine (NAC prevented the increase in global DNA methylation, as well as the increase in dnmti and 3b expression, observed during melan-a detachment. Interestingly, both L-NAME and NAC did not inhibit nitric oxide (NO production in these cells, but abrogated superoxide anion production during anchorage blockade. In conclusion, oxidative stress observed during melanocyte anchorage blockade seems to modulate DNA methylation levels and may directly contribute to the acquisition of an anoikis-resistant phenotype through an epigenetic mechanism.

  7. DNA甲基化、单核苷酸多态性与肿瘤%DNA methylation, single nucleotide polymorphism, and tumor

    Institute of Scientific and Technical Information of China (English)

    蒋逸群; 胡多沙; 段芝

    2011-01-01

    DNA甲基化是表观遗传修饰的重要部分,基因异常甲基化在肿瘤发生、发展中扮演重要角色.众多研究结果表明单核苷酸多态性(single nucleotide polymorphism,SNP)与DNA甲基化之间存在密切联系,可能通过改变甲基化位点或对DNA甲基化相关酶类产生影响,继而影响到相关基因的甲基化状态,改变表达水平,导致肿瘤易感性发生变化.DNA甲基化代表的表观遗传与SNP代表的遗传突变能为癌变的分子机制研究提供更多的理论支持.%DNA methylation is one of the most important part in epigenetic modifications, and aberrant gene methylation plays a critical role in tumorigenesis. Many studies have indicated that DNA methylation is related to single nucleotide polymorphism ( SNP) which may modify the methylation sites or influence the biological function of enzymes related to DNA methylation, change the status of genes' methylation, which in turn, contribute to tumorigenesis. The role of epigenetic (such as DNA mehtyla-tion) and mutation (such as SNP) can provide more information for molecular metabolism in the carcin-ogensis.

  8. Characteristic DNA methylation profiles in peripheral blood monocytes are associated with inflammatory phenotypes of asthma

    OpenAIRE

    Gunawardhana, Lakshitha P; Gibson, Peter G; Simpson, Jodie L.; Benton, Miles C.; Rodney A. Lea; Baines, Katherine J

    2014-01-01

    Epigenetic changes including DNA methylation caused by environmental exposures may contribute to the heterogeneous inflammatory response in asthma. Here we investigate alterations in DNA methylation of purified blood monocytes that are associated with inflammatory phenotypes of asthma. Peripheral blood was collected from adults with eosinophilic asthma (EA; n = 21), paucigranulocytic asthma (PGA; n = 22), neutrophilic asthma (NA; n = 9), and healthy controls (n = 10). Blood monocytes were iso...

  9. Epigenetic targets and drug discovery Part 2: Histone demethylation and DNA methylation.

    Science.gov (United States)

    Liu, Ke; Liu, Yanli; Lau, Johnathan L; Min, Jinrong

    2015-07-01

    Chromatin structure is dynamically modulated by various chromatin modifications, such as histone/DNA methylation and demethylation. We have reviewed histone methyltransferases and methyllysine binders in terms of small molecule screening and drug discovery in the first part of this review series. In this part, we will summarize recent progress in chemical probe and drug discovery of histone demethylases and DNA methyltransferases. Histone demethylation and DNA methylation have attracted a lot of attention regarding their biology and disease implications. Correspondingly, many small molecule compounds have been designed to modulate the activity of histone demethylases and DNA methyltransferases, and some of them have been developed into therapeutic drugs or put into clinical trials.

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

    Science.gov (United States)

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

    2015-04-01

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

  11. Assessment of DNA methylation changes in tissue culture of Brassica napus.

    Science.gov (United States)

    Gao, Y; Ran, L; Kong, Y; Jiang, J; Sokolov, V; Wang, Y

    2014-11-01

    Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA methylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue:cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rape-seed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture. PMID:25739287

  12. Altered DNA methylation patterns of the H19 differentially methylated region and the DAZL gene promoter are associated with defective human sperm.

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    Bo Li

    Full Text Available DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ and asthenozoospermia (AZ usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control, with only complete methylation and mild hypomethylation(0.05. However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05% and AZ (0.58±1.77% (P20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05. In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.

  13. Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    During the process of alien germplasm introduced into wheat genome by chromosome engineering,extensive genetic variations of genome structure and gene expression in recipient could be induced.In this study,we performed GISH(genome in situ hybridization)and AFLP(amplified fragment length polymorphism) on wheat-rye chromosome transIocation lines and their parents to detect the identity in genomic structure of different translocation lines.The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation.Methylation sensitive amplification polymorphism(MSAP)analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines(CN12,20.15%;CN17,20.91%;CN18,22.42%),but the ratios of hemimethylated sites were significantly lowered(CN12,21.41%;CN17,23.43%;CN18,22.42%),whereas 16.37%were fully-methylated and 25.44%were hemimethylated in case of their wheat parent.Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent,including 13 hypermethylation patterns(33.74%),9 demethylation patterns(22.76%)and 7 uncertain patterns(4.07%).In further sequence analysis,the alterations of methylation pattern affected both repetitive DNA sequences,such as retrotransposons and tandem repetitive sequences,and low-copy DNA.

  14. Widespread Epigenetic Abnormalities Suggest a Broad DNA Methylation Erasure Defect in Abnormal Human Sperm

    Science.gov (United States)

    Siegmund, Kimberly; Yang, Allen; Laird, Peter W.; Sokol, Rebecca Z.

    2007-01-01

    Background Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis. Methodology/Principal Finding We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm. Conclusions This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line. PMID:18074014

  15. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm.

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    Sahar Houshdaran

    Full Text Available BACKGROUND: Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis. METHODOLOGY/PRINCIPAL FINDING: We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm. CONCLUSIONS: This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.

  16. Different Levels of DNA Methylation Detected in Human Sperms after Morphological Selection Using High Magnification Microscopy

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    Nino Guy Cassuto

    2016-01-01

    Full Text Available Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p<10-6, OR = 2.4; and p<0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI.

  17. Aberrant gene methylation in the peritoneal fluid is a risk factor predicting peritoneal recurrence in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Masatsugu; Hiraki; Yoshihiko; Kitajima; Seiji; Sato; Jun; Nakamura; Kazuyoshi; Hashiguchi; Hirokazu; Noshiro; Kohji; Miyazaki

    2010-01-01

    AIM:To investigate whether gene methylation in the peritoneal fluid (PF) predicts peritoneal recurrence in gastric cancer patients.METHODS: The gene methylation of CHFR (checkpoint with forkhead and ring finger domains), p16, RUNX3 (runt-related transcription factor 3), E-cadherin, hMLH1 (mutL homolog 1), ABCG2 (ATP-binding cassette, sub-family G, member 2) and BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) were analyzed in 80 specimens of PF by quantitative methylation-specific polymerase chain r...

  18. The Dynamics of DNA Methylation in Maize Roots under Pb Stress

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    Haiping Ding

    2014-12-01

    Full Text Available Plants adapt to adverse conditions through a series of physiological, cellular, and molecular processes, culminating in stress tolerance. However, little is known about the associated regulatory mechanisms at the epigenetic level in maize under lead (Pb stress. Therefore, in this study, we aimed to compare DNA methylation profiles during the dynamic development of maize roots following Pb treatment to identify candidate genes involved in the response to Pb stress. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq was used to investigate the genome-wide DNA methylation patterns in maize roots under normal condition (A1 and 3 mM Pb(NO32 stress for 12 h (K2, 24 h (K3 and 48 h (K4. The results showed that the average methylation density was the highest in CpG islands (CGIs, followed by the intergenic regions. Within the gene body, the methylation density of the introns was higher than those of the UTRs and exons. In total, 3857 methylated genes were found in 4 tested samples, including 1805 differentially methylated genes for K2 versus A1, 1508 for K3 versus A1, and 1660 for K4 versus A1. Further analysis showed that 140 genes exhibited altered DNA methylation in all three comparisons, including some well-known stress-responsive transcription factors and proteins, such as MYB, AP2/ERF, bZIP, serine-threonine/tyrosine-proteins, pentatricopeptide repeat proteins, RING zinc finger proteins, F-box proteins, leucine-rich repeat proteins and tetratricopeptide repeat proteins. This study revealed the genome-scale DNA methylation patterns of maize roots in response to Pb exposure and identified candidate genes that potentially regulate root dynamic development under Pb stress at the methylation level.

  19. DNA methylation mediates persistent epileptiform activity in vitro and in vivo.

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    Ziv M Machnes

    Full Text Available Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5' regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5' region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5' region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5' region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5' region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.

  20. Genome-wide DNA methylation profiling of non-small cell lung carcinomas

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    Carvalho Rejane

    2012-06-01

    Full Text Available Abstract Background Non-small cell lung carcinoma (NSCLC is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions. Results Analysis of the MethylCap-seq data revealed a strong positive correlation between replicate experiments and between paired tumor/lung samples. We identified 57 differentially methylated regions (DMRs present in all NSCLC tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. We also identified DMRs that were specific to two of the major subtypes of NSCLC, adenocarcinomas and squamous cell carcinomas. Conclusions Collectively, we provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC.

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

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    Árpád V Patai

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

  2. Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips.

    Science.gov (United States)

    Shi, Xiao Wei; Shi, Bo Hui; Lyu, Ai Li; Zhang, Feng; Zhou, Tian Tian; Guo, Xiong

    2016-07-01

    To understand how differentially methylated genes (DMGs) might affect the pathogenesis of Kashin-Beck disease (KBD). Genome-wide methylation profiling of whole blood from 12 matched KBD and controls pairs was performed using a high-resolution Infinium 450 K methylation array. In total, 97 CpG sites were differentially methylated in KBD compared to the normal controls; of these sites, 36 sites were significantly hypermethylated (covering 22 genes) and 61 sites were significantly hypomethylated (covering 34 genes). Of these genes, 14 significant pathways were identified, the most significant P value pathway was type I diabetes mellitus pathway and pathways associated with autoimmune diseases and inflammatory diseases were included in this study. Subsequently, 4 CpG sites in HLA-DRB1 were validated using bisulfite sequencing polymerase chain reaction (BSP) in articular cartilage, and the results showed significant differences in the methylation status between KBD and controls, consistent with the results of the high-resolution array. These results suggested that differences in genome-wide DNA methylation exist between KBD and the controls, and the biological pathways support the autoimmune disease and inflammatory disease hypothesis of KBD.

  3. Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips.

    Science.gov (United States)

    Shi, Xiao Wei; Shi, Bo Hui; Lyu, Ai Li; Zhang, Feng; Zhou, Tian Tian; Guo, Xiong

    2016-07-01

    To understand how differentially methylated genes (DMGs) might affect the pathogenesis of Kashin-Beck disease (KBD). Genome-wide methylation profiling of whole blood from 12 matched KBD and controls pairs was performed using a high-resolution Infinium 450 K methylation array. In total, 97 CpG sites were differentially methylated in KBD compared to the normal controls; of these sites, 36 sites were significantly hypermethylated (covering 22 genes) and 61 sites were significantly hypomethylated (covering 34 genes). Of these genes, 14 significant pathways were identified, the most significant P value pathway was type I diabetes mellitus pathway and pathways associated with autoimmune diseases and inflammatory diseases were included in this study. Subsequently, 4 CpG sites in HLA-DRB1 were validated using bisulfite sequencing polymerase chain reaction (BSP) in articular cartilage, and the results showed significant differences in the methylation status between KBD and controls, consistent with the results of the high-resolution array. These results suggested that differences in genome-wide DNA methylation exist between KBD and the controls, and the biological pathways support the autoimmune disease and inflammatory disease hypothesis of KBD. PMID:27554126

  4. DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.

    Directory of Open Access Journals (Sweden)

    Sahar Houshdaran

    Full Text Available BACKGROUND: Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. METHODOLOGY/PRINCIPAL FINDINGS: We obtained DNA methylation profiles of 1,505 CpG sites (808 genes in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes that exhibited 'subtype-specific' DNA methylation patterns (FDR<1% among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene. SIGNIFICANCE: The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data

  5. The Role of Cytosine Methylation on Charge Transport through a DNA Strand

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Jianqing [Univ. of Washington, Seattle, WA (United States); Govind, Niranjan [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Anantram, M. P. [Univ. of Washington, Seattle, WA (United States)

    2015-09-04

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modifi-cation remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Buttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. Specifically, we compare the results generated with the widely used B3LYP exchange-correlation (XC) functional and CAM-B3LYP based tuned range-separated hybrid density functional. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that with both functionals, the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and interstrand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital (HOMO) level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with both functionals. We also study the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit. Our results suggest that the effect of the two different functionals is to alter the on-site energies of the DNA bases at the HOMO level, while the transport properties don't depend much on the two

  6. Genome-wide DNA methylation profiling in cultured eutopic and ectopic endometrial stromal cells.

    Directory of Open Access Journals (Sweden)

    Yoshiaki Yamagata

    Full Text Available The objective of this study was to characterize the genome-wide DNA methylation profiles of isolated endometrial stromal cells obtained from eutopic endometria with (euESCa and without endometriosis (euESCb and ovarian endometrial cysts (choESC. Three samples were analyzed in each group. The infinium methylation array identified more hypermethylated and hypomethylated CpGs in choESC than in euESCa, and only a few genes were methylated differently in euESCa and euESCb. A functional analysis revealed that signal transduction, developmental processes, immunity, etc. were different in choESC and euESCa. A clustering analysis and a principal component analysis performed based on the methylation levels segregated choESC from euESC, while euESCa and euESCb were identical. A transcriptome analysis was then conducted and the results were compared with those of the DNA methylation analysis. Interestingly, the hierarchical clustering and principal component analyses showed that choESC were segregated from euESCa and euESCb in the DNA methylation analysis, while no segregation was recognized in the transcriptome analysis. The mRNA expression levels of the epigenetic modification enzymes, including DNA methyltransferases, obtained from the specimens were not significantly different between the groups. Some of the differentially methylated and/or expressed genes (NR5A1, STAR, STRA6 and HSD17B2, which are related with steroidogenesis, were validated by independent methods in a larger number of samples. Our findings indicate that different DNA methylation profiles exist in ectopic ESC, highlighting the benefits of genome wide DNA methylation analyses over transcriptome analyses in clarifying the development and characterization of endometriosis.

  7. 5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley

    OpenAIRE

    Solís, María-Teresa; El-Tantawy, Ahmed-Abdalla; Cano, Vanesa; Risueño, María C.; Testillano, Pilar S.

    2015-01-01

    Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application ...

  8. Modulation of DNA methylation machineries in japanese rice fish (Oryzias latipes) embryogenesis by ethanol and 5-azacytidine

    Science.gov (United States)

    As a sequel of our investigations on the impact of epigenome in inducing fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish, we investigated on several DNA methylation machinery genes including DNA methyl transferase 3ba (dnmt3ba) and methyl binding proteins (MBPs), namely, mbdl...

  9. Expression and DNA methylation analysis of SNRPN in Prader-Willi patients

    Energy Technology Data Exchange (ETDEWEB)

    Glenn, C.C.; Jong, M.T.C.; Driscoll, D.J. [Univ. of Florida, Gainesville, FL (United States)] [and others

    1994-09-01

    The human SNRPN gene is one of a gene family that encode proteins involved in pre-mRNA splicing and maps to the Prader-Willi syndrome critical region in 15q11-q13. We have previously demonstrated functional imprinting of SNRPN, with absent expression in PWS skin fibroblasts and lymphoblasts. We now show a similar lack of expression in blood of PWS patients, which appear to correlate with DNA methylation of NotI sites in the 5{prime} region of the gene. RNA and DNA was extracted from peripheral blood of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) deletion patients to be used for RT-PCR with SNRPN gene-specific primers and DNA methylation analysis. Either no or highly reduced levels of SNRPN RT-PCR product were detected in nine PWS samples but was present in normals, AS patients, and one clinically typical PWS patient. Parent-of-origin DNA methylation imprints are also present within the SNRPN gene. PWS patients having only a maternal contribution of SNRPN have several NotI restriction sites near the transcription start site which are methylated, while these same sites are unmethylated on the paternal chromosome (i.e., AS samples). Several CpG sites approximately 22 kb downstream of the transcription start site are methylated preferentially on the paternal allele. These observations for human SNRPN are similar to those of the mouse imprinted gene Igf2r, which exhibits DNA methylation of a CpG island 27 kb from the transcription start site on the expressed allele, and DNA methylation in the promoter region of the repressed allele. We suggest that RT-PCR and/or DNA methylation analysis from blood of PWS patients may be the most accurate means of diagnosing classical PWS. These results further indicate a role for SNRPN in the pathogenesis of PWS, and may serve as a model to study other human imprinted genes.

  10. Environmental Impact on DNA Methylation in the Germline: State of the Art and Gaps of Knowledge

    Directory of Open Access Journals (Sweden)

    Francesca Pacchierotti

    2015-01-01

    Full Text Available The epigenome consists of chemical changes in DNA and chromatin that without modifying the DNA sequence modulate gene expression and cellular phenotype. The epigenome is highly plastic and reacts to changing external conditions with modifications that can be inherited to daughter cells and across generations. Whereas this innate plasticity allows for adaptation to a changing environment, it also implies the potential of epigenetic derailment leading to so-called epimutations. DNA methylation is the most studied epigenetic mark. DNA methylation changes have been associated with cancer, infertility, cardiovascular, respiratory, metabolic, immunologic, and neurodegenerative pathologies. Experiments in rodents demonstrate that exposure to a variety of chemical stressors, occurring during the prenatal or the adult life, may induce DNA methylation changes in germ cells, which may be transmitted across generations with phenotypic consequences. An increasing number of human biomonitoring studies show environmentally related DNA methylation changes mainly in blood leukocytes, whereas very few data have been so far collected on possible epigenetic changes induced in the germline, even by the analysis of easily accessible sperm. In this paper, we review the state of the art on factors impinging on DNA methylation in the germline, highlight gaps of knowledge, and propose priorities for future studies.

  11. Glycolic Acid Silences Inflammasome Complex Genes, NLRC4 and ASC, by Inducing DNA Methylation in HaCaT Cells.

    Science.gov (United States)

    Tang, Sheau-Chung; Yeh, Jih-I; Hung, Sung-Jen; Hsiao, Yu-Ping; Liu, Fu-Tong; Yang, Jen-Hung

    2016-03-01

    AHAs (α-hydroxy acids), including glycolic acid (GA), have been widely used in cosmetic products and superficial chemical peels. Inflammasome complex has been shown to play critical roles in inflammatory pathways in human keratinocytes. However, the anti-inflammatory mechanism of GA is still unknown. The aim of this study is to investigate the relationship between the expression of the inflammasome complex and epigenetic modification to elucidate the molecular mechanism of the anti-inflammatory effect of GA in HaCaT cells. We evaluated NLRP3, NLRC4, AIM2, and ASC inflammasome complex gene expression on real-time polymerase chain reaction (PCR). Methylation changes were detected in these genes following treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza) with or without the addition of GA using methylation-specific PCR (MSP). GA inhibited the expressions of these inflammasome complex genes, and the decreases in the expressions of mRNA were reversed by 5-Aza treatment. Methylation was detected in NLRC4 and ASC on MSP, but not in NLRP3 or AIM2. GA decreased NLRC4 and ASC gene expression by increasing not only DNA methyltransferase 3B (DNMT-3B) protein level, but also total DNMT activity. Furthermore, silencing of DNMT-3B (shDNMT-3B) increased the expressions of NLRC4 and ASC. Our data demonstrated that GA treatment induces hypermethylation of promoters of NLRC4 and ASC genes, which may subsequently lead to the hindering of the assembly of the inflammasome complex in HaCaT cells. These results highlight the anti-inflammatory potential of GA-containing cosmetic agents in human skin cells and demonstrate for the first time the role of aberrant hypermethylation in this process.

  12. Divergent DNA Methylation Patterns Associated with Abiotic Stress in Hevea brasiliensis

    Institute of Scientific and Technical Information of China (English)

    Thomas K. Uthup; Mlnlmol Ravindran; K. Bini; Saha Thakurdas

    2011-01-01

    Cytosine methylation is a fundamental epigenetic mechanism for gene-expression regulation and development in plants.Here,we report for the first time the identification of DNA methylation patterns and their putative relationship with abiotic stress in the tree crop Hevea brasiliensis (source of 99% of natural rubber in the world).Regulatory sequences of four major genes involved in the mevalonate pathway (rubber biosynthesis pathway) and one general defense-related gene of three high-yielding popular rubber clones grown at two different agroclimatic conditions were analyzed for the presence of methylation.We found several significant variations in the methylation pattern at core DNA binding motifs within all the five genes.Several consistent clone-specific and location-specific methylation patterns were identified.The differences in methylation pattern observed at certain pivotal cis-regulatory sites indicate the direct impact of stress on the genome and support the hypothesis of site-specific stress-induced DNA methylation.It is assumed that some of the methylation patterns observed may be involved in the stress-responsive mechanism in plants by which they adapt to extreme conditions.The study also provide clues towards the existence of highly divergent phenotypic characters among Hevea clones despite their very similar genetic make-up.Altogether,the observations from this study prove beyond doubt that there exist epigenetic variations in Hevea and environmental factors play a significant role in the induction of site-specific epigenetic mutations in its genome.

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

    Science.gov (United States)

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

    2016-05-27

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

  14. Genome and Metagenome Sequencing: Using the Human Methyl-Binding Domain to Partition Genomic DNA Derived from Plant Tissues

    Directory of Open Access Journals (Sweden)

    Erbay Yigit

    2014-11-01

    Full Text Available Premise of the study: Variation in the distribution of methylated CpG (methyl-CpG in genomic DNA (gDNA across the tree of life is biologically interesting and useful in genomic studies. We illustrate the use of human methyl-CpG-binding domain (MBD2 to fractionate angiosperm DNA into eukaryotic nuclear (methyl-CpG-rich vs. organellar and prokaryotic (methyl-CpG-poor elements for genomic and metagenomic sequencing projects. Methods: MBD2 has been used to enrich prokaryotic DNA in animal systems. Using gDNA from five model angiosperm species, we apply a similar approach to identify whether MBD2 can fractionate plant gDNA into methyl-CpG-depleted vs. enriched methyl-CpG elements. For each sample, three gDNA libraries were sequenced: (1 untreated gDNA, (2 a methyl-CpG-depleted fraction, and (3 a methyl-CpG-enriched fraction. Results: Relative to untreated gDNA, the methyl-depleted libraries showed a 3.2–11.2-fold and 3.4–11.3-fold increase in chloroplast DNA (cpDNA and mitochondrial DNA (mtDNA, respectively. Methyl-enriched fractions showed a 1.8–31.3-fold and 1.3–29.0-fold decrease in cpDNA and mtDNA, respectively. Discussion: The application of MBD2 enabled fractionation of plant gDNA. The effectiveness was particularly striking for monocot gDNA (Poaceae. When sufficiently effective on a sample, this approach can increase the cost efficiency of sequencing plant genomes as well as prokaryotes living in or on plant tissues.

  15. Methylation analysis of multiple genes in blood DNA of Alzheimer's disease and healthy individuals.

    Science.gov (United States)

    Tannorella, Pierpaola; Stoccoro, Andrea; Tognoni, Gloria; Petrozzi, Lucia; Salluzzo, Maria Grazia; Ragalmuto, Alda; Siciliano, Gabriele; Haslberger, Alexander; Bosco, Paolo; Bonuccelli, Ubaldo; Migliore, Lucia; Coppedè, Fabio

    2015-07-23

    We collected blood DNA from 120 late-onset Alzheimer's disease (AD) patients and 115 healthy matched controls and analysed the methylation levels of genes involved in amyloid-beta peptide production (PSEN1 and BACE1), in DNA methylation (DNMT1, DNMT3A and DNMT3B), and in one-carbon metabolism (MTHFR), searching for correlation with age and gender, with biomarkers of one-carbon metabolism (plasma homocysteine, and serum folate and vitamin B12 levels), and with disease status (being healthy or having AD). We also evaluated the contribution of the APOE ϵ4 allele, the major late-onset AD genetic risk factor, to the studied gene methylation levels. All the genes showed low mean methylation levels (<5%) in both AD and control DNA, no difference between groups, and no correlation with the studied biomarkers, except for MTHFR that showed methylation levels ranging from 5% to 75%, and correlation with circulating biomarkers of one-carbon metabolism. However, mean MTHFR methylation levels were similar between groups (31.1% in AD and 30.7% in controls, P=0.58). Overall, present data suggest that none of the studied regions is differently methylated in blood DNA between AD and control subjects. PMID:26079324

  16. Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements

    Science.gov (United States)

    Liang, Gangning; Chan, Matilda F.; Tomigahara, Yoshitaka; Tsai, Yvonne C.; Gonzales, Felicidad A.; Li, En; Laird, Peter W.; Jones, Peter A.

    2002-01-01

    We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells. PMID:11756544

  17. HP1 Is Involved in Regulating the Global Impact of DNA Methylation on Alternative Splicing

    Directory of Open Access Journals (Sweden)

    Ahuvi Yearim

    2015-02-01

    Full Text Available The global impact of DNA methylation on alternative splicing is largely unknown. Using a genome-wide approach in wild-type and methylation-deficient embryonic stem cells, we found that DNA methylation can either enhance or silence exon recognition and affects the splicing of more than 20% of alternative exons. These exons are characterized by distinct genetic and epigenetic signatures. Alternative splicing regulation of a subset of these exons can be explained by heterochromatin protein 1 (HP1, which silences or enhances exon recognition in a position-dependent manner. We constructed an experimental system using site-specific targeting of a methylated/unmethylated gene and demonstrate a direct causal relationship between DNA methylation and alternative splicing. HP1 regulates this gene’s alternative splicing in a methylation-dependent manner by recruiting splicing factors to its methylated form. Our results demonstrate DNA methylation’s significant global influence on mRNA splicing and identify a specific mechanism of splicing regulation mediated by HP1.

  18. Genomic DNA methylation patterns in bovine preimplantation embryos derived from in vitro fertilization

    Institute of Scientific and Technical Information of China (English)

    HOU Jian; LIU Lei; LEI TingHua; CUI XiuHong; AN XiaoRong; CHEN YongFu

    2007-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplantation embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methylation level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

  19. Genomic DNA methylation patterns in bovine preim-plantation embryos derived from in vitro fertilization

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplanta-tion embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methyla-tion level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

  20. Methylated genes as new cancer biomarkers.

    LENUS (Irish Health Repository)

    Duffy, M J

    2012-02-01

    Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2 for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene methylation need to be standardised, simplified and evaluated in external quality assurance programmes. It is concluded that methylated genes have the potential to provide a new generation of cancer biomarkers.

  1. Methylation plotter: a web tool for dynamic visualization of DNA methylation data.

    Science.gov (United States)

    Mallona, Izaskun; Díez-Villanueva, Anna; Peinado, Miguel A

    2014-01-01

    Methylation plotter is a Web tool that allows the visualization of methylation data in a user-friendly manner and with publication-ready quality. The user is asked to introduce a file containing the methylation status of a genomic region. This file can contain up to 100 samples and 100 CpGs. Optionally, the user can assign a group for each sample (i.e. whether a sample is a tumoral or normal tissue). After the data upload, the tool produces different graphical representations of the results following the most commonly used styles to display this type of data. They include an interactive plot that summarizes the status of every CpG site and for every sample in lollipop or grid styles. Methylation values ranging from 0 (unmethylated) to 1 (fully methylated) are represented using a gray color gradient. A practical feature of the tool allows the user to choose from different types of arrangement of the samples in the display: for instance, sorting by overall methylation level, by group, by unsupervised clustering or just following the order in which data were entered. In addition to the detailed plot, Methylation plotter produces a methylation profile plot that summarizes the status of the scrutinized region, a boxplot that sums up the differences between groups (if any) and a dendrogram that classifies the data by unsupervised clustering. Coupled with this analysis, descriptive statistics and testing for differences at both CpG and group levels are provided. The implementation is based in R/shiny, providing a highly dynamic user interface that generates quality graphics without the need of writing R code. Methylation plotter is freely available at http://gattaca.imppc.org:3838/methylation_plotter/. PMID:25260021

  2. Identification of a quantitative MINT locus methylation profile predicting local regional recurrence of rectal cancer.

    NARCIS (Netherlands)

    Maat, M.F. de; Velde, C.J. van de; Benard, A.; Putter, H.; Morreau, H.; Krieken, J.H.J.M. van; Meershoek Klein-Kranenbarg, E.; Graaf, E.J. de; Tollenaar, R.A.E.M.; Hoon, D.S.

    2010-01-01

    PURPOSE: Risk assessment for locoregional disease recurrence would be highly valuable in preoperative treatment planning for patients undergoing primary rectal tumor resection. Epigenetic aberrations such as DNA methylation have been shown to be significant prognostic biomarkers of disease outcome.

  3. The landscape of DNA methylation amid a perfect storm of autism aetiologies.

    Science.gov (United States)

    Ciernia, Annie Vogel; LaSalle, Janine

    2016-07-01

    Increasing evidence points to a complex interplay between genes and the environment in autism spectrum disorder (ASD), including rare de novo mutations in chromatin genes such as methyl-CpG binding protein 2 (MECP2) in Rett syndrome. Epigenetic mechanisms such as DNA methylation act at this interface, reflecting the plasticity in metabolic and neurodevelopmentally regulated gene pathways. Genome-wide studies of gene sequences, gene pathways and DNA methylation are providing valuable mechanistic insights into ASD. The dynamic developmental landscape of DNA methylation is vulnerable to numerous genetic and environmental insults: therefore, understanding pathways that are central to this 'perfect storm' will be crucial to improving the diagnosis and treatment of ASD. PMID:27150399

  4. More questions than answers about the potential anticancer agents: DNA methylation inhibitors

    Institute of Scientific and Technical Information of China (English)

    ZOU Xiao-ping; ZHANG Bin; LIU Ying

    2010-01-01

    Objective To review the emerging role of DNA methylation inhibitors in cancer therapy and make a serious reflection on their current status and future perspectives. Data sources The data used in the present article were mainly from PubMed with relevant English papers published from April 1988 to January 2010. The search terms were "DNA methylation", "demethylation" and "cancer". Study selection Studies involed in the DNA methylation in carcinogenesis and DNA methylation inhibitors for cancer therapy were selected. The original milestone articles were also included. Results Treatment with DNA methylation inhibitors leads to demethylation of a panel of tumour suppressor genes and reverse the expression in different tumors, thus making them potential cancer therapeutics. However, we cannot be very optimistic about their future perspectives because we still have a long way to go before they function well like specific targeted anticancer drugs as we expected.Conclusion The best way forward is to further clarify the exact methylation profiles of tumors and to develop novel agents targeting the specific genes.

  5. Detection of Epigenetic Modifications During Microspore Embryogenesis: Analysis of DNA Methylation Patterns Dynamics.

    Science.gov (United States)

    Testillano, Pilar S; Risueño, María Carmen

    2016-01-01

    Methylation of 5-deoxy-cytidines of DNA constitutes a prominent epigenetic modification of the chromatin fiber which is locked in a transcriptionally inactive conformation. Changes in global DNA methylation are involved in many plant developmental processes during proliferation and differentiation events. The analysis of the changes of global DNA methylation distribution patterns during microspore embryogenesis induction and progression will inform on the regulatory mechanisms of the process, helping in the design of protocols to improve its efficiency in different species. To investigate the DNA methylation dynamics during microspore embryogenesis in the different cell types present in the cultures, the analysis of spatial and temporal pattern of nuclear distribution of 5-methyl-deoxy-cytidine (5mdC) constitutes a potent approach. The immunolocalization of 5mdC on sections and subsequent confocal laser microscopy analysis have been developed for in situ cellular analysis of a variety of plant samples, including embryogenic microspore and anther cultures. Quantification of 5mdC immunofluorescence intensity by image analysis software also permits to estimate differences in global DNA methylation levels among different cell types during development. PMID:26619883

  6. The role of DNA methylation in directing the functional organization of the cancer epigenome

    Science.gov (United States)

    Lay, Fides D.; Liu, Yaping; Kelly, Theresa K.; Witt, Heather; Farnham, Peggy J.

    2015-01-01

    The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. Here we perform a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation-deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised, and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome-depleted regions, respectively. At higher-order genomic scales, we found that long, H3K9me3-marked domains had lower accessibility, consistent with a more compact chromatin structure. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multiscale organization of cancer epigenomes. PMID:25747664

  7. The association between global DNA methylation and telomere length in a longitudinal study of boilermakers.

    Science.gov (United States)

    Wong, Jason Y Y; De Vivo, Immaculata; Lin, Xihong; Grashow, Rachel; Cavallari, Jennifer; Christiani, David C

    2014-04-01

    The objectives of this study were to determine if global DNA methylation, as reflected in LINE-1 and Alu elements, is associated with telomere length and whether it modifies the rate of telomeric change. A repeated-measures longitudinal study was performed with a panel of 87 boilermaker subjects. The follow-up period was 29 months. LINE-1 and Alu methylation was determined using pyrosequencing. Leukocyte relative telomere length was assessed via real-time qPCR. Linear-mixed models were used to estimate the association between DNA methylation and telomere length. A structural equation model (SEM) was used to explore the hypothesized relationship between DNA methylation, proxies of particulate matter exposure, and telomere length at baseline. There appeared to be a positive association between both LINE-1 and Alu methylation levels, and telomere length. For every incremental increase in LINE-1 methylation, there was a statistically significant 1.0 × 10(-1) (95% CI: 4.6 × 10(-2), 1.5 × 10(-1), P telomere length, controlling for age at baseline, current and past smoking status, work history, BMI (log kg/m(2) ) and leukocyte differentials. Furthermore, for every incremental increase in Alu methylation, there was a statistically significant 6.2 × 10(-2) (95% CI: 1.0 × 10(-2), 1.1 × 10(-1), P = 0.02) unit increase in relative telomere length. The interaction between LINE-1 methylation and follow-up time was statistically significant with an estimate -9.8 × 10(-3) (95% CI: -1.8 × 10(-2), -1.9 × 10(-3), P = 0.02); suggesting that the rate of telomeric change was modified by the degree of LINE-1 methylation. No statistically significant association was found between the cumulative PM exposure construct, with global DNA methylation and telomere length at baseline.

  8. DNA methylation profiles delineate etiologic heterogeneity and clinically important subgroups of bladder cancer.

    Science.gov (United States)

    Wilhelm-Benartzi, C S; Koestler, D C; Houseman, E A; Christensen, B C; Wiencke, John K; Schned, A R; Karagas, M R; Kelsey, K T; Marsit, C J

    2010-11-01

    DNA methylation profiles can be used to define molecular cancer subtypes that may better inform disease etiology and clinical decision-making. This investigation aimed to create DNA methylation profiles of bladder cancer based on CpG methylation from almost 800 cancer-related genes and to then examine the relationship of those profiles with exposures related to risk and clinical characteristics. DNA, derived from formalin-fixed paraffin-embedded tumor samples obtained from incident cases involved in a population-based case-control study of bladder cancer in New Hampshire, was used for methylation profiling on the Illumina GoldenGate Methylation Bead Array. Unsupervised clustering of those loci with the greatest change in methylation between tumor and non-diseased tissue was performed to defined molecular subgroups of disease, and univariate tests of association followed by multinomial logistic regression was used to examine the association between these classes, bladder cancer risk factors and clinical phenotypes. Membership in the two most methylated classes was significantly associated with invasive disease (P class 3 and 4). Male gender (P = 0.04) and age >70 years (P = 0.05) was associated with membership in one of the most methylated classes. Finally, average water arsenic levels in the highest percentile predicted membership in an intermediately methylated class of tumors (P = 0.02 for both classes). Exposures and demographic associated with increased risk of bladder cancer specifically associate with particular subgroups of tumors defined by DNA methylation profiling and these subgroups may define more aggressive disease.

  9. DNA Methylation is Associated with an Increased Level of Conservation at Nondegenerate Nucleotides in Mammals

    Science.gov (United States)

    Chuang, Trees-Juen; Chen, Feng-Chi

    2014-01-01

    DNA methylation at CpG dinucleotides can significantly increase the rate of cytosine-to-thymine mutations and the level of sequence divergence. Although the correlations between DNA methylation and genomic sequence evolution have been widely studied, an unaddressed yet fundamental question is how DNA methylation is associated with the conservation of individual nucleotides in different sequence contexts. Here, we demonstrate that in mammalian exons, the correlations between DNA methylation and the conservation of individual nucleotides are dependent on the type of exonic sequence (coding or untranslated), the degeneracy of coding nucleotides, background selection pressure, and the relative position (first or nonfirst exon in the transcript) where the nucleotides are located. For untranslated and nonzero-fold degenerate nucleotides, methylated sites are less conserved than unmethylated sites regardless of background selection pressure and the relative position of the exon. For zero-fold degenerate (or nondegenerate) nucleotides, however, the reverse trend is observed in nonfirst coding exons and first coding exons that are under stringent background selection pressure. Furthermore, cytosine-to-thymine mutations at methylated zero-fold degenerate nucleotides are predicted to be more detrimental than those that occur at unmethylated nucleotides. As zero-fold and nonzero-fold degenerate nucleotides are very close to each other, our results suggest that the “functional resolution” of DNA methylation may be finer than previously recognized. In addition, the positive correlation between CpG methylation and the level of conservation at zero-fold degenerate nucleotides implies that CpG methylation may serve as an “indicator” of functional importance of these nucleotides. PMID:24157417

  10. Association of postmenopausal endogenous sex hormones with global methylation level of leukocyte DNA among Japanese women

    Directory of Open Access Journals (Sweden)

    Iwasaki Motoki

    2012-07-01

    Full Text Available Abstract Background Although global hypomethylation of leukocyte DNA has been associated with an increased risk of several sites of cancer, including breast cancer, determinants of global methylation level among healthy individuals remain largely unexplored. Here, we examined whether postmenopausal endogenous sex hormones were associated with the global methylation level of leukocyte DNA. Methods A cross-sectional study was conducted using the control group of a breast cancer case–control study in Nagano, Japan. Subjects were postmenopausal women aged 55 years or over who provided blood samples. We measured global methylation level of peripheral blood leukocyte DNA by luminometric methylation assay; estradiol, estrone, androstenedione, dehydroepiandrosterone sulfate, testosterone and free testosterone by radioimmunoassay; bioavailable estradiol by the ammonium sulfate precipitation method; and sex-hormone binding globulin by immunoradiometric assay. A linear trend of association between methylation and hormone levels was evaluated by regression coefficients in a multivariable liner regression model. A total of 185 women were included in the analyses. Results Mean global methylation level (standard deviation was 70.3% (3.1 and range was from 60.3% to 79.2%. Global methylation level decreased 0.27% per quartile category for estradiol and 0.39% per quartile category for estrone while it increased 0.41% per quartile category for bioavailable estradiol. However, we found no statistically significant association of any sex hormone level measured in the present study with global methylation level of leukocyte DNA. Conclusions Our findings suggest that endogenous sex hormones are not major determinants of the global methylation level of leukocyte DNA.

  11. An integrative analysis of DNA methylation and RNA-Seq data for human heart, kidney and liver

    Directory of Open Access Journals (Sweden)

    Xie Linglin

    2011-12-01

    Full Text Available Abstract Background Many groups, including our own, have proposed the use of DNA methylation profiles as biomarkers for various disease states. While much research has been done identifying DNA methylation signatures in cancer vs. normal etc., we still lack sufficient knowledge of the role that differential methylation plays during normal cellular differentiation and tissue specification. We also need thorough, genome level studies to determine the meaning of methylation of individual CpG dinucleotides in terms of gene expression. Results In this study, we have used (insert statistical method here to compile unique DNA methylation signatures from normal human heart, lung, and kidney using the Illumina Infinium 27 K methylation arraysand compared those to gene expression by RNA sequencing. We have identified unique signatures of global DNA methylation for human heart, kidney and liver, and showed that DNA methylation data can be used to correctly classify various tissues. It indicates that DNA methylation reflects tissue specificity and may play an important role in tissue differentiation. The integrative analysis of methylation and RNA-Seq data showed that gene methylation and its transcriptional levels were comprehensively correlated. The location of methylation markers in terms of distance to transcription start site and CpG island showed no effects on the regulation of gene expression by DNA methylation in normal tissues. Conclusions This study showed that an integrative analysis of methylation array and RNA-Seq data can be utilized to discover the global regulation of gene expression by DNA methylation and suggests that DNA methylation plays an important role in normal tissue differentiation via modulation of gene expression.

  12. DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.

    Science.gov (United States)

    Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng

    2015-03-01

    DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. PMID:25461675

  13. DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance

    Institute of Scientific and Technical Information of China (English)

    Jie-hong ZHAO; Ji-shun ZHANG; Yi WANG; Ren-gang WANG; Chun WU; Long-jiang FAN; Xue-liang REN

    2011-01-01

    DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth,development,and polyploidization.However,there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics.We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco,Nicotiana tabacum,using a methylation-sensitive amplified polymorphism (MSAP) technique.The results showed that methylation existed at a high level among tobacco accessions,among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic.A cluster analysis revealed distinct patterns of geography-specific groups.In addition,three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored.This suggests that tobacco breeders should pay more attention to epigenetic traits.

  14. DNA methylation analysis using CpG microarrays is impaired in benzopyrene exposed cells

    International Nuclear Information System (INIS)

    Epigenetic alterations have emerged as a key mechanism involved in tumorigenesis. These disruptions are partly due to environmental factors that change normal DNA methylation patterns necessary for transcriptional regulation and chromatin compaction. Microarray technologies are allowing environmentally susceptible epigenetic patterns to be mapped and the precise targets of environmentally induced alterations to be identified. Previously, we observed BaP-induced epigenetic events and cell cycle disruptions in breast cancer cell lines that included time- and concentration-dependent loss of proliferation as well as sequence-specific hypo- and hypermethylation events. In this present report, we further characterized epigenetic changes in BaP-exposed MCF-7 cells. We analyzed DNA methylation on a CpG island microarray platform with over 5400 unique genomic regions. Depleted and enriched microarray targets, representative of putative DNA methylation changes, were identified across the genome; however, subsequent sodium bisulfite analyses revealed no changes in DNA methylation at a number of these loci. Instead, we found that the identification of DNA methylation changes using this restriction enzyme-based microarray approach corresponded with the regions of DNA bound by the BaP derived DNA adducts. This DNA adduct formation occurs at both methylated and unmethylated CpG dinucleotides and affects PCR amplification during sample preparation. Our data suggest that caution should be exercised when interpreting data from comparative microarray experiments that rely on enzymatic reactions. These results are relevant to genome screening approaches involving environmental exposures in which DNA adduct formation at specific nucleotide sites may bias target acquisition and compromise the correct identification of epigenetically responsive genes

  15. Novel insights into DNA methylation features in spermatozoa: stability and peculiarities.

    Directory of Open Access Journals (Sweden)

    Csilla Krausz

    Full Text Available Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic and cancer cells. Our study, based on the largest number of subjects (n = 8 ever considered for such a large number of CpGs (n = 487,517, provided clear evidence for i a highly conserved DNA methylation profile among normozoospermic subjects; ii a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development

  16. Methylation plotter: a web tool for dynamic visualization of DNA methylation data

    OpenAIRE

    Mallona, Izaskun; Díez-Villanueva, Anna; Peinado, Miguel A

    2014-01-01

    Methylation plotter is a Web tool that allows the visualization of methylation data in a user-friendly manner and with publication-ready quality. The user is asked to introduce a file containing the methylation status of a genomic region. This file can contain up to 100 samples and 100 CpGs. Optionally, the user can assign a group for each sample (i.e. whether a sample is a tumoral or normal tissue). After the data upload, the tool produces different graphical representations of the results f...

  17. Longitudinal Analysis of DNA Methylation in CD34+ Hematopoietic Progenitors in Myelodysplastic Syndrome

    DEFF Research Database (Denmark)

    Wong, Yan Fung; Micklem, Chris N; Taguchi, Masataka;

    2014-01-01

    Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5-azacytidine [AZA], 5-aza-2'-deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease pr...

  18. DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Nordlund, Jessica; Bäcklin, Christofer L; Zachariadis, Vasilios;

    2015-01-01

    BACKGROUND: We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA...

  19. Epigenetic Vestiges of Early Developmental Adversity: Childhood Stress Exposure and DNA Methylation in Adolescence

    Science.gov (United States)

    Essex, Marilyn J.; Boyce, W. Thomas; Hertzman, Clyde; Lam, Lucia L.; Armstrong, Jeffrey M.; Neumann, Sarah M. A.; Kobor, Michael S.

    2013-01-01

    Fifteen-year-old adolescents (N = 109) in a longitudinal study of child development were recruited to examine differences in DNA methylation in relation to parent reports of adversity during the adolescents' infancy and preschool periods. Microarray technology applied to 28,000 cytosine-guanine dinucleotide sites within DNA derived from buccal…

  20. Global Methylation and Hydroxymethylation in DNA from Blood and Saliva in Healthy Volunteers.

    Science.gov (United States)

    Godderis, Lode; Schouteden, Caroline; Tabish, Ali; Poels, Katrien; Hoet, Peter; Baccarelli, Andrea A; Van Landuyt, Kirsten

    2015-01-01

    Aims. We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify and compare simultaneously global methylation and hydroxymethylation in human DNA of different tissues. Materials and Methods. Blood and saliva DNA from fourteen volunteers was processed for epigenetic endpoints using LC-MS/MS and PCR-pyrosequencing technology. Results. Global DNA methylation was significantly lower in saliva (mean 4.61% ±  0.80%), compared to blood samples (5.70% ± 0.22%). In contrast, saliva (0.036% ± 0.011%) revealed significantly higher hydroxymethylation compared to blood samples (mean 0.027% ± 0.004%). Whereas we did not find significant correlations for both epigenetic measures between the tissues, a significant association was observed between global methylation and global hydroxymethylation in saliva DNA. Neither LINE-1 nor Alu elements of blood and saliva correlated, nor were they correlated with the DNA hydroxymethylation of blood or saliva, respectively. Conclusion. Global DNA methylation and hydroxymethylation of cytosine can be quantified simultaneously by LC-MS/MS. Saliva DNA cannot be considered as a surrogate for blood DNA to study epigenetic endpoints.

  1. Vitamin and antioxidant rich diet increases MLH1 promoter DNA methylation in DMT2 subjects

    Directory of Open Access Journals (Sweden)

    Switzeny Olivier J

    2012-10-01

    Full Text Available Abstract Background Oxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1 gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS, we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2 and impaired fasting glucose (IFG. Methods In this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student’s two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount. Results The intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate. Conclusion This vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a

  2. Dynamics of DNA methylation and Histone H4 acetylation during floral bud differentiation in azalea

    Directory of Open Access Journals (Sweden)

    Valledor Luis

    2010-01-01

    Full Text Available Abstract Background The ability to control the timing of flowering is a key strategy for planning production in ornamental species such as azalea, however it requires a thorough understanding of floral transition. Floral transition is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Dynamic changes between chromatin states facilitating or inhibiting DNA transcription regulate the expression of floral induction pathways in response to environmental and developmental signals. DNA methylation and histone modifications are involved in controlling the functional state of chromatin and gene expression. Results The results of this work indicate that epigenetic mechanisms such as DNA methylation and histone H4 acetylation have opposite and particular dynamics during the transition from vegetative to reproductive development in the apical shoots of azalea. Global levels of DNA methylation and histone H4 acetylation as well as immunodetection of 5-mdC and acetylated H4, in addition to a morphological study have permitted the delimitation of four basic phases in the development of the azalea bud and allowed the identification of a stage of epigenetic reprogramming which showed a sharp decrease of whole DNA methylation similar to that is defined in other developmental processes in plants and in mammals. Conclusion The epigenetic control and reorganization of chromatin seem to be decisive for coordinating floral development in azalea. DNA methylation and H4 deacetylation act simultaneously and co-ordinately, restructuring the chromatin and regulating the gene expression during soot apical meristem development and floral differentiation.

  3. Whole-Genome Saliva and Blood DNA Methylation Profiling in Individuals with a Respiratory Allergy.

    Science.gov (United States)

    Langie, Sabine A S; Szarc Vel Szic, Katarzyna; Declerck, Ken; Traen, Sophie; Koppen, Gudrun; Van Camp, Guy; Schoeters, Greet; Vanden Berghe, Wim; De Boever, Patrick

    2016-01-01

    The etiology of respiratory allergies (RA) can be partly explained by DNA methylation changes caused by adverse environmental and lifestyle factors experienced early in life. Longitudinal, prospective studies can aid in the unravelment of the epigenetic mechanisms involved in the disease development. High compliance rates can be expected in these studies when data is collected using non-invasive and convenient procedures. Saliva is an attractive biofluid to analyze changes in DNA methylation patterns. We investigated in a pilot study the differential methylation in saliva of RA (n = 5) compared to healthy controls (n = 5) using the Illumina Methylation 450K BeadChip platform. We evaluated the results against the results obtained in mononuclear blood cells from the same individuals. Differences in methylation patterns from saliva and mononuclear blood cells were clearly distinguishable (PAdj0.2), though the methylation status of about 96% of the cg-sites was comparable between peripheral blood mononuclear cells and saliva. When comparing RA cases with healthy controls, the number of differentially methylated sites (DMS) in saliva and blood were 485 and 437 (P0.1), respectively, of which 216 were in common. The methylation levels of these sites were significantly correlated between blood and saliva. The absolute levels of methylation in blood and saliva were confirmed for 3 selected DMS in the PM20D1, STK32C, and FGFR2 genes using pyrosequencing analysis. The differential methylation could only be confirmed for DMS in PM20D1 and STK32C genes in saliva. We show that saliva can be used for genome-wide methylation analysis and that it is possible to identify DMS when comparing RA cases and healthy controls. The results were replicated in blood cells of the same individuals and confirmed by pyrosequencing analysis. This study provides proof-of-concept for the applicability of saliva-based whole-genome methylation analysis in the field of respiratory allergy.

  4. Identification of GABRA1 and LAMA2 as new DNA methylation markers in colorectal cancer.

    Science.gov (United States)

    Lee, Sunwoo; Oh, Taejeong; Chung, Hyuncheol; Rha, Sunyoung; Kim, Changjin; Moon, Youngho; Hoehn, Benjamin D; Jeong, Dongjun; Lee, Seunghoon; Kim, Namkyu; Park, Chanhee; Yoo, Miae; An, Sungwhan

    2012-03-01

    Aberrant methylation of CpG islands in the promoter region of genes is a common epigenetic phenomenon found in early cancers. Therefore conducting genome-scale methylation studies will enhance our understanding of the epigenetic etiology behind carcinogenesis by providing reliable biomarkers for early detection of cancer. To discover novel hypermethylated genes in colorectal cancer by genome-wide search, we first defined a subset of genes epigenetically reactivated in colon cancer cells after treatment with a demethylating agent. Next, we identified another subset of genes with relatively down-regulated expression patterns in colorectal primary tumors when compared with normal appearing-adjacent regions. Among 29 genes obtained by cross-comparison of the two gene-sets, we subsequently selected, through stepwise subtraction processes, two novel genes, GABRA1 and LAMA2, as methylation targets in colorectal cancer. For clinical validation pyrosequencing was used to assess methylation in 134 matched tissue samples from CRC patients. Aberrant methylation at target CpG sites in GABRA1 and LAMA2 was observed with high frequency in tumor tissues (92.5% and 80.6%, respectively), while less frequently in matched tumor-adjacent normal tissues (33.6% for GABRA1 and 13.4% for LAMA2). Methylation levels in primary tumors were not significantly correlated with clinico-pathological features including age, sex, survival and TNM stage. Additionally, we found that ectopic overexpression of GABRA1 in colon cancer cell lines resulted in strong inhibition of cell growth. These results suggest that two novel hypermethylated genes in colorectal cancer, GABRA1 and LAMA2, may have roles in colorectal tumorigenesis and could be potential biomarkers for the screening and the detection of colorectal cancer in clinical practice. PMID:22038115

  5. Identification of tissue-specific cell death using methylation patterns of circulating DNA

    Science.gov (United States)

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J.; Wasserfall, Clive H.; Schatz, Desmond A.; Greenbaum, Carla J.; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K.; Golan, Talia; Ben Sasson, Shmuel A.; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A. M. James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-01-01

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

  6. Identification of tissue-specific cell death using methylation patterns of circulating DNA.

    Science.gov (United States)

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J; Wasserfall, Clive H; Schatz, Desmond A; Greenbaum, Carla J; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K; Golan, Talia; Ben Sasson, Shmuel A; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A M James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-03-29

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

  7. Methylation pattern of the O6-methylguanine-DNA methyltransferase gene in colon during progressive colorectal tumorigenesis

    OpenAIRE

    NAGASAKA, Takeshi; Goel, Ajay; Notohara, Kenji; Takahata, Takaomi; Sasamoto, Hiromi; Uchida, Takuyuki; Nishida, Naoshi; Tanaka, Noriaki; Boland, Clement Richard; Matsubara, Nagahide

    2008-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair gene which is frequently methylated in colorectal cancer (CRC). However, it remains controversial whether methylation of specific CpG sequences within MGMT promoter leads to loss of its protein expression, and if MGMT methylation correlates with G to A transition mutations in KRAS. Two methylation sensitive regions (Mp and Eh region) of MGMT promoter were investigated in 593 specimens of colorectal tissue: 233 CRCs, 104 adenomatous...

  8. NLRP3 Activation Was Regulated by DNA Methylation Modification during Mycobacterium tuberculosis Infection

    Science.gov (United States)

    Wei, Meili; Wang, Lu; Wu, Tao; Xi, Jun; Han, Yuze; Yang, Xingxiang; Zhang, Ding; Fang, Qiang

    2016-01-01

    Mycobacterium tuberculosis (Mtb) infection activates the NLRP3 inflammasome in macrophages and dendritic cells. Much attention has been paid to the mechanisms for regulation of NLRP3 against Mtb. However, whether epigenetic mechanisms participated in NLRP3 activation is still little known. Here we showed that NLRP3 activation was regulated by DNA methylation modification. Mtb infection promoted NLRP3 activation and inflammatory cytokines expression. NLRP3 promoter was cloned and subsequently identified by Dual-Luciferase Reporter System. The results showed that NLRP3 promoter activity was decreased after methylation by DNA methylase Sss I in vitro. Meanwhile, DNA methyltransferases inhibitor DAC could upregulate the expression of NLRP3. Furthermore, promoter region of NLRP3 gene was demethylated after Mtb H37Rv strain infection. These data revealed that DNA methylation was involved in NLRP3 inflammasome activation during Mtb infection and provided a new insight into the relationship between host and pathogens. PMID:27366746

  9. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation

    DEFF Research Database (Denmark)

    Liu, Si-Yang; Lin, Jian-Qing; Wu, Hong-Long;

    2012-01-01

    data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase......Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status......, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing...

  10. DNA methylation and tumor%DNA甲基化与肿瘤

    Institute of Scientific and Technical Information of China (English)

    王震凯

    2011-01-01

    DNA methylaion, one of the earliest found modification ways control the gene expression through changing chromatin structer,DNA conformation, DNA stability and DNA-protein interaction. The changes of DNA methylation status are discovered in tumor. Therefore ,the role of DNA methylation in the tumor' s occurrence and develapment is becoming a hot spot of study in current.%DNA甲基化是最早发现的基因修饰途径之一,可引起染色质结构、DNA构象、DNA稳定性及DNA与蛋白质相互作用方式的改变,控制基因表达.肿瘤中普遍存在DNA甲基化状态的改变,DNA甲基化在肿瘤的发生发展中的作用是当前的研究热点.

  11. Blood DNA methylation markers in potentially identified Chinese patients with hepatocellular carcinoma.

    Science.gov (United States)

    Liu, Zongying; Yan, Haixiu; Zhang, Jinshu

    2016-07-01

    To determine whether blood DNA methylation is associated with hepatocellular carcinoma (HCC) for Chinese patients, we used genome-wide DNA methylation detection to access the blood samples of Chinese patients by Illumina Human methylation 450K arrays. Sixty potentially gene locis which had different methylated levels significantly among tumor and adjacent normal tissues would be tested in this study. A previous study was conducted in China communities and followed with 7 years. The DNA from white blood cells (WBC) from 192 patients with HCC and 215 matched controls were assayed in this study. The χ2 test was used to measure data to categorize variables and t -test was used to evaluate the different characteristics among groups. Besides, odds ratios (OR) and 95%CI was calculated for matching factors by conditional logistic regression models. We found that high methylation in WNK2 was related to increased risk of HCC, and high methylation in TPO were related to decreased risk of HCC. In our multivariable conditional logistic regression models, these results all exist. Those findings support the methylated changes of WNK2 and TPO may become a new detection index for HCC patients in clinical laboratory. However, the results should be replicated in additional prospective studies with lager samples. PMID:27592479

  12. DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

    Directory of Open Access Journals (Sweden)

    Gaëlle Demarre

    2010-05-01

    Full Text Available DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

  13. Epigenome-Wide Association Analysis Identified Nine Skin DNA Methylation Loci for Psoriasis.

    Science.gov (United States)

    Zhou, Fusheng; Wang, Wenjun; Shen, Changbing; Li, Hui; Zuo, Xianbo; Zheng, Xiaodong; Yue, Min; Zhang, Cuicui; Yu, Liang; Chen, Mengyun; Zhu, Caihong; Yin, Xianyong; Tang, Mingjun; Li, Yongjiang; Chen, Gang; Wang, Zaixing; Liu, Shengxiu; Zhou, Yi; Zhang, Fengyu; Zhang, Weijia; Li, Caihua; Yang, Sen; Sun, Liangdan; Zhang, Xuejun

    2016-04-01

    Psoriasis is a chronic hyperproliferative and inflammatory skin disease caused by the interplay of genetic and environmental factors. DNA methylation has been linked to psoriasis, but the manner in which this process contributes to the disease is not fully understood. In this study, we carried out a three-stage epigenome-wide association study to identify disease-associated differentially methylated sites using a combination of 262 skin and 48 peripheral blood mononuclear cell samples. We not only revealed genome-wide methylation patterns for psoriasis but also identified strong associations between the skin-specific DNA methylation of nine disease-associated differentially methylated sites and psoriasis (Wilcoxon ranked PBonferroni 0.10). Further analysis revealed that these nine disease-associated differentially methylated sites were not significantly affected by genetic variations, supporting their remarkable contributions to disease status. The expression of CYP2S1, ECE1, EIF2C2, MAN1C1, and DLGAP4 was negatively correlated with DNA methylation. These findings will help us to better understand the molecular mechanism of psoriasis. PMID:26743604

  14. Epigenome-Wide Association Analysis Identified Nine Skin DNA Methylation Loci for Psoriasis.

    Science.gov (United States)

    Zhou, Fusheng; Wang, Wenjun; Shen, Changbing; Li, Hui; Zuo, Xianbo; Zheng, Xiaodong; Yue, Min; Zhang, Cuicui; Yu, Liang; Chen, Mengyun; Zhu, Caihong; Yin, Xianyong; Tang, Mingjun; Li, Yongjiang; Chen, Gang; Wang, Zaixing; Liu, Shengxiu; Zhou, Yi; Zhang, Fengyu; Zhang, Weijia; Li, Caihua; Yang, Sen; Sun, Liangdan; Zhang, Xuejun

    2016-04-01

    Psoriasis is a chronic hyperproliferative and inflammatory skin disease caused by the interplay of genetic and environmental factors. DNA methylation has been linked to psoriasis, but the manner in which this process contributes to the disease is not fully understood. In this study, we carried out a three-stage epigenome-wide association study to identify disease-associated differentially methylated sites using a combination of 262 skin and 48 peripheral blood mononuclear cell samples. We not only revealed genome-wide methylation patterns for psoriasis but also identified strong associations between the skin-specific DNA methylation of nine disease-associated differentially methylated sites and psoriasis (Wilcoxon ranked PBonferroni 0.10). Further analysis revealed that these nine disease-associated differentially methylated sites were not significantly affected by genetic variations, supporting their remarkable contributions to disease status. The expression of CYP2S1, ECE1, EIF2C2, MAN1C1, and DLGAP4 was negatively correlated with DNA methylation. These findings will help us to better understand the molecular mechanism of psoriasis.

  15. HIF3A DNA Methylation Is Associated with Childhood Obesity and ALT.

    Directory of Open Access Journals (Sweden)

    Shuo Wang

    Full Text Available Gene polymorphisms associated so far with body mass index (BMI can explain only 1.18-1.45% of observed variation in BMI. Recent studies suggest that epigenetic modifications, especially DNA methylation, could contribute to explain part of the missing heritability, and two epigenetic genome-wide analysis studies (EWAS have reported that Hypoxia Inducible Factor 3 Alpha Subunit (HIF3A methylation was associated with BMI or BMI change. We therefore assessed whether the HIF3A methylation is associated with obesity and other obesity-related phenotypes in Chinese children. The subjects included 110 severe obese cases aged 7-17y and 110 normal-weight controls matched by age and gender for measurement of blood DNA methylation levels at the HIF3A gene locus using the Sequenom's MassARRAY system. We observed significantly higher methylation levels in obese children than in controls at positions 46801642 and 46801699 in HIF3A gene (P<0.05, and found positive associations between methylation and alanine aminotransferase (ALT levels adjusted by gender, age and BMI at the position 46801699 (r = 0.226, P = 0.007. These results suggest that HIF3A DNA methylation is associated with childhood obesity, and has a BMI-independent association with ALT. The results provide evidence for identifying epigenetic factors of elivated ALT and may be useful for risk assessment and personalized medicine of liver diseases such as non-alcoholic fatty liver disease (NAFLD.

  16. DNA methylation profiles at precancerous stages associated with recurrence of lung adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Takashi Sato

    Full Text Available The aim of this study was to clarify the significance of DNA methylation alterations at precancerous stages of lung adenocarcinoma. Using single-CpG resolution Infinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue obtained from patients without any primary lung tumor, 145 samples of non-cancerous lung tissue (N obtained