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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...... into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential...

  4. Mechanisms for the induction of gastric cancer by Helicobacter pylori infection: aberrant DNA methylation pathway.

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    Maeda, Masahiro; Moro, Hiroshi; Ushijima, Toshikazu

    2017-03-01

    Multiple pathogenic mechanisms by which Helicobacter pylori infection induces gastric cancer have been established in the last two decades. In particular, aberrant DNA methylation is induced in multiple driver genes, which inactivates them. Methylation profiles in gastric cancer are associated with specific subtypes, such as microsatellite instability. Recent comprehensive and integrated analyses showed that many cancer-related pathways are more frequently altered by aberrant DNA methylation than by mutations. Aberrant DNA methylation can even be present in noncancerous gastric mucosae, producing an "epigenetic field for cancerization." Mechanistically, H. pylori-induced chronic inflammation, but not H. pylori itself, plays a direct role in the induction of aberrant DNA methylation. The expression of three inflammation-related genes, Il1b, Nos2, and Tnf, is highly associated with the induction of aberrant DNA methylation. Importantly, the degree of accumulated aberrant DNA methylation is strongly correlated with gastric cancer risk. A recent multicenter prospective cohort study demonstrated the utility of epigenetic cancer risk diagnosis for metachronous gastric cancer. Suppression of aberrant DNA methylation by a demethylating agent was shown to inhibit gastric cancer development in an animal model. Induction of aberrant DNA methylation is the major pathway by which H. pylori infection induces gastric cancer, and this can be utilized for translational opportunities.

  5. Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model.

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    Borinstein, Scott C; Conerly, Melissa; Dzieciatkowski, Slavomir; Biswas, Swati; Washington, M Kay; Trobridge, Patty; Henikoff, Steve; Grady, William M

    2010-01-01

    Mouse models of intestinal tumors have advanced our understanding of the role of gene mutations in colorectal malignancy. However, the utility of these systems for studying the role of epigenetic alterations in intestinal neoplasms remains to be defined. Consequently, we assessed the role of aberrant DNA methylation in the azoxymethane (AOM) rodent model of colon cancer. AOM induced tumors display global DNA hypomethylation, which is similar to human colorectal cancer. We next assessed the methylation status of a panel of candidate genes previously shown to be aberrantly methylated in human cancer or in mouse models of malignant neoplasms. This analysis revealed different patterns of DNA methylation that were gene specific. Zik1 and Gja9 demonstrated cancer-specific aberrant DNA methylation, whereas, Cdkn2a/p16, Igfbp3, Mgmt, Id4, and Cxcr4 were methylated in both the AOM tumors and normal colon mucosa. No aberrant methylation of Dapk1 or Mlt1 was detected in the neoplasms, but normal colon mucosa samples displayed methylation of these genes. Finally, p19(Arf), Tslc1, Hltf, and Mlh1 were unmethylated in both the AOM tumors and normal colon mucosa. Thus, aberrant DNA methylation does occur in AOM tumors, although the frequency of aberrantly methylated genes appears to be less common than in human colorectal cancer. Additional studies are necessary to further characterize the patterns of aberrantly methylated genes in AOM tumors.

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

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    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.

  7. 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.

  8. 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.

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

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    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.

  10. 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.

  11. 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.

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

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    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.

  13. A DNA methylation signature associated with aberrant promoter DNA hypermethylation of DNMT3B in human colorectal cancer.

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    Huidobro, Covadonga; Urdinguio, Rocío G; Rodríguez, Ramón María; Mangas, Cristina; Calvanese, Vincenzo; Martínez-Camblor, Pablo; Ferrero, Cecilia; Parra-Blanco, Adolfo; Rodrigo, Luis; Obaya, Alvaro J; Suárez-Fernández, Laura; Astudillo, Aurora; Hernando, Henar; Ballestar, Esteban; Fernández, Agustín F; Fraga, Mario F

    2012-09-01

    Altered promoter DNA methylation, one of the most important molecular alterations in cancer, is proposed to correlate with deregulation of DNA methyltransferases, although the molecular mechanisms implicated are still poorly understood. Here we show that the de novo DNA methyltransferase DNMT3B is frequently repressed in human colorectal cancer cell lines (CCL) and primary tumours by aberrant DNA hypermethylation of its distal promoter. At the epigenome level, DNMT3B promoter hypermethylation was associated with the hypomethylation of gene promoters usually hypermethylated in the healthy colon. Forced DNMT3B overexpression in cancer cells restored the methylation levels of these promoters in the healthy colon. Our results show a new molecular mechanism of aberrant DNMT3B regulation in colon cancer and suggest that its expression is associated with the methylation of constitutively hypermethylated promoters in the healthy colon.

  14. Aberrant DNA Methylation Is Associated with a Poor Outcome in Juvenile Myelomonocytic Leukemia.

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    Hirotoshi Sakaguchi

    Full Text Available Juvenile myelomonocytic leukemia (JMML, an overlap of myelodysplastic / myeloproliferative neoplasm, is an intractable pediatric myeloid neoplasm. Epigenetic regulation of transcription, particularly by CpG methylation, plays an important role in tumor progression, mainly by repressing tumor-suppressor genes. To clarify the clinical importance of aberrant DNA methylation, we studied the hypermethylation status of 16 target genes in the genomes of 92 patients with JMML by bisulfite conversion and the pryosequencing technique. Among 16 candidate genes, BMP4, CALCA, CDKN2A, and RARB exhibited significant hypermethylation in 72% (67/92 of patients. Based on the number of hypermethylated genes, patients were stratified into three cohorts based on an aberrant methylation score (AMS of 0, 1-2, or 3-4. In the AMS 0 cohort, the 5-year overall survival (OS and transplantation-free survival (TFS were good (69% and 76%, respectively. In the AMS 1-2 cohort, the 5-year OS was comparable to that in the AMS 0 cohort (68%, whereas TFS was poor (6%. In the AMS 3-4 cohort, 5-year OS and TFS were markedly low (8% and 0%, respectively. Epigenetic analysis provides helpful information for clinicians to select treatment strategies for patients with JMML. For patients with AMS 3-4 in whom hematopoietic stem cell transplantation does not improve the prognosis, alternative therapies, including DNA methyltransferase inhibitors and new molecular-targeting agents, should be established as treatment options.

  15. ∆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.

  16. Aberrant DNA methylation profiles in the premature aging disorders Hutchinson-Gilford Progeria and Werner syndrome

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    Heyn, Holger; Moran, Sebastian; Esteller, Manel

    2013-01-01

    DNA methylation gradiently changes with age and is likely to be involved in aging-related processes with subsequent phenotype changes and increased susceptibility to certain diseases. The Hutchinson-Gilford Progeria (HGP) and Werner Syndrome (WS) are two premature aging diseases showing features of common natural aging early in life. Mutations in the LMNA and WRN genes were associated to disease onset; however, for a subset of patients the underlying causative mechanisms remain elusive. We aimed to evaluate the role of epigenetic alteration on premature aging diseases by performing comprehensive DNA methylation profiling of HGP and WS patients. We observed profound changes in the DNA methylation landscapes of WRN and LMNA mutant patients, which were narrowed down to a set of aging related genes and processes. Although of low overall variance, non-mutant patients revealed differential DNA methylation at distinct loci. Hence, we propose DNA methylation to have an impact on premature aging diseases. PMID:23257959

  17. Aberrant DNA methylation profiles in the premature aging disorders Hutchinson-Gilford Progeria and Werner syndrome.

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    Heyn, Holger; Moran, Sebastian; Esteller, Manel

    2013-01-01

    DNA methylation gradiently changes with age and is likely to be involved in aging-related processes with subsequent phenotype changes and increased susceptibility to certain diseases. The Hutchinson-Gilford Progeria (HGP) and Werner Syndrome (WS) are two premature aging diseases showing features of common natural aging early in life. Mutations in the LMNA and WRN genes were associated to disease onset; however, for a subset of patients the underlying causative mechanisms remain elusive. We aimed to evaluate the role of epigenetic alteration on premature aging diseases by performing comprehensive DNA methylation profiling of HGP and WS patients. We observed profound changes in the DNA methylation landscapes of WRN and LMNA mutant patients, which were narrowed down to a set of aging related genes and processes. Although of low overall variance, non-mutant patients revealed differential DNA methylation at distinct loci. Hence, we propose DNA methylation to have an impact on premature aging diseases.

  18. 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.

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

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

    2016-08-02

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

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

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    Ma, Mark Z.; Ruxian Lin; José Carrillo; Manisha Bhutani; Ashutosh Pathak; Hening Ren; Yaokun Li; Jiuzhou Song; Li Mao

    2015-01-01

    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/...

  1. Aberrant DNA methylation of ESR1 and p14ARF genes could be useful as prognostic indicators in osteosarcoma

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    Sonaglio V

    2013-06-01

    Full Text Available Viviane Sonaglio,1 Ana C de Carvalho,2 Silvia R C Toledo,3,4 Carolina Salinas-Souza,3,4 André L Carvalho,5 Antonio S Petrilli,3 Beatriz de Camargo,6 André L Vettore21Pediatrics Department, A C Camargo Hospital, São Paulo, Brazil; 2Biological Science Department, Federal University of São Paulo, Diadema, Brazil; 3Department of Pediatrics, Pediatric Oncology Institute, GRAACC/Federal University of São Paulo, São Paulo, Brazil; 4Department of Morphology and Genetics, Federal University of São Paulo, São Paulo, Brazil; 5Department of Head and Neck Surgery, PIO XII Foundation, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; 6Research Program Pediatric Oncology Program, CPNq, Instituto Nacional do Cancer, Rio de Janeiro, BrazilAbstract: Osteosarcoma (OS is the eighth most common form of childhood and adolescence cancer. Approximately 10%–20% of patients present metastatic disease at diagnosis and the 5-year overall survival remains around 70% for nonmetastatic patients and around 30% for metastatic patients. Metastatic disease at diagnosis and the necrosis grade induced by preoperative treatment are the only well-established prognostic factors for osteosarcoma. The DNA aberrant methylation is a frequent epigenetic alteration in humans and has been described as a molecular marker in different tumor types. This study evaluated the DNA aberrant methylation status of 18 genes in 34 OS samples without previous chemotherapy treatment and in four normal bone specimens and compared the methylation profile with clinicopathological characteristics of the patients. We were able to define a three-gene panel (AIM1, p14ARF, and ESR1 in which methylation was correlated with OS cases. The hypermethylation of p14ARF showed a significant association with the absence of metastases at diagnoses, while ESR1 hypermethylation was marginally associated with worse overall survival. This study demonstrated that aberrant promoter methylation is a common event

  2. Aberrant DNA methylation profile in pleural fluid for differential diagnosis of malignant pleural mesothelioma.

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    Fujii, Masanori; Fujimoto, Nobukazu; Hiraki, Akio; Gemba, Kenichi; Aoe, Keisuke; Umemura, Shigeki; Katayama, Hideki; Takigawa, Nagio; Kiura, Katsuyuki; Tanimoto, Mitsune; Kishimoto, Takumi

    2012-03-01

    Malignant pleural mesothelioma (MPM) usually develops pleural fluid. We investigated the value of DNA methylation in the pleural fluid for differentiating MPM from lung cancer (LC). Pleural fluid was collected from 39 patients with MPM, 46 with LC, 25 with benign asbestos pleurisy (BAP) and 30 with other causes. The methylation of O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a) , ras association domain family 1A (RASSF1A), death-associated protein kinase (DAPK), and retinoic acid receptor β (RARβ) was examined using quantitative real-time PCR. DNA methylation of RASSF1A, p16(INK4a), RARβ, MGMT and DAPK was detected in 12 (30.8%), 3 (7.7%), 11 (28.2%), 0 (0.0%) and five patients (12.8%) with MPM, and in 22 (47.8%), 14 (30.4%), 24 (52.2%), 1 (2.2%) and six patients (13.0%) with LC, respectively. The mean methylation ratios of RASSF1A, p16(INK4a) and RARβ were 0.37 (range 0.0-2.84), 0.11 (0.0-2.67) and 0.44 (0.0-3.32) in MPM, and 0.87 (0.0-3.14), 1.16 (0.0-5.35) and 1.69 (0.0-6.49) in LC, respectively. The methylation ratios for the three genes were significantly higher in LC than in MPM (RASSF1A, P = 0.039; p16(INK4a), P = 0.005; and RARβ, P = 0.002). Patients with methylation in at least one gene were 3.51 (95% confidence interval, 1.09-11.34) times more likely to have LC. Hypermethylation seemed no greater with MPM than with BAP. Extended exposure to asbestos (≧30 years) was correlated with an increased methylation frequency (P = 0.020). Hypermethylation of tumor suppressor genes in pleural fluid DNA has the potential to be a valuable marker for differentiating MPM from LC.

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

    Science.gov (United States)

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

    2014-04-15

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

  4. DNA Methylation

    OpenAIRE

    Alokail, Majed S.; Alenad, Amal M.

    2015-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication e...

  5. Aberrant DNA methylation of cancer-associated genes in gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC-EURGAST)

    NARCIS (Netherlands)

    Balassiano, Karen; Lima, Sheila; Jenab, Mazda; Overvad, Kim; Tjonneland, Anne; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Francoise; Canzian, Federico; Kaaks, Rudolf; Boeing, Heiner; Meidtner, Karina; Trichopoulou, Antonia; Laglou, Pagona; Vineis, Paolo; Panico, Salvatore; Palli, Domenico; Grioni, Sara; Tumino, Rosario; Lund, Eiliv; Bueno-de-Mesquita, H. Bas; Numans, Mattjis E.; Peeters, Petra H. M.; Ramon Quiros, J.; Sanchez, Maria-Jose; Navarro, Carmen; Ardanaz, Eva; Dorronsoro, Miren; Hallmans, Goran; Stenling, Roger; Ehrnstrom, Roy; Regner, Sara; Allen, Naomi E.; Travis, Ruth C.; Khaw, Kay-Tee; Offerhaus, G. Johan A.; Sala, Nuria; Riboli, Elio; Hainaut, Pierre; Scoazec, Jean-Yves; Sylla, Bakary S.; Gonzalez, Carlos A.; Herceg, Zdenko

    2011-01-01

    Epigenetic events have emerged as key mechanisms in the regulation of critical biological processes and in the development of a wide variety of human malignancies, including gastric cancer (GC), however precise gene targets of aberrant DNA methylation in GC remain largely unknown. Here, we have comb

  6. Similarity of aberrant DNA methylation in Barrett's esophagus and esophageal adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Gotley David C

    2008-10-01

    Full Text Available Abstract Background Barrett's esophagus (BE is the metaplastic replacement of squamous with columnar epithelium in the esophagus, as a result of reflux. It is the major risk factor for the development of esophageal adenocarcinoma (EAC. Methylation of CpG dinucleotides of normally unmethylated genes is associated with silencing of their expression, and is common in EAC. This study was designed to determine at what stage, in the progression from BE to EAC, methylation of key genes occurs. Results We examined nine genes (APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3, and TMEFF2, frequently methylated in multiple cancer types, in a panel of squamous (19 biopsies from patients without BE or EAC, 16 from patients with BE, 21 from patients with EAC, BE (40 metaplastic, seven high grade dysplastic and 37 EAC tissues. The methylation frequency, the percentage of samples that had any extent of methylation, for each of the nine genes in the EAC (95%, 59%, 76%, 57%, 70%, 73%, 95%, 74% and 83% respectively was significantly higher than in any of the squamous groups. The methylation frequency for each of the nine genes in the metaplastic BE (95%, 28%, 78%, 48%, 58%, 48%, 93%, 88% and 75% respectively was significantly higher than in the squamous samples except for CDKN2A and RBP1. The methylation frequency did not differ between BE and EAC samples, except for CDKN2A and RUNX3 which were significantly higher in EAC. The methylation extent was an estimate of both the number of methylated alleles and the density of methylation on these alleles. This was significantly greater in EAC than in metaplastic BE for all genes except APC, MGMT and TIMP3. There was no significant difference in methylation extent for any gene between high grade dysplastic BE and EAC. Conclusion We found significant methylation in metaplastic BE, which for seven of the nine genes studied did not differ in frequency from that found in EAC. This is also the first report of gene silencing

  7. DNA methylation signature in peripheral blood reveals distinct characteristics of human X chromosome numerical aberrations

    OpenAIRE

    2015-01-01

    Background Abnormal sex chromosome numbers in humans are observed in Turner (45,X) and Klinefelter (47,XXY) syndromes. Both syndromes are associated with several clinical phenotypes, whose molecular mechanisms are obscure, and show a range of inter-individual penetrance. In order to understand the effect of abnormal numbers of X chromosome on the methylome and its correlation to the variable clinical phenotype, we performed a genome-wide methylation analysis using MeDIP and Illumina’s Infiniu...

  8. 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...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  9. 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.

  10. 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.

  11. 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.

  12. Quantitative DNA Methylation Profiling in Cancer.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  14. Inferring a role for methylation of intergenic DNA in the regulation of genes aberrantly expressed in precursor B-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Almamun, Md; Kholod, Olha; Stuckel, Alexei J; Levinson, Benjamin T; Johnson, Nathan T; Arthur, Gerald L; Davis, J Wade; Taylor, Kristen H

    2017-01-17

    A complete understanding of the mechanisms involved in the development of pre-B ALL is lacking. In this study, we integrated DNA methylation data and gene expression data to elucidate the impact of aberrant intergenic DNA methylation on gene expression in pre-B ALL. We found a subset of differentially methylated intergenic loci that were associated with altered gene expression in pre-B ALL patients. Notably, 84% of these regions were also bound by transcription factors (TF) known to play roles in differentiation and B-cell development in a lymphoblastoid cell line. Further, an overall downregulation of eRNA transcripts was observed in pre-B ALL patients and these transcripts were associated with the downregulation of putative target genes involved in B-cell migration, proliferation, and apoptosis. The identification of novel putative regulatory regions highlights the significance of intergenic DNA sequences and may contribute to the identification of new therapeutic targets for the treatment of pre-B ALL.

  15. 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.

  16. DNA methylation profiling of sorted cells from myelofibrosis patients reveals aberrant epigenetic regulation of immune pathways and identifies early MPN driver genes

    DEFF Research Database (Denmark)

    Nielsen, H. M.; Andersen, C. L.; Kristensen, L. S.;

    2015-01-01

    Methylation 450K BeadChip. Candidate genes were validated by pyrosequencing in a second cohort of 30 MF patients where DNA was extracted from full blood (PB). To identify potential driver genes, the DNA methylation status of candidate genes was likewise analyzed in PB from a larger cohort consisting of 60 ET...

  17. Factors associated with aberrant imprint methylation and oligozoospermia

    Science.gov (United States)

    Kobayashi, Norio; Miyauchi, Naoko; Tatsuta, Nozomi; Kitamura, Akane; Okae, Hiroaki; Hiura, Hitoshi; Sato, Akiko; Utsunomiya, Takafumi; Yaegashi, Nobuo; Nakai, Kunihiko; Arima, Takahiro

    2017-01-01

    Disturbingly, the number of patients with oligozoospermia (low sperm count) has been gradually increasing in industrialized countries. Epigenetic alterations are believed to be involved in this condition. Recent studies have clarified that intrinsic and extrinsic factors can induce epigenetic transgenerational phenotypes through apparent reprogramming of the male germ line. Here we examined DNA methylation levels of 22 human imprinted loci in a total of 221 purified sperm samples from infertile couples and found methylation alterations in 24.8% of the patients. Structural equation model suggested that the cause of imprint methylation errors in sperm might have been environmental factors. More specifically, aberrant methylation and a particular lifestyle (current smoking, excess consumption of carbonated drinks) were associated with severe oligozoospermia, while aging probably affected this pathology indirectly through the accumulation of PCB in the patients. Next we examined the pregnancy outcomes for patients when the sperm had abnormal imprint methylation. The live-birth rate decreased and the miscarriage rate increased with the methylation errors. Our research will be useful for the prevention of methylation errors in sperm from infertile men, and sperm with normal imprint methylation might increase the safety of assisted reproduction technology (ART) by reducing methylation-induced diseases of children conceived via ART. PMID:28186187

  18. [DNA methylation and epigenetics].

    Science.gov (United States)

    Vaniushin, B F

    2006-09-01

    In eukaryotic cells, nuclear DNA is subject to enzymatic methylation with the formation of 5-methylcytosine residues, mostly within the CG and CNG sequences. In plants and animals this DNA methylation is species-, tissue-, and organelle-specific. It changes (decreases) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. Replicative and post-replicative DNA methylation types are distinguished. They are mediated by multiple DNA methyltransferases with different site-specificity. Replication is accompanied by the appearance of hemimethylated DNA sites. Pronounced asymmetry of the DNA strand methylation disappears to the end of the cell cycle. A model of methylation-regulated DNA replication is proposed. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, and gene transposition). It is the mechanism of cell differentiation, gene discrimination and silencing. In animals, suppression of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Disruption of DNA methylation pattern results in the malignant cell transformation and serves as one of the early diagnostic features of carcinogenesis. In malignant cell the pattern of DNA methylation, as well as the set of DNA methyltransferase activities, differs from that in normal cell. In plants inhibition of DNA methylation is accompanied by the induction of seed storage and florescence genes. In eukaryotes one and the same gene can be simultaneously methylated both at cytosine and adenine residues. It can be thus suggested, that the plant cell contains at least two different, and probably, interdependent systems of DNA methylation. The first eukaryotic adenine DNA methyltransferase was isolated from plants. This enzyme methylates DNA with the formation of N6-methyladenine residues in the sequence TGATCA (TGATCA-->TGm6ATCA). Plants possess AdoMet-dependent endonucleases

  19. Epigenome-wide DNA methylation landscape of melanoma progression to brain metastasis reveals aberrations on homeobox D cluster associated with prognosis.

    Science.gov (United States)

    Marzese, Diego M; Scolyer, Richard A; Huynh, Jamie L; Huang, Sharon K; Hirose, Hajime; Chong, Kelly K; Kiyohara, Eiji; Wang, Jinhua; Kawas, Neal P; Donovan, Nicholas C; Hata, Keisuke; Wilmott, James S; Murali, Rajmohan; Buckland, Michael E; Shivalingam, Brindha; Thompson, John F; Morton, Donald L; Kelly, Daniel F; Hoon, Dave S B

    2014-01-01

    Melanoma brain metastasis (MBM) represents a frequent complication of cutaneous melanoma. Despite aggressive multi-modality therapy, patients with MBM often have a survival rate of MBM. In this study, we generated a comprehensive DNA methylation landscape through the use of genome-wide copy number, DNA methylation and gene expression data integrative analysis of melanoma progression to MBM. A progressive genome-wide demethylation in low CpG density and an increase in methylation level of CpG islands according to melanoma progression were observed. MBM-specific partially methylated domains (PMDs) affecting key brain developmental processes were identified. Differentially methylated CpG sites between MBM and lymph node metastasis (LNM) from patients with good prognosis were identified. Among the most significantly affected genes were the HOX family members. DNA methylation of HOXD9 gene promoter affected transcript and protein expression and was significantly higher in MBM than that in early stages. A MBM-specific PMD was identified in this region. Low methylation level of this region was associated with active HOXD9 expression, open chromatin and histone modifications associated with active transcription. Demethylating agent induced HOXD9 expression in melanoma cell lines. The clinical relevance of this finding was verified in an independent large cohort of melanomas (n = 145). Patients with HOXD9 hypermethylation in LNM had poorer disease-free and overall survival. This epigenome-wide study identified novel methylated genes with functional and clinical implications for MBM patients.

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

    Science.gov (United States)

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

    2015-12-01

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

  1. Late-occurring chromosome aberrations and global DNA methylation in hematopoietic stem/progenitor cells of CBA/CaJ mice exposed to silicon ({sup 28}Si) ions

    Energy Technology Data Exchange (ETDEWEB)

    Rithidech, Kanokporn Noy, E-mail: kanokporn.rithidech@stonybrookmedicine.edu [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Honikel, Louise M. [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Reungpathanaphong, Paiboon [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Department of Applied Radiation and Isotopes, Faculty of Sciences, Kasetsart University, Chatuchuck, Bangkok 10900 (Thailand); Tungjai, Montree [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Department of Radiologic Technology, Faculty of Associated Medical Sciences, Center of Excellence for Molecular Imaging, Chiang Mai University, Chiang Mai 50200 (Thailand); Jangiam, Witawat [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Department of Chemical Engineering, Faculty of Engineering, Burapha University, Chonburi 20131 (Thailand); Whorton, Elbert B. [StatCom, PO Box 3041, Galveston, TX 77551 (United States)

    2015-11-15

    Highlights: • Late-occurring chromosome aberrations were found in HSPCs of exposed CBA/CaJ mice. • A dose-dependent reduction in the level of global 5hmC was detected in HSPCs. • There is a link between reduced global 5hmC levels and genomic instability in vivo. • The level of global 5hmC is a better marker of radiation exposure than that of 5mC. - Abstract: Although myeloid leukemia (ML) is one of the major health concerns from exposure to space radiation, the risk prediction for developing ML is unsatisfactory. To increase the reliability of predicting ML risk, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is important. We focused on the in vivo induction of late-occurring damage in HSPCs of mice exposed to {sup 28}Si ions since such damage is associated with radiation-induced genomic instability (a key event of carcinogenesis). We gave adult male CBA/CaJ mice, known to be sensitive to radiation-induced ML, a whole-body exposure (2 fractionated exposures, 15 days apart, that totaled each selected dose, delivered at the dose-rate of 1 cGy/min) to various doses of 300 MeV/n {sup 28}Si ions, i.e. 0 (sham controls), 0.1, 0.25, or 0.5 Gy. At 6 months post-irradiation, we collected bone marrow cells from each mouse (five mice per treatment-group) for obtaining the myeloid-lineage of HSPC-derived clones for analyses. We measured the frequencies of late-occurring chromosome aberrations (CAs), using the genome-wide multicolor fluorescence in situ hybridization method. The measurement of CAs was coupled with the characterization of the global DNA methylation patterns, i.e. 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). A dose-dependent increase in the frequencies of CAs was detected (Analysis of Variance or ANOVA, p < 0.01), indicating the induction of genomic instability after exposure of mice to 300 MeV/n {sup 28}Si ions. Slight increases in the levels of 5m

  2. Aberrant promoter hypermethylation in serum DNA from patients with silicosis.

    Science.gov (United States)

    Umemura, Shigeki; Fujimoto, Nobukazu; Hiraki, Akio; Gemba, Kenichi; Takigawa, Nagio; Fujiwara, Keiichi; Fujii, Masanori; Umemura, Hiroshi; Satoh, Mamoru; Tabata, Masahiro; Ueoka, Hiroshi; Kiura, Katsuyuki; Kishimoto, Takumi; Tanimoto, Mitsune

    2008-09-01

    It is well established that patients with silicosis are at high risk for lung cancer; however, it is difficult to detect lung cancer by chest radiography during follow-up treatment of patients with silicosis because of preexisting diffuse pulmonary shadows. The purpose of this study is to evaluate the usefulness of detection of serum DNA methylation for early detection of lung cancer in silicosis. Serum samples from healthy controls (n = 20) and silicosis patients with (n = 11) and without (n = 67) lung cancer were tested for aberrant hypermethylation at the promoters of the DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a), ras association domain family 1A (RASSF1A), the apoptosis-related gene death-associated protein kinase (DAPK) and retinoic acid receptor beta (RARbeta) by methylation-specific polymerase chain reaction. Aberrant promoter methylation in at least one of five tumor suppressor genes was detected more frequently in the serum DNA of silicosis patients with lung cancer than in that of patients without it (P = 0.006). Furthermore, the odds ratio of having lung cancer was 9.77 (P = 0.009) for those silicosis patients with methylation of at least one gene. Extended exposure to silica (>30 years) was correlated with an increased methylation frequency (P = 0.017); however, methylation status did not correlate with age, smoking history or radiographic findings of silicosis. These results suggest that testing for aberrant promoter methylation of tumor suppressor genes using serum DNA may facilitate early detection of lung cancer in patients with silicosis.

  3. Electrochemical biosensing strategies for DNA methylation analysis.

    Science.gov (United States)

    Hossain, Tanvir; Mahmudunnabi, Golam; Masud, Mostafa Kamal; Islam, Md Nazmul; Ooi, Lezanne; Konstantinov, Konstantin; Hossain, Md Shahriar Al; Martinac, Boris; Alici, Gursel; Nguyen, Nam-Trung; Shiddiky, Muhammad J A

    2017-02-17

    DNA methylation is one of the key epigenetic modifications of DNA that results from the enzymatic addition of a methyl group at the fifth carbon of the cytosine base. It plays a crucial role in cellular development, genomic stability and gene expression. Aberrant DNA methylation is responsible for the pathogenesis of many diseases including cancers. Over the past several decades, many methodologies have been developed to detect DNA methylation. These methodologies range from classical molecular biology and optical approaches, such as bisulfite sequencing, microarrays, quantitative real-time PCR, colorimetry, Raman spectroscopy to the more recent electrochemical approaches. Among these, electrochemical approaches offer sensitive, simple, specific, rapid, and cost-effective analysis of DNA methylation. Additionally, electrochemical methods are highly amenable to miniaturization and possess the potential to be multiplexed. In recent years, several reviews have provided information on the detection strategies of DNA methylation. However, to date, there is no comprehensive evaluation of electrochemical DNA methylation detection strategies. Herein, we address the recent developments of electrochemical DNA methylation detection approaches. Furthermore, we highlight the major technical and biological challenges involved in these strategies and provide suggestions for the future direction of this important field.

  4. 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.

  5. 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.

  6. 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

  7. 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.

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

    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

  9. Targeting DNA methylation with green tea catechins.

    Science.gov (United States)

    Yiannakopoulou, Eugenia C

    2015-01-01

    Aberrant epigenetic alterations in the genome such as DNA methylation play a significant role in cancer development. Green tea catechins have been reported to modulate epigenetic processes. This review aims to synthesize evidence on the modulation of DNA methylation by green tea catechins. Green tea catechins have been reported to reverse DNA methylation of tumor suppressor genes and increase transcription of these genes. Green tea catechins and especially epigallocatechin gallate modulate DNA methylation by attenuating the effect of DNA methyltransferase 1 (DNMT1). However, the exact mechanism of DNMT1 inhibition is not delineated. Suggested mechanisms include direct enzymatic inhibition, indirect enzymatic inhibition, reduced DNMT1 expression and translation. The possible effect of green tea catechins on other pathways of DNA methylation, i.e. methyl-CpG binding domain proteins, has not been investigated. Furthermore, the link between redox properties and epigenetic modulation by green tea catechins has not been defined either. Since green tea catechins are natural compounds with a rather acceptable safety profile, further research on their action as inhibitors of DNA methylation seems worthwhile.

  10. DNA methylation abnormalities in congenital heart disease.

    Science.gov (United States)

    Serra-Juhé, Clara; Cuscó, Ivon; Homs, Aïda; Flores, Raquel; Torán, Núria; Pérez-Jurado, Luis A

    2015-01-01

    Congenital heart defects represent the most common malformation at birth, occurring also in ∼50% of individuals with Down syndrome. Congenital heart defects are thought to have multifactorial etiology, but the main causes are largely unknown. We have explored the global methylation profile of fetal heart DNA in comparison to blood DNA from control subjects: an absolute correlation with the type of tissue was detected. Pathway analysis revealed a significant enrichment of differential methylation at genes related to muscle contraction and cardiomyopathies in the developing heart DNA. We have also searched for abnormal methylation profiles on developing heart-tissue DNA of syndromic and non-syndromic congenital heart defects. On average, 3 regions with aberrant methylation were detected per sample and 18 regions were found differentially methylated between groups. Several epimutations were detected in candidate genes involved in growth regulation, apoptosis and folate pathway. A likely pathogenic hypermethylation of several intragenic sites at the MSX1 gene, involved in outflow tract morphogenesis, was found in a fetus with isolated heart malformation. In addition, hypermethylation of the GATA4 gene was present in fetuses with Down syndrome with or without congenital heart defects, as well as in fetuses with isolated heart malformations. Expression deregulation of the abnormally methylated genes was detected. Our data indicate that epigenetic alterations of relevant genes are present in developing heart DNA in fetuses with both isolated and syndromic heart malformations. These epimutations likely contribute to the pathogenesis of the malformation by cis-acting effects on gene expression.

  11. Event extraction for DNA methylation

    Directory of Open Access Journals (Sweden)

    Ohta Tomoko

    2011-10-01

    Full Text Available 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 including a representative sample of all PubMed citations relevant to DNA methylation, and introduce manual annotation for this corpus marking nearly 3000 gene/protein mentions and 1500 DNA methylation and demethylation events. We retrain a state-of-the-art event extraction system on the corpus and find that automatic extraction of DNA methylation events, the methylated genes, and their methylation sites can be performed at 78% precision and 76% recall. Conclusions Our results demonstrate that reliable extraction methods for DNA methylation events can be created through corpus annotation and straightforward retraining of a general event extraction system. The introduced resources are freely available for use in research from the GENIA project homepage http://www-tsujii.is.s.u-tokyo.ac.jp/GENIA.

  12. 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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. DNA methylation profiling using bisulfite-based epityping of pooled genomic DNA.

    Science.gov (United States)

    Docherty, Sophia J; Davis, Oliver S P; Haworth, Claire M A; Plomin, Robert; Mill, Jonathan

    2010-11-01

    DNA methylation plays a vital role in normal cellular function, with aberrant methylation signatures being implicated in a growing number of human pathologies and complex human traits. Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however they require large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. DNA pooling approaches are already widely used in large-scale studies of DNA sequence and gene expression. In this paper, we describe the application of this economical DNA pooling technique to the study of DNA methylation profiles. This method generates accurate quantitative assessments of group DNA methylation averages, reducing the time, cost and amount of DNA starting material required for large-scale epigenetic investigation of disease phenotypes.

  16. 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....

  17. 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

  18. Pathway Implications of Aberrant Global Methylation in Adrenocortical Cancer.

    Directory of Open Access Journals (Sweden)

    Christophe R Legendre

    Full Text Available Adrenocortical carcinomas (ACC are a rare tumor type with a poor five-year survival rate and limited treatment options.Understanding of the molecular pathogenesis of this disease has been aided by genomic analyses highlighting alterations in TP53, WNT, and IGF signaling pathways. Further elucidation is needed to reveal therapeutically actionable targets in ACC.In this study, global DNA methylation levels were assessed by the Infinium HumanMethylation450 BeadChip Array on 18 ACC tumors and 6 normal adrenal tissues. A new, non-linear correlation approach, the discretization method, assessed the relationship between DNA methylation/gene expression across ACC tumors.This correlation analysis revealed epigenetic regulation of genes known to modulate TP53, WNT, and IGF signaling, as well as silencing of the tumor suppressor MARCKS, previously unreported in ACC.DNA methylation may regulate genes known to play a role in ACC pathogenesis as well as known tumor suppressors.

  19. 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.

  20. 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.

  1. 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.

  2. The emerging role of DNA methylation in epileptogenesis.

    Science.gov (United States)

    Kobow, Katja; Blümcke, Ingmar

    2012-12-01

    DNA methylation is a covalent chromatin modification, characterized by the biochemical addition of a methyl group (-CH3) to cytosine nucleotides via a DNA methyltransferase enzyme. 5'-Methylcytosine (5-mC), frequently called the fifth base, has been implicated in genome stability, silencing of transposable elements, and repression of gene expression. Through the latter, DNA methylation dynamics broadly influence brain development, function, and aging. Aberrant DNA methylation patterns, either localized to specific gene regions or scattered throughout the genome, are associated with many neurologic disorders. Herein, we discuss the emerging role of DNA methylation in epileptogenesis and the perspectives arising from epigenetic medicine as new therapeutic strategy in difficult-to-treat epilepsies.

  3. Aberrant gene promoter methylation in sputum from individuals exposed to smoky coal emissions

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.; Lan, Q.; Shen, M.; Jin, J.; Mumford, J.; Ren, D.X.; Keohavong, P. [University of Pittsburgh, Pittsburgh, PA (United States). Dept. of Environment and Occupational Health

    2008-07-15

    Recent studies suggested the potential for aberrant gene promoter methylation in sputum as a predictive marker for lung cancer. Here, the promoter methylation of p16, MGMT, RASSF1A and DAPK genes was investigated in sputum of individuals exposed to smoky coal emissions in Xuan Wei, China, where the lung cancer rate is more than 6 times the Chinese national average. Sputum DNA of 107 noncancer individuals and 58 lung cancer patients was screened for promoter methylation using methylation-specific PCR. Promoter methylation of the p16 gene was detected in about half (51.4% (551107)) of sputum DNA from noncancer individuals, a frequency higher than that observed for the RASSF1A (29.9%), MGMT (17.8%) and DAPK (15.9%) genes. Furthermore, the p16 gene was affected by promoter methylation at a frequency even higher among the lung cancer group, compared with the noncancer group (70.7% (41/58) versus 51.7% (55/107), p=0.017). Individuals exposed to smoky coal emissions in this region harbored frequent promoter methylation of these genes in their sputum and some of such alterations may be involved in lung tumor development.

  4. 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

  5. Clinical applications of DNA methylation in gastrointestinal cancer

    NARCIS (Netherlands)

    Maat, Michiel Frank Gerard de

    2010-01-01

    Survival rates after surgical treatment of gastric, colon and rectal cancer can improve with preoperative and/or postoperative adjuvant treatment with chemo- and/or radiotherapy. The role of epigenetic aberrancies such as DNA methylation is established to play a pivotal role in gastrointestinal carc

  6. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

    Science.gov (United States)

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells.

  7. 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.

  8. 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].

  9. A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

    Science.gov (United States)

    Claus, Rainer; Wilop, Stefan; Hielscher, Thomas; Sonnet, Miriam; Dahl, Edgar; Galm, Oliver; Jost, Edgar; Plass, Christoph

    2012-01-01

    Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R2 = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly. PMID:22647397

  10. Neuronal DNA Methylation Profiling of Blast-Related Traumatic Brain Injury

    OpenAIRE

    Haghighi, Fatemeh; Ge, Yongchao; Chen, Sean; Xin, Yurong; Umali, Michelle U.; De Gasperi, Rita; Gama Sosa, Miguel A.; Ahlers, Stephen T.; Elder, Gregory A.

    2015-01-01

    Long-term molecular changes in the brain resulting from blast exposure may be mediated by epigenetic changes, such as deoxyribonucleic acid (DNA) methylation, that regulate gene expression. Aberrant regulation of gene expression is associated with behavioral abnormalities, where DNA methylation bridges environmental signals to sustained changes in gene expression. We assessed DNA methylation changes in the brains of rats exposed to three 74.5 kPa blast overpressure events, conditions that hav...

  11. 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.

  12. Methods in DNA methylation profiling.

    Science.gov (United States)

    Zuo, Tao; Tycko, Benjamin; Liu, Ta-Ming; Lin, Juey-Jen L; Huang, Tim H-M

    2009-12-01

    Metastable and somatically heritable patterns of DNA methylation provide an important level of genomic regulation. In this article, we review methods for analyzing these genome-wide epigenetic patterns and offer a perspective on the ever-expanding literature, which we hope will be useful for investigators who are new to this area. The historical aspects that we cover will be helpful in interpreting this literature and we hope that our discussion of the newest analytical methods will stimulate future progress. We emphasize that no single approach can provide a complete view of the overall methylome, and that combinations of several modalities applied to the same sample set will give the clearest picture. Given the unexpected epigenomic patterns and new biological principles, as well as new disease markers, that have been uncovered in recent studies, it is likely that important discoveries will continue to be made using genome-wide DNA methylation profiling.

  13. 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.

  14. 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.

  15. DNA methylation profiling of hematopoietic stem cells.

    Science.gov (United States)

    Begtrup, Amber Hogart

    2014-01-01

    DNA methylation is a key epigenetic mark that is essential for properly functioning hematopoietic stem cells. Determining where functionally relevant DNA methylation marks exist in the genome is crucial to understanding the role that methylation plays in hematopoiesis. This chapter describes a method to profile DNA methylation by selectively enriching methylated DNA sequences that are bound in vitro by methyl-binding domain (MBD) proteins. The MBD-pulldown approach selects for DNA sequences that have the potential to be "read" by the endogenous machinery involved in epigenetic regulation. Furthermore, this approach is feasible with very small quantities of DNA, and is compatible with the use of any downstream high-throughput sequencing approach. This technique offers a reliable, simple, and powerful tool for exploration of the role of DNA methylation in hematopoietic stem cells.

  16. 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.

  17. 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.

  18. Methylation profiling using methylated DNA immunoprecipitation and tiling array hybridization.

    Science.gov (United States)

    Cheung, Hoi-Hung; Lee, Tin-Lap; Rennert, Owen M; Chan, Wai-Yee

    2012-01-01

    DNA methylation is an important epigenetic modification that regulates development and plays a role in the pathophysiology of many diseases. It is dynamically changed during germline development. Methylated DNA immunoprecipitation (MeDIP) is an efficient, cost-effective method for locus-specific and genome-wide analysis. Methylated DNA fragments are enriched by a 5-methylcytidine-recognizing antibody, therefore allowing the analysis of both CpG and non-CpG methylation. The enriched DNA fragments can be amplified and hybridized to tiling arrays covering CpG islands, promoters, or the entire genome. Comparison of different methylomes permits the discovery of differentially methylated regions that might be important in disease- or tissue-specific expression. Here, we describe an established MeDIP protocol and tiling array hybridization method for profiling methylation of testicular germ cells.

  19. 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

  20. Relationship between nucleosome positioning and DNA methylation

    Science.gov (United States)

    Chodavarapu, Ramakrishna K.; Feng, Suhua; Bernatavichute, Yana V.; Chen, Pao-Yang; Stroud, Hume; Yu, Yanchun; Hetzel, Jonathan; Kuo, Frank; Kim, Jin; Cokus, Shawn J.; Casero, David; Bernal, Maria; Huijser, Peter; Clark, Amander T.; Krämer, Ute; Merchant, Sabeeha S.; Zhang, Xiaoyu; Jacobsen, Steven E.; Pellegrini, Matteo

    2010-01-01

    Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer1, 2. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana utilizing massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified ten base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results suggest that nucleosome positioning strongly influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA suggesting that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA Pol II was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition. PMID:20512117

  1. Aberrant CBFA2T3B gene promoter methylation in breast tumors

    Directory of Open Access Journals (Sweden)

    Bais Anthony J

    2004-08-01

    Full Text Available Abstract Background The CBFA2T3 locus located on the human chromosome region 16q24.3 is frequently deleted in breast tumors. CBFA2T3 gene expression levels are aberrant in breast tumor cell lines and the CBFA2T3B isoform is a potential tumor suppressor gene. In the absence of identified mutations to further support a role for this gene in tumorigenesis, we explored whether the CBFA2T3B promoter region is aberrantly methylated and whether this correlates with expression. Results Aberrant hypo and hypermethylation of the CBFA2T3B promoter was detected in breast tumor cell lines and primary breast tumor samples relative to methylation index interquartile ranges in normal breast counterpart and normal whole blood samples. A statistically significant inverse correlation between aberrant CBFA2T3B promoter methylation and gene expression was established. Conclusion CBFA2T3B is a potential breast tumor suppressor gene affected by aberrant promoter methylation and gene expression. The methylation levels were quantitated using a second-round real-time methylation-specific PCR assay. The detection of both hypo and hypermethylation is a technicality regarding the methylation methodology.

  2. 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.

  3. 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

  4. Tumour class prediction and discovery by microarray-based DNA methylation analysis

    OpenAIRE

    Adorján, Péter; Distler, Jürgen; Lipscher, Evelyne; Model, Fabian; Müller, Jürgen; Pelet, Cécile; Braun, Aron; Florl, Andrea R.; Gütig, David; Grabs, Gabi; Howe, André; Kursar, Mischo; Lesche, Ralf; Leu, Erik; Lewin, André

    2002-01-01

    Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at e...

  5. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  7. 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.

  8. DNA Methylation: Bisulphite Modification and Analysis

    OpenAIRE

    Patterson, Kate; Molloy, Laura; Qu, Wenjia; Clark, Susan

    2011-01-01

    Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expres...

  9. 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.

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

    Science.gov (United States)

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

  11. 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.

  12. Conventional and nanotechniques for DNA methylation profiling.

    Science.gov (United States)

    Shanmuganathan, Rajasree; Basheer, Nazeema B; Amirthalingam, Laxmi; Muthukumar, Harshiny; Kaliaperumal, Rajendran; Shanmugam, Kumaran

    2013-01-01

    DNA methylation is critical for gene silencing and is associated with the incidence of many diseases, including cancer. Underlying molecular mechanisms of human diseases and tissue-specific gene expression have been elucidated based on DNA methylation studies. This review highlights the advantages and drawbacks of various methylation screening techniques: blotting, genomic sequencing, bisulfite sequencing, methylation-specific PCR, methylated DNA immunoprecipitation, microarray analysis, matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, nanowire transistor detection procedure, quantum dot-based nanoassay, single-molecule real-time detection, fluorimetric assay, electrochemical detection, and atomic force spectroscopy. The review provides insight for selecting a method or a combination of methods for DNA methylation analysis. Convergence of conventional and contemporary nanotechniques to enumerate methylation at specific CpG sites of oncogene would fill the gap in diagnosis of cancer.

  13. DNA Methylation Modulates Nociceptive Sensitization after Incision.

    Directory of Open Access Journals (Sweden)

    Yuan Sun

    Full Text Available DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT inhibitor 5-Aza-2'-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2'-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.

  14. 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.

  15. 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.

  16. 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

    The origin of aberrant DNA methylation in cancer remains largely unknown. In the present study, we elucidated the DNA methylome in primary acute promyelocytic leukemia (APL) and the role of promyelocytic leukemia-retinoic acid receptor α (PML-RARα) in establishing these patterns. Cells from APL...... patients showed increased genome-wide DNA methylation with higher variability than healthy CD34(+) cells, promyelocytes, and remission BM cells. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score, and Flt3-mutation status characterized methylation subtypes......-trans retinoic acid also did not result in immediate DNA methylation changes. The results of the present study suggest that aberrant DNA methylation is associated with leukemia phenotype but is not required for PML-RARα-mediated initiation of leukemogenesis....

  17. Profiling genome-wide DNA methylation.

    Science.gov (United States)

    Yong, Wai-Shin; Hsu, Fei-Man; Chen, Pao-Yang

    2016-01-01

    DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.

  18. Maintenance of DNA methylation: Dnmt3b joins the dance.

    Science.gov (United States)

    Walton, Emma L; Francastel, Claire; Velasco, Guillaume

    2011-11-01

    DNA methylation mostly occurs within the context of CpG dinucleotides and is essential for embryonic development and gene repression. It is generally accepted that DNA methyltransferases carry out specific and non-overlapping functions, Dnmt3a and Dnmt3b being responsible for the establishment of methylation around the time of implantation and Dnmt1 ensuring that methylation is faithfully copied to daughter cells via what has come to be known as "maintenance methylation." This longstanding view has been challenged over the years with the observation that Dnmt1 alone is incapable of perfect maintenance methylation. A new model is emerging that takes into account a contribution of the de novo enzymes Dnmt3a and Dnmt3b in the maintenance of the DNA methylation. We recently showed that certain germ line genes are specific targets of Dnmt3b, and that Dnmt3b remains bound to their promoter regions in somatic cells via interaction with the transcriptional repressor E2F6. It is tempting to consider an ongoing role for Dnmt3b in the methylation of germ line genes in somatic cells. We propose here observations in support of the hypothesis that the maintenance of methylation and subsequent silencing of a handful of germ line genes requires Dnmt3b but not Dnmt1. In addition to suggesting a new role for Dnmt3b in the protection of somatic cells against the promiscuous expression of the germ line program, these observations are of particular interest in the field of carcinogenesis, given that the expression of catalytically inactive Dnmt3b isoforms and aberrant expression of germ line genes are commonly observed in cancer cells.

  19. 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...... contributes to the regulation of DNA methylation fidelity....

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

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    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.

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

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    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

  2. DNA Methylation Biomarkers: Cancer and Beyond

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    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.

  3. Whole genome methylation profiling by immunoprecipitation of methylated DNA.

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    Sharp, Andrew J

    2012-01-01

    I provide a protocol for DNA methylation profiling based on immunoprecipitation of methylated DNA using commercially available monoclonal antibodies that specifically recognize 5-methylcytosine. Quantification of the level of enrichment of the resulting DNA enables DNA methylation to be assayed for any genomic locus, including entire chromosomes or genomes if appropriate microarray or high-throughput sequencing platforms are used. In previous studies (1, 2), I have used hybridization to oligonucleotide arrays from Roche Nimblegen Inc, which allow any genomic region of interest to be interrogated, dependent on the array design. For example, using modern tiling arrays comprising millions of oligonucleotide probes, several complete human chromosomes can be assayed at densities of one probe per 100 bp or greater, sufficient to yield high-quality data. However, other methods such as quantitative real-time PCR or high-throughput sequencing can be used, giving either measurement of methylation at a single locus or across the entire genome, respectively. While the data produced by single locus assays is relatively simple to analyze and interpret, global assays such as microarrays or high-throughput sequencing require more complex statistical approaches in order to effectively identify regions of differential methylation, and a brief outline of some approaches is given.

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

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    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.

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

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    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.

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

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    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.

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

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    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.

  8. Conservation and divergence of DNA methylation in eukaryotes: new insights from single base-resolution DNA methylomes.

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    Su, Zhixi; Han, Leng; Zhao, Zhongming

    2011-02-01

    DNA methylation is one of the most important heritable epigenetic modifications of the genome and is involved in the regulation of many cellular processes. Aberrant DNA methylation has been frequently reported to influence gene expression and subsequently cause various human diseases, including cancer. Recent rapid advances in next-generation sequencing technologies have enabled investigators to profile genome methylation patterns at single-base resolution. Remarkably, more than 20 eukaryotic methylomes have been generated thus far, with a majority published since November 2009. Analysis of this vast amount of data has dramatically enriched our knowledge of biological function, conservation and divergence of DNA methylation in eukaryotes. Even so, many specific functions of DNA methylation and their underlying regulatory systems still remain unknown to us. Here, we briefly introduce current approaches for DNA methylation profiling and then systematically review the features of whole genome DNA methylation patterns in eight animals, six plants and five fungi. Our systematic comparison provides new insights into the conservation and divergence of DNA methylation in eukaryotes and their regulation of gene expression. This work aims to summarize the current state of available methylome data and features informatively.

  9. Detection of DNA methylation in eucaryotic cells.

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    Lech Chyczewski

    2008-01-01

    Full Text Available The methods of molecular biology allow for analyzing the methylation pattern in the whole genome and in particular genes. We differentiate methylated sequences from unmethylated ones by means of cutting the genomic template with methylation-sensitive restriction enzymes or by sodium bisulfite DNA modification. Chemical modification precedes most quantitative and qualitative PCR techniques: MS-PCR, MS-nested PCR, Real-Time PCR, QAMA, HeavyMethyl, MSHRM. Restriction enzymes, on the other hand, may be used together with PCR or hybridisation methods (Southern blot and microarrays. PCRs are conducted with primers specific for methylated and unmethylated sequences and sometimes, similarly to hybridisation techniques, with specifically labeled probes or dyes intercalating to double-stranded nucleic acids. The most advanced methylation detection techniques (MALDI-TOF MS and HPLC significantly reduce the amount of biological material used for tests, but they require specialist equipment.

  10. Bisulfite-based epityping on pooled genomic DNA provides an accurate estimate of average group DNA methylation

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    Docherty Sophia J

    2009-03-01

    Full Text Available Abstract Background DNA methylation plays a vital role in normal cellular function, with aberrant methylation signatures being implicated in a growing number of human pathologies and complex human traits. Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however, they require relatively large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. We propose that a high-throughput DNA pooling approach will facilitate the use of emerging methylomic profiling techniques in large samples. Results Compared with data generated from 89 individual samples, our analysis of 205 CpG sites spanning nine independent regions of the genome demonstrates that DNA pools can be used to provide an accurate and reliable quantitative estimate of average group DNA methylation. Comparison of data generated from the pooled DNA samples with results averaged across the individual samples comprising each pool revealed highly significant correlations for individual CpG sites across all nine regions, with an average overall correlation across all regions and pools of 0.95 (95% bootstrapped confidence intervals: 0.94 to 0.96. Conclusion In this study we demonstrate the validity of using pooled DNA samples to accurately assess group DNA methylation averages. Such an approach can be readily applied to the assessment of disease phenotypes reducing the time, cost and amount of DNA starting material required for large-scale epigenetic analyses.

  11. Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug

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    Pickard, Amanda J.; Diaz, Anthony Joseph; Mura, Hugo; Nyuwen, Lila; Coello, Daniel; Sheva, Saif; Maria, Nava; Gallo, James M.; Wang, Tieli

    2017-01-01

    Background/Aim The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas –known as glioblastoma multiforme (GBM)– an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins. Materials and Methods Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis. Results Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications. Conclusion The possibility that TMZ can methylate histones involved with epigenetic regulation of protein indicates a potentially unique mechanism of action. The study will contribute to the understanding the anticancer activity of TMZ in order to develop novel targeted molecular strategies to advance the cancer treatment. PMID:27354585

  12. Cell-type specific DNA methylation patterns define human breast cellular identity.

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    Petr Novak

    Full Text Available DNA methylation plays a role in a variety of biological processes including embryonic development, imprinting, X-chromosome inactivation, and stem cell differentiation. Tissue specific differential methylation has also been well characterized. We sought to extend these studies to create a map of differential DNA methylation between different cell types derived from a single tissue. Using three pairs of isogenic human mammary epithelial and fibroblast cells, promoter region DNA methylation was characterized using MeDIP coupled to microarray analysis. Comparison of DNA methylation between these cell types revealed nearly three thousand cell-type specific differentially methylated regions (ctDMRs. MassARRAY was performed upon 87 ctDMRs to confirm and quantify differential DNA methylation. Each of the examined regions exhibited statistically significant differences ranging from 10-70%. Gene ontology analysis revealed the overrepresentation of many transcription factors involved in developmental processes. Additionally, we have shown that ctDMRs are associated with histone related epigenetic marks and are often aberrantly methylated in breast cancer. Overall, our data suggest that there are thousands of ctDMRs which consistently exhibit differential DNA methylation and may underlie cell type specificity in human breast tissue. In addition, we describe the pathways affected by these differences and provide insight into the molecular mechanisms and physiological overlap between normal cellular differentiation and breast carcinogenesis.

  13. Genome-wide DNA methylation analysis reveals a potential mechanism for the pathogenesis and development of uterine leiomyomas.

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    Ryo Maekawa

    Full Text Available BACKGROUND: The pathogenesis of uterine leiomyomas, the most common benign tumor in women, remains unclear. Since acquired factors such as obesity, hypertension and early menarche place women at greater risk for uterine leiomyomas, uterine leiomyomas may be associated with epigenetic abnormalities that are caused by unfavorable environmental exposures. PRINCIPAL FINDINGS: Profiles of genome-wide DNA methylation and mRNA expression were investigated in leiomyomas and in myometrium with and without leiomyomas. Profiles of DNA methylation and mRNA expression in the myometrium with and without leiomyomas were quite similar while those in leiomyomas were distinct. We identified 120 genes whose DNA methylation and mRNA expression patterns differed between leiomyomas and the adjacent myometrium. The biological relevance of the aberrantly methylated and expressed genes was cancer process, including IRS1 that is related to transformation, and collagen-related genes such as COL4A1, COL4A2 and COL6A3. We also detected 22 target genes of estrogen receptor (ER alpha, including apoptosis-related genes, that have aberrant DNA methylation in the promoter, suggesting that the aberrant epigenetic regulation of ER alpha-target genes contributes to the aberrant response to estrogen. CONCLUSIONS: Aberrant DNA methylation and its related transcriptional aberration were associated with cancer processes, which may represent a critical initial mechanism that triggers transformation of a single tumor stem cell that will eventually develop into a monoclonal leiomyoma tumor. The aberrant epigenetic regulation of ER alpha-target genes also may contribute to the aberrant response to estrogen, which is involved in the development of uterine leiomyomas after menarche.

  14. Prognostic DNA Methylation Markers for Prostate Cancer

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    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.

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

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    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. Polyploidy and DNA methylation: new tools available.

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    Salmon, Armel; Ainouche, Malika L

    2010-01-01

    Most plant species are recent or ancient polyploids (displaying at least one round of genome duplication in their history). Cultivated species (e.g. wheat, cotton, canola, sugarcane, coffee) and invasive species are often relatively recent polyploids, and frequently of hybrid origin (i.e. allopolyploids). Despite the genetic bottleneck occurring during the allopolyploid speciation process, the formation of such species from two divergent lineages leads to fixed heterozygosity decisive to their success. New phenotypes and new niche occupation are usually associated with this mode of speciation, as a result of both genomic rearrangements and gene expression changes of different magnitudes depending on the different polyploid species investigated. These gene expression changes affecting newly formed polyploid species may result from various, interconnected mechanisms, including (i) functional interactions between the homoeologous copies and between their products, that are reunited in the same nucleus and cell; (ii) the fate of duplicated copies, selective pressure on one of the parental copy being released which could lead to gene loss, pseudogenization, or alternatively, to subfunctionalization or neofunctionalization; and (iii) epigenetic landscape changes that in turn affect gene expression. As one of the interrelated processes leading to epigenetic regulation of gene expression, the DNA methylation status of newly formed species appears to be consistently affected following both hybridization and genome doubling. In this issue, Verhoeven et al. have investigated the fate of DNA methylation patterns that could affect naturally occurring new asexual triploid lineages of dandelions. As a result of such a ploidy level change, the authors demonstrate stably transmitted DNA methylation changes leading to unique DNA methylation patterns in each newly formed lineage. Most studies published to date on plant DNA methylation polymorphism were performed using restriction

  17. DNA methylation and healthy human aging.

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    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.

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

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    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.

  19. Global DNA methylation of ischemic stroke subtypes.

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    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.

  20. Information Thermodynamics of Cytosine DNA Methylation.

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    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

  1. Information Thermodynamics of Cytosine DNA Methylation.

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    Robersy Sanchez

    Full Text Available 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

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

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    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.

  3. Changes in DNA methylation patterns in subjects exposed to low-dose benzene.

    Science.gov (United States)

    Bollati, Valentina; Baccarelli, Andrea; Hou, Lifang; Bonzini, Matteo; Fustinoni, Silvia; Cavallo, Domenico; Byun, Hyang-Min; Jiang, Jiayi; Marinelli, Barbara; Pesatori, Angela C; Bertazzi, Pier A; Yang, Allen S

    2007-02-01

    Aberrant DNA methylation patterns, including global hypomethylation, gene-specific hypermethylation/hypomethylation, and loss of imprinting (LOI), are common in acute myelogenous leukemia (AML) and other cancer tissues. We investigated for the first time whether such epigenetic changes are induced in healthy subjects by low-level exposure to benzene, a widespread pollutant associated with AML risk. Blood DNA samples and exposure data were obtained from subjects with different levels of benzene exposure, including 78 gas station attendants, 77 traffic police officers, and 58 unexposed referents in Milan, Italy (personal airborne benzene range, 0.20). This is the first human study to link altered DNA methylation, reproducing the aberrant epigenetic patterns found in malignant cells, to low-level carcinogen exposure.

  4. 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.

  5. 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...

  6. 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.

  7. 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

  8. Aberrant methylation of the M-type phospholipase A2 receptor gene in leukemic cells

    Directory of Open Access Journals (Sweden)

    Menschikowski Mario

    2012-12-01

    Full Text Available Abstract Background The M-type phospholipase A2 receptor (PLA2R1 plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS or acute leukemia. Methods Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. Results Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. Conclusions The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis.

  9. DNA methylation profiling of transcription factor genes in normal lymphocyte development and lymphomas.

    Science.gov (United States)

    Ivascu, Claudia; Wasserkort, Reinhold; Lesche, Ralf; Dong, Jun; Stein, Harald; Thiel, Andreas; Eckhardt, Florian

    2007-01-01

    Transcription factors play a crucial role during hematopoiesis by orchestrating lineage commitment and determining cellular fate. Although tight regulation of transcription factor expression appears to be essential, little is known about the epigenetic mechanisms involved in transcription factor gene regulation. We have analyzed DNA methylation profiles of 13 key transcription factor genes in primary cells of the hematopoietic cascade, lymphoma cell lines and lymph node biopsies of diffuse large B-cell- and T-cell-non-Hodgkin lymphoma patients. Several of the transcription factor genes (SPI1, GATA3, TCF-7, Etv5, c-maf and TBX21) are differentially methylated in specific cell lineages and stages of the hematopoietic cascade. For some genes, such as SPI1, Etv5 and Eomes, we found an inverse correlation between the methylation of the 5' untranslated region and expression of the associated gene suggesting that these genes are regulated by DNA methylation. Differential methylation is not limited to cells of the healthy hematopoietic cascade, as we observed aberrant methylation of c-maf, TCF7, Eomes and SPI1 in diffuse large B-cell lymphomas. Our results suggest that epigenetic remodelling of transcription factor genes is a frequent mechanism during hematopoietic development. Aberrant methylation of transcription factor genes is frequently observed in diffuse large B-cell lymphomas and might have a functional role during tumorigenesis.

  10. Neuronal DNA Methylation Profiling of Blast-Related Traumatic Brain Injury

    Science.gov (United States)

    Ge, Yongchao; Chen, Sean; Xin, Yurong; Umali, Michelle U.; De Gasperi, Rita; Gama Sosa, Miguel A.; Ahlers, Stephen T.; Elder, Gregory A.

    2015-01-01

    Abstract Long-term molecular changes in the brain resulting from blast exposure may be mediated by epigenetic changes, such as deoxyribonucleic acid (DNA) methylation, that regulate gene expression. Aberrant regulation of gene expression is associated with behavioral abnormalities, where DNA methylation bridges environmental signals to sustained changes in gene expression. We assessed DNA methylation changes in the brains of rats exposed to three 74.5 kPa blast overpressure events, conditions that have been associated with long-term anxiogenic manifestations weeks or months following the initial exposures. Rat frontal cortex eight months post-exposure was used for cell sorting of whole brain tissue into neurons and glia. We interrogated DNA methylation profiles in these cells using Expanded Reduced Representation Bisulfite Sequencing. We obtained data for millions of cytosines, showing distinct methylation profiles for neurons and glia and an increase in global methylation in neuronal versus glial cells (p<10−7). We detected DNA methylation perturbations in blast overpressure–exposed animals, compared with sham blast controls, within 458 and 379 genes in neurons and glia, respectively. Differentially methylated neuronal genes showed enrichment in cell death and survival and nervous system development and function, including genes involved in transforming growth factor β and nitric oxide signaling. Functional validation via gene expression analysis of 30 differentially methylated neuronal and glial genes showed a 1.2 fold change in gene expression of the serotonin N-acetyltransferase gene (Aanat) in blast animals (p<0.05). These data provide the first genome-based evidence for changes in DNA methylation induced in response to multiple blast overpressure exposures. In particular, increased methylation and decreased gene expression were observed in the Aanat gene, which is involved in converting serotonin to the circadian hormone melatonin and is implicated in

  11. Epigenomic analysis reveals 23 breast cancer pathogenic genes induced by aberrant DNA methylation%表观基因组研究揭示23个由 DNA 甲基化异常介导的乳腺癌致病基因的研究

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Objective To identify and analyze the DNA methylation induced pathogenic genes in breast cancer.Methods Using genomic data provided by the international cancer genome,we carried out systematic a-nalysis of the abnormal gene expression in breast cancer and epigenetic regulation mechanism by bioinformatics methods.Results We identified 428 genes with abnormal expressions in breast cancer by t test.Functional enrich-ment analysis revealed genes up-regulation in breast cancer were closely related to the cell cycle,and those down-regulated were significantly enriched in the hormone response function.Study on DNA methylation revealed that breast cancer showed an unique DNA methylation pattern.Further analysis reveals 23 breast cancer genes induced by abnormal DNA methylation.Conclusion DNA methylation can mediate the abnormal expression of breast canc-er genes,and is an important biological marker for early diagnosis and prognosis of breast cancer.%目的:筛选并系统分析由DNA甲基化介导的乳腺癌致病基因。方法本研究利用国际癌症基因组计划提供的表观基因组数据,通过生物信息学方法筛选并系统分析了乳腺癌中异常表达的基因及其表观遗传调控机制。结果通过t检验筛选了428个乳腺癌异常表达的基因,功能富集分析揭示乳腺癌中上调的基因与细胞周期密切相关,而下调的基因则显著富集在激素反应相关的功能上。对DNA甲基化组的研究揭示乳腺癌呈现了独特的DNA甲基化模式,并筛选到23个由DNA甲基化异常介导的乳腺癌致病基因。结论 DNA甲基化可介导乳腺癌致病基因的异常表达,是乳腺癌早期诊断和预后的重要生物标记。

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. DNA Methylation Dynamics During Differentiation, Proliferation, and Tumorigenesis in the Intestinal Tract

    Science.gov (United States)

    Huang, Can-Ze

    2015-01-01

    DNA methylation, an epigenetic control mechanism in mammals, is widely present in the intestinal tract during the differentiation and proliferation of epithelial cells. Cells in stem cell pools or villi have different patterns of DNA methylation. The process of DNA methylation is dynamic and occurs at many relevant regulatory elements during the rapid transition of stem cells into fully mature, differentiated epithelial cells. Changes in DNA methylation patterns most often take place in enhancer and promoter regions and are associated with transcription factor binding. During differentiation, enhancer regions associated with genes important to enterocyte differentiation are demethylated, activating gene expression. Abnormal patterns of DNA methylation during differentiation and proliferation in the intestinal tract can lead to the formation of aberrant crypt foci and destroy the barrier and absorptive functions of the intestinal epithelium. Accumulation of these epigenetic changes may even result in tumorigenesis. In the current review, we discuss recent findings on the association between DNA methylation and cell differentiation and proliferation in the small intestine and highlight the possible links between dysregulation of this process and tumorigenesis. PMID:26413818

  18. 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

  19. 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....

  20. DNA methylation profiling of human chromosomes 6, 20 and 22

    OpenAIRE

    Eckhardt, Florian; Lewin, Joern; Cortese, Rene; Rakyan, Vardhman K.; Attwood, John; Burger, Matthias; Burton, John; Cox, Tony V.; Davies, Rob; Down, Thomas A; Haefliger, Carolina; Horton, Roger; Howe, Kevin; Jackson, David K.; Kunde, Jan

    2006-01-01

    DNA methylation constitutes the most stable type of epigenetic modifications modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation reference profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of 6 annotation categories, revealed evolutionary conserved regions to be the predominant sites for differential DNA methyl...

  1. 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

  2. DNA methylation in mouse embryonic stem cells and development.

    Science.gov (United States)

    Latham, Tom; Gilbert, Nick; Ramsahoye, Bernard

    2008-01-01

    Mammalian development is associated with considerable changes in global DNA methylation levels at times of genomic reprogramming. Normal DNA methylation is essential for development but, despite considerable advances in our understanding of the DNA methyltransferases, the reason that development fails when DNA methylation is deficient remains unclear. Furthermore, although much is known about the enzymes that cause DNA methylation, comparatively little is known about the mechanisms or significance of active demethylation in early development. In this review, we discuss the roles of the various DNA methyltransferases and their likely functions in development.

  3. Deoxyribonucleic acid (DNA methylation and its impact in generation of Cancer

    Directory of Open Access Journals (Sweden)

    Rajeswari J

    2014-07-01

    Full Text Available The arrangement of genes in the chromosome is dependent on histone modifications, deoxyribonucleic acid (DNA binding proteins and methylation of cytosines within 51–cytosine-phosphate-Guanine–31 (CpG dinucleotides. DNA methylation can modify the gene activity without changing the gene sequence. Aberrant hypomethylation and hypermethylations, causal or heritable gene expressions play an important role in tumour initiation and progression. Global hypomethylation at some part of genome and hypermethylation at the promoter regions of the tumour suppressor genes could generate mutations in several types of cancers. Reversal or inhibition of DNA methylation mechanism provides a promising improvement in the treatment of cancer along with chemotherapy. A combined approach utilising epigenetic treatment along with standard chemotherapy appears to hold promise as a future therapy.

  4. DNA methylation profiling identifies novel markers of progression in hepatitis B-related chronic liver disease

    OpenAIRE

    Zeybel, Müjdat; Karahüseyinoğlu, Serçin; Vatansever, Sezgin; Hardy, Timothy; Sarı, Aysegül Akder; Çakalağaoğlu, Fulya; Avcı, Arzu; Zeybel, Gemma Louise; Bashton, Matthew; Mathers, John C.; Ünsal, Belkis; Mann, Jelena

    2016-01-01

    Background: Chronic hepatitis B infection is characterized by hepatic immune and inflammatory response with considerable variation in the rates of progression to cirrhosis. Genetic variants and environmental cues influence predisposition to the development of chronic liver disease; however, it remains unknown if aberrant DNA methylation is associated with fibrosis progression in chronic hepatitis B. Results: To identify epigenetic marks associated with inflammatory and fibrotic processes of t...

  5. 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.

  6. 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...

  7. 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.

  8. 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.

  9. 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.

  10. DNA methylation in the pathophysiology of hyperphenylalaninemia in the PAH(enu2) mouse model of phenylketonuria.

    Science.gov (United States)

    Dobrowolski, S F; Lyons-Weiler, J; Spridik, K; Vockley, J; Skvorak, K; Biery, A

    2016-09-01

    Phenylalanine hydroxylase deficient phenylketonuria (PKU) is the paradigm for a treatable inborn error of metabolism where maintaining plasma phenylalanine (Phe) in the therapeutic range relates to improved clinical outcomes. While Phe is the presumed intoxicating analyte causal in neurologic damage, the mechanism(s) of Phe toxicity has remained elusive. Altered DNA methylation is a recognized response associated with exposure to numerous small molecule toxic agents. Paralleling this effect, we hypothesized that chronic Phe over-exposure in the brain would lead to aberrant DNA methylation with secondary influence upon gene regulation that would ultimately contribute to PKU neuropathology. The PAH(enu2) mouse models human PKU with intrinsic hyperphenylalaninemia, abnormal response to Phe challenge, and neurologic deficit. To examine this hypothesis, we assessed DNA methylation patterns in brain tissues using methylated DNA immunoprecipitation and paired end sequencing in adult PAH(enu2) animals maintained under either continuous dietary Phe restriction or chronic hyperphenylalaninemia. Heterozygous PAH(enu2/WT) litter mates served as controls for normal Phe exposure. Extensive repatterning of DNA methylation was observed in brain tissue of hyperphenylalaninemic animals while Phe restricted animals displayed an attenuated pattern of aberrant DNA methylation. Affected gene coding regions displayed aberrant hypermethylation and hypomethylation. Gene body methylation of noncoding RNA genes was observed and among these microRNA genes were prominent. Of particular note, observed only in hyperphenylalaninemic animals, was hypomethylation of miRNA genes within the imprinted Dlk1-Dio3 locus on chromosome 12. Aberrant methylation of microRNA genes influenced their expression which has secondary effects upon the expression of targeted protein coding genes. Differential hypermethylation of gene promoters was exclusive to hyperphenylalaninemic PAH(enu2) animals. Genes with

  11. 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

  12. Fetal DNA hypermethylation in tight junction pathway is associated with neural tube defects: A genome-wide DNA methylation analysis.

    Science.gov (United States)

    Wang, Linlin; Lin, Shanshan; Zhang, Ji; Tian, Tian; Jin, Lei; Ren, Aiguo

    2017-02-01

    Neural tube defects (NTDs) are a spectrum of severe congenital malformations of fusion failure of the neural tube during early embryogenesis. Evidence on aberrant DNA methylation in NTD development remains scarce, especially when exposure to environmental pollutant is taken into consideration. DNA methylation profiling was quantified using the Infinium HumanMethylation450 array in neural tissues from 10 NTD cases and 8 non-malformed controls (stage 1). Subsequent validation was performed using a Sequenom MassARRAY system in neural tissues from 20 NTD cases and 20 non-malformed controls (stage 2). Correlation analysis of differentially methylated CpG sites in fetal neural tissues and polycyclic aromatic hydrocarbons concentrations in fetal neural tissues and maternal serum was conducted. Differentially methylated CpG sites of neural tissues were further validated in fetal mice with NTDs induced by benzo(a)pyrene given to pregnant mice. Differentially hypermethylated CpG sites in neural tissues from 17 genes and 6 pathways were identified in stage 1. Subsequently, differentially hypermethylated CpG sites in neural tissues from 6 genes (BDKRB2, CTNNA1, CYFIP2, MMP7, MYH2, and TIAM2) were confirmed in stage 2. Correlation analysis showed that methylated CpG sites in CTNNA1 and MYH2 from NTD cases were positively correlated to polycyclic aromatic hydrocarbon level in fetal neural tissues and maternal serum. The correlation was confirmed in NTD-affected fetal mice that were exposed to benzo(a)pyrene in utero. In conclusion, hypermethylation of the CTNNA1 and MYH2 genes in tight junction pathway is associated with the risk for NTDs, and the DNA methylation aberration may be caused by exposure to benzo(a)pyrene.

  13. 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

  14. 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

  15. 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.

  16. CpG methylation increases the DNA binding of 9-aminoacridine carboxamide Pt analogues.

    Science.gov (United States)

    Kava, Hieronimus W; Murray, Vincent

    2016-10-01

    This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5'-CpG sequences. Their binding to methylated and non-methylated 5'-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5'-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5'-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell's genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use.

  17. DNA methylation profiling using the methylated-CpG island recovery assay (MIRA).

    Science.gov (United States)

    Rauch, Tibor A; Pfeifer, Gerd P

    2010-11-01

    The methylated-CpG island recovery assay (MIRA) exploits the intrinsic specificity and the high affinity of a methylated-CpG-binding protein complex (MBD2B and MBD3L1) to methylated CpG dinucleotides in genomic DNA. The MIRA approach works on double-stranded DNA and does not depend on the application of methylation-sensitive restriction enzymes. It can be performed on a few hundred nanograms of genomic DNA. Recently, the MIRA technique has been used to profile DNA methylation patterns at a resolution of 100 base pairs along the entire genome of normal human B-lymphocytes. The MIRA method is compatible with microarray and next generation DNA sequencing approaches. We describe the principles and details of this method applied for methylation profiling of genomes containing methylated CpG sequences.

  18. Whole-genome DNA methylation profiling using MethylCap-seq.

    Science.gov (United States)

    Brinkman, Arie B; Simmer, Femke; Ma, Kelong; Kaan, Anita; Zhu, Jingde; Stunnenberg, Hendrik G

    2010-11-01

    MethylCap-seq is a robust procedure for genome-wide profiling of DNA methylation. The approach consists of the capture of methylated DNA using the MBD domain of MeCP2, and subsequent next-generation sequencing of eluted DNA. Elution of the captured methylated DNA is done in steps using a salt gradient, which stratifies the genome into fractions with different CpG density. The enrichment reached within the individual eluates allows for cost-effective deep sequence coverage. The profiles together yield a detailed genome-wide map of methylated regions and readily allows detection of DNA methylation in known and novel regions. Here, we describe principles and details of the MethylCap-seq procedure using different sources of starting material.

  19. Aberrant promoter methylation and gene expression of H-cadherin gene is associated with tumor progression and recurrence in epithelial ovarian carcinoma

    Directory of Open Access Journals (Sweden)

    Rahul Bhagat

    2014-01-01

    Full Text Available Background: Loss of expression of cadherins by promoter hypermethylation has been described in many epithelial cancers, and it may play a role in tumor cell invasion and metastasis. Previously, we reported that E-cadherin gene is frequently methylated in epithelial ovarian cancer. Aim: The aim of this study was to compare the promoter hypermethylation of H-cadherin gene in ovarian epithelial neoplasms to better understand the role of epigenetic silencing in carcinogenesis. Materials and Methods: We examined the promoter methylation of the H-cadherin gene in 134 epithelial ovarian carcinomas (EOC, 23 low malignant potential (LMP tumors, 26 benign cystadenomas and 15 normal ovarian tissues. Methylation was investigated by methylation specific polymerase chain reaction (MSP and the results confirmed by bisulfite DNA sequencing. Relative gene expression of H-cadherin was done using quantitative reverse transcriptase PCR on 51 EOC cases, 9 LMP tumors, 7 benign cystadenomas with 5 normal ovarian tissues. Results: Aberrant methylation of H-cadherin was present in 20 of 134 (15% carcinoma cases, 2 of 23 (09% LMP tumors and 1 of 26 (4% benign cystadenomas. No methylation was observed in any of the normal ovarian tissues. The mRNA expression level of H-cadherin was significantly down-regulated in EOC and LMP tumors than the corresponding normal tissues, whereas the expression level was normal in benign cystadenomas. A significant correlation of H-cadherin promoter methylation was observed with reduced gene expression in EOC. The prevalence of H-cadherin methylation was associated significantly with stage, histopathological grade, and menopausal status of the patient. H-cadherin methylation also had significant association with recurrence and differentiation of tumor. Conclusion: Our findings suggest an association between H-cadherin methylation, tumor progression and recurrence in EOC.

  20. A DNA methylation biomarker of alcohol consumption.

    Science.gov (United States)

    Liu, C; Marioni, R E; Hedman, Å K; Pfeiffer, L; Tsai, P-C; Reynolds, L M; Just, A C; Duan, Q; Boer, C G; Tanaka, T; Elks, C E; Aslibekyan, S; Brody, J A; Kühnel, B; Herder, C; Almli, L M; Zhi, D; Wang, Y; Huan, T; Yao, C; Mendelson, M M; Joehanes, R; Liang, L; Love, S-A; Guan, W; Shah, S; McRae, A F; Kretschmer, A; Prokisch, H; Strauch, K; Peters, A; Visscher, P M; Wray, N R; Guo, X; Wiggins, K L; Smith, A K; Binder, E B; Ressler, K J; Irvin, M R; Absher, D M; Hernandez, D; Ferrucci, L; Bandinelli, S; Lohman, K; Ding, J; Trevisi, L; Gustafsson, S; Sandling, J H; Stolk, L; Uitterlinden, A G; Yet, I; Castillo-Fernandez, J E; Spector, T D; Schwartz, J D; Vokonas, P; Lind, L; Li, Y; Fornage, M; Arnett, D K; Wareham, N J; Sotoodehnia, N; Ong, K K; van Meurs, J B J; Conneely, K N; Baccarelli, A A; Deary, I J; Bell, J T; North, K E; Liu, Y; Waldenberger, M; London, S J; Ingelsson, E; Levy, D

    2016-11-15

    The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis and treatment of alcohol-related diseases. Epigenetic modifications such as DNA methylation may provide novel biomarkers of alcohol use. To examine this possibility, we performed an epigenome-wide association study of methylation of cytosine-phosphate-guanine dinucleotide (CpG) sites in relation to alcohol intake in 13 population-based cohorts (ntotal=13 317; 54% women; mean age across cohorts 42-76 years) using whole blood (9643 European and 2423 African ancestries) or monocyte-derived DNA (588 European, 263 African and 400 Hispanic ancestry) samples. We performed meta-analysis and variable selection in whole-blood samples of people of European ancestry (n=6926) and identified 144 CpGs that provided substantial discrimination (area under the curve=0.90-0.99) for current heavy alcohol intake (⩾42 g per day in men and ⩾28 g per day in women) in four replication cohorts. The ancestry-stratified meta-analysis in whole blood identified 328 (9643 European ancestry samples) and 165 (2423 African ancestry samples) alcohol-related CpGs at Bonferroni-adjusted PMolecular Psychiatry advance online publication, 15 November 2016; doi:10.1038/mp.2016.192.

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

    Science.gov (United States)

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

    2016-01-01

    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

  2. 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].

  3. Dynamic changes in DNA methylation in ischemic tolerance

    Directory of Open Access Journals (Sweden)

    Robert eMeller

    2015-05-01

    Full Text Available Epigenetic mediators of gene expression are hypothesized to regulate transcriptomic responses to preconditioning ischemia and ischemic tolerance. Here we utilized a methyl-DNA enrichment protocol and sequencing (ChIP-seq to identify patterns of DNA methylation in an established model of ischemic tolerance in neuronal cultures (oxygen and glucose deprivation: OGD. We observed an overall decrease in global DNA methylation at 4h following preconditioning ischemia (30min OGD, harmful ischemia (120min OGD and in ischemic tolerant neuronal cultures (30min OGD, 24h recovery, 120min OGD. We detected a smaller cohort of hypermethylated regions following ischemic conditions, which were further analyzed revealing differential chromosomal localization of methylation, and a differential concentration of methylation on genomic regions. Together these data show that the temporal profiles of DNA methylation with respect to chromatin hyper – and hypo-methylation following various ischemic conditions, are highly dynamic and may reveal novel targets for neuroprotection.

  4. 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...... and by strengthening the interaction between mDNA and MBD proteins. Free-energy perturbation calculations also show that methylation yields favorable contribution to the binding free energy for MBD-mDNA complex....

  5. 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.

  6. 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.

  7. Methylated DNA for monitoring tumor growth and regression

    DEFF Research Database (Denmark)

    Kristiansen, Søren; Nielsen, Dorte; Söletormos, Georg

    2014-01-01

    with benign diseases. In contrast, evidence has accumulated that suggests that modified methylated DNA is strongly related to the cancer phenotype. The modifications found in modified methylated DNA include a global loss of methylation in the genomes of the tumor cells as well as focal hypermethylation...... of gene promoters. Because tumor cells naturally secrete DNA and upon cell death leak DNA, modified methylated DNA can be detected in blood, urine, sputum and other body fluids. At present international guidelines do not include recommendations for monitoring modified methylated DNA. The low level...... of evidence can partly be explained by incomplete collection of serial blood samples, by analytical challenges, and by lack of knowledge of how monitoring studies should be designed and how serial marker data obtained from individual patients should be interpreted. Here, we review the clinical validity...

  8. DNA methylation: the future of crime scene investigation?

    Science.gov (United States)

    Gršković, Branka; Zrnec, Dario; Vicković, Sanja; Popović, Maja; Mršić, Gordan

    2013-07-01

    Proper detection and subsequent analysis of biological evidence is crucial for crime scene reconstruction. The number of different criminal acts is increasing rapidly. Therefore, forensic geneticists are constantly on the battlefield, trying hard to find solutions how to solve them. One of the essential defensive lines in the fight against the invasion of crime is relying on DNA methylation. In this review, the role of DNA methylation in body fluid identification and other DNA methylation applications are discussed. Among other applications of DNA methylation, age determination of the donor of biological evidence, analysis of the parent-of-origin specific DNA methylation markers at imprinted loci for parentage testing and personal identification, differentiation between monozygotic twins due to their different DNA methylation patterns, artificial DNA detection and analyses of DNA methylation patterns in the promoter regions of circadian clock genes are the most important ones. Nevertheless, there are still a lot of open chapters in DNA methylation research that need to be closed before its final implementation in routine forensic casework.

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

    Science.gov (United States)

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

    2016-05-01

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

  10. 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.

  11. Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma

    OpenAIRE

    Ostler, Kelly R.; Yang, Qiwei; Looney, Timothy J.; Li ZHANG; Vasanthakumar, Aparna; Tian, Yufeng; Kocherginsky, Masha; Stacey L. Raimondi; DeMaio, Jessica G.; Salwen, Helen R.; Gu, Song; Chlenski, Alexandre; Naranjo, Arlene; Gill, Amy; Peddinti, Radhika

    2012-01-01

    Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7 which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tum...

  12. Detection of regional DNA methylation using DNA-graphene affinity interactions.

    Science.gov (United States)

    Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A

    2017-01-15

    We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues.

  13. 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...

  14. 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.

  15. Comprehensive DNA methylation analysis of the Aedes aegypti genome

    Science.gov (United States)

    Falckenhayn, Cassandra; Carneiro, Vitor Coutinho; de Mendonça Amarante, Anderson; Schmid, Katharina; Hanna, Katharina; Kang, Seokyoung; Helm, Mark; Dimopoulos, George; Fantappié, Marcelo Rosado; Lyko, Frank

    2016-01-01

    Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation. PMID:27805064

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Goldberg José

    2008-08-01

    Full Text Available Abstract Background 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. Methods 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. Results 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 RASSF1A gene, respectively, in relation to the control group. Conclusion 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 panel of

  18. DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

    DEFF Research Database (Denmark)

    Shaknovich, Rita; Cerchietti, Leandro; Tsikitas, Lucas;

    2011-01-01

    The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and t......, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.......The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation...... and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression...

  19. 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...

  20. Qualitatively predicting acetylation and methylation areas in DNA sequences.

    Science.gov (United States)

    Pham, Tho Hoan; Tran, Dang Hung; Ho, Tu Bao; Satou, Kenji; Valiente, Gabriel

    2005-01-01

    Eukaryotic genomes are packaged by the wrapping of DNA around histone octamers to form nucleosomes. Nucleosome occupancy, acetylation, and methylation, which have a major impact on all nuclear processes involving DNA, have been recently mapped across the yeast genome using chromatin immunoprecipitation and DNA microarrays. However, this experimental protocol is laborious and expensive. Moreover, experimental methods often produce noisy results. In this paper, we introduce a computational approach to the qualitative prediction of nucleosome occupancy, acetylation, and methylation areas in DNA sequences. Our method uses support vector machines to discriminate between DNA areas with high and low relative occupancy, acetylation, or methylation, and rank k-gram features based on their support for these DNA modifications. Experimental results on the yeast genome reveal genetic area preferences of nucleosome occupancy, acetylation, and methylation that are consistent with previous studies. Supplementary files are available from http://www.jaist.ac.jp/~tran/nucleosome/.

  1. Epigenetics in Alzheimer's Disease: Perspective of DNA Methylation.

    Science.gov (United States)

    Qazi, Talal Jamil; Quan, Zhenzhen; Mir, Asif; Qing, Hong

    2017-01-14

    Research over the years has shown that causes of Alzheimer's disease are not well understood, but over the past years, the involvement of epigenetic mechanisms in the developing memory formation either under pathological or physiological conditions has become clear. The term epigenetics represents the heredity of changes in phenotype that are independent of altered DNA sequences. Different studies validated that cytosine methylation of genomic DNA decreases with age in different tissues of mammals, and therefore, the role of epigenetic factors in developing neurological disorders in aging has been under focus. In this review, we summarized and reviewed the involvement of different epigenetic mechanisms especially the DNA methylation in Alzheimer's disease (AD), late-onset Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and autosomal dominant Alzheimer's disease (ADAD). Down to the minutest of details, we tried to discuss the methylation patterns like mitochondrial DNA methylation and ribosomal DNA (rDNA) methylation. Additionally, we mentioned some therapeutic approaches related to epigenetics, which could provide a potential cure for AD. Moreover, we reviewed some recent studies that validate DNA methylation as a potential biomarker and its role in AD. We hope that this review will provide new insights into the understanding of AD pathogenesis from the epigenetic perspective especially from the perspective of DNA methylation.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas.

    Science.gov (United States)

    Virmani, A K; Rathi, A; Sathyanarayana, U G; Padar, A; Huang, C X; Cunnigham, H T; Farinas, A J; Milchgrub, S; Euhus, D M; Gilcrease, M; Herman, J; Minna, J D; Gazdar, A F

    2001-07-01

    The adenomatous polyposis coli (APC) gene is a tumor suppressor gene associated with both familial and sporadic cancer. Despite high rates of allelic loss in lung and breast cancers, point mutations of the APC gene are infrequent in these cancer types. Aberrant methylation of the APC promoter 1A occurs in some colorectal and gastric malignancies, and we investigated whether the same mechanism occurs in lung and breast cancers. The methylation status of the APC gene promoter 1A was analyzed in 77 breast, 50 small cell (SCLC), and 106 non-small cell (NSCLC) lung cancer tumors and cell lines and in 68 nonmalignant tissues by methylation-specific PCR. Expression of the APC promoter 1A transcript was examined in a subset of cell lines by reverse transcription-PCR, and loss of heterozygosity at the gene locus was analyzed by the use of 12 microsatellite and polymorphic markers. Statistical tests were two-sided. Promoter 1A was methylated in 34 of 77 breast cancer tumors and cell lines (44%), in 56 of 106 NSCLC tumors and cell lines (53%), in 13 of 50 SCLC cell lines (26%), and in 3 of 68 nonmalignant samples (4%). Most cell lines tested contained the unmethylated or methylated form exclusively. In 27 cell lines tested, there was complete concordance between promoter methylation and silencing of its transcript. Demethylation with 5-aza-2'-deoxycytidine treatment restored transcript 1A expression in all eight methylated cell lines tested. Loss of heterozygosity at the APC locus was observed in 85% of SCLCs, 83% of NSCLCs, and 63% of breast cancer cell lines. The frequency of methylation in breast cancers increased with tumor stage and size. In summary, aberrant methylation of the 1A promoter of the APC gene and loss of its specific transcript is frequently present in breast and NSCLC cancers and cell lines and, to a lesser extent, in SCLC cell lines. Our findings may be of biological and clinical importance.

  5. Methylation-induced blocks to in vitro DNA replication.

    Science.gov (United States)

    Larson, K; Sahm, J; Shenkar, R; Strauss, B

    1985-01-01

    Single-stranded primed M13mp2 templates and double-stranded templates were treated with either dimethyl sulfate (DMS) or N-methyl-N'-nitro-N-nitrosoguanidine and used for DNA synthesis in vitro. Methylation inhibits the ability of the molecules to serve as templates. When either E. coli DNA polymerase I or AMV reverse transcriptase were used as polymerases, DNA synthesis terminated one nucleotide 3' to the site of adenine residues in the template. Heating of the templates resulted in the appearance of additional termination bands one nucleotide before the site of G's in the template. We assume that methylated A's but not methylated G's are blocks to in vitro DNA synthesis and that heating converts a portion of the sites of methylated G to AP sites which are blocks to synthesis.

  6. DNA methylation of SPARC and chronic low back pain

    Directory of Open Access Journals (Sweden)

    Dashwood Thomas

    2011-08-01

    Full Text Available Abstract Background The extracellular matrix protein SPARC (Secreted Protein, Acidic, Rich in Cysteine has been linked to degeneration of the intervertebral discs and chronic low back pain (LBP. In humans, SPARC protein expression is decreased as a function of age and disc degeneration. In mice, inactivation of the SPARC gene results in the development of accelerated age-dependent disc degeneration concurrent with age-dependent behavioral signs of chronic LBP. DNA methylation is the covalent modification of DNA by addition of methyl moieties to cytosines in DNA. DNA methylation plays an important role in programming of gene expression, including in the dynamic regulation of changes in gene expression in response to aging and environmental signals. We tested the hypothesis that DNA methylation down-regulates SPARC expression in chronic LBP in pre-clinical models and in patients with chronic LBP. Results Our data shows that aging mice develop anatomical and behavioral signs of disc degeneration and back pain, decreased SPARC expression and increased methylation of the SPARC promoter. In parallel, we show that human subjects with back pain exhibit signs of disc degeneration and increased methylation of the SPARC promoter. Methylation of either the human or mouse SPARC promoter silences its activity in transient transfection assays. Conclusions This study provides the first evidence that DNA methylation of a single gene plays a role in chronic pain in humans and animal models. This has important implications for understanding the mechanisms involved in chronic pain and for pain therapy.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Epigenome-wide DNA methylation changes with development of arsenic-induced skin lesions in Bangladesh: a case-control follow-up study

    OpenAIRE

    2014-01-01

    Studies have found an association between aberrant DNA methylation and arsenic-induced skin lesions. Yet, little is known about DNA methylation changes over time in people who develop arsenic-induced skin lesions. We sought to investigate epigenome-wide changes of DNA methylation in people who developed arsenic-induced skin lesions in a ten year period. In 2009–2011, we conducted a follow-up study of 900 skin lesion cases and 900 controls and identified 10 people who developed skin lesions si...

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

    Directory of Open Access Journals (Sweden)

    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.

  11. META2: Intercellular DNA Methylation Pairwise Annotation and Integrative Analysis

    Directory of Open Access Journals (Sweden)

    Binhua Tang

    2016-01-01

    Full Text Available Genome-wide deciphering intercellular differential DNA methylation as well as its roles in transcriptional regulation remains elusive in cancer epigenetics. Here we developed a toolkit META2 for DNA methylation annotation and analysis, which aims to perform integrative analysis on differentially methylated loci and regions through deep mining and statistical comparison methods. META2 contains multiple versatile functions for investigating and annotating DNA methylation profiles. Benchmarked with T-47D cell, we interrogated the association within differentially methylated CpG (DMC and region (DMR candidate count and region length and identified major transition zones as clues for inferring statistically significant DMRs; together we validated those DMRs with the functional annotation. Thus META2 can provide a comprehensive analysis approach for epigenetic research and clinical study.

  12. 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.

  13. 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.

  14. 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

  15. DNA methylation supports intrinsic epigenetic memory in mammalian cells.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation.

  16. Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping

    Directory of Open Access Journals (Sweden)

    Maruoka Shuichiro

    2008-12-01

    Full Text Available Abstract Background The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK, a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci. Results Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05 after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes. Conclusion The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models

  17. Identification of Predictive DNA Methylation Biomarkers for Chemotherapy Response in Colorectal Cancer

    Science.gov (United States)

    Baharudin, Rashidah; Ab Mutalib, Nurul-Syakima; Othman, Sri N.; Sagap, Ismail; Rose, Isa M.; Mohd Mokhtar, Norfilza; Jamal, Rahman

    2017-01-01

    Resistance to 5-Fluorouracil (5-FU) is a major obstacle to the successful treatment of colorectal cancer (CRC) and posed an increased risk of recurrence. DNA methylation has been suggested as one of the underlying mechanisms for recurrent disease and its contribution to the development of drug resistance remains to be clarified. This study aimed to determine the methylation phenotype in CRC for identification of predictive markers for chemotherapy response. We performed DNA methylation profiling on 43 non-recurrent and five recurrent CRC patients using the Illumina Infinium HumanMethylation450 Beadchip assay. In addition, CRC cells with different genetic backgrounds, response to 5-FU and global methylation levels (HT29 and SW48) were treated with 5-FU and DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC). The singular and combined effects of these two drug classes on cell viability and global methylation profiles were investigated. Our genome-wide methylation study on the clinical specimens showed that recurrent CRCs exhibited higher methylation levels compared to non-recurrent CRCs. We identified 4787 significantly differentially methylated genes (P < 0.05); 3112 genes were hyper- while 1675 genes were hypomethylated in the recurrent group compared to the non-recurrent. Fifty eight and 47 of the significantly hypermethylated and hypomethylated genes have an absolute recurrent/non-recurrent methylation difference of ≥20%. Most of the hypermethylated genes were involved in the MAPK signaling pathway which is a key regulator for apoptosis while the hypomethylated genes were involved in the PI3K-AKT signaling pathway and proliferation process. We also demonstrate that 5-azadC treatment enhanced response to 5-FU which resulted in significant growth inhibition compared to 5-FU alone in hypermethylated cell lines SW48. In conclusion, we found the evidence of five potentially biologically important genes in recurrent CRCs that could possibly serve as a new

  18. Oxidative Stress and DNA Methylation in Prostate Cancer

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    Krishna Vanaja Donkena

    2010-01-01

    Full Text Available The protective effects of fruits, vegetables, and other foods on prostate cancer may be due to their antioxidant properties. An imbalance in the oxidative stress/antioxidant status is observed in prostate cancer patients. Genome oxidative damage in prostate cancer patients is associated with higher lipid peroxidation and lower antioxidant levels. Oxygen radicals are associated with different steps of carcinogenesis, including structural DNA damage, epigenetic changes, and protein and lipid alterations. Epigenetics affects genetic regulation, cellular differentiation, embryology, aging, cancer, and other diseases. DNA methylation is perhaps the most extensively studied epigenetic modification, which plays an important role in the regulation of gene expression and chromatin architecture, in association with histone modification and other chromatin-associated proteins. This review will provide a broad overview of the interplay of oxidative stress and DNA methylation, DNA methylation changes in regulation of gene expression, lifestyle changes for prostate cancer prevention, DNA methylation as biomarkers for prostate cancer, methods for detection of methylation, and clinical application of DNA methylation inhibitors for epigenetic therapy.

  19. EPIGENETIC EFFECTS OF SHIFTWORK ON BLOOD DNA METHYLATION

    Science.gov (United States)

    Bollati, Valentina; Baccarelli, Andrea; Sartori, Samantha; Tarantini, Letizia; Motta, Valeria; Rota, Federica; Costa, Giovanni

    2012-01-01

    In the present study, the authors investigated the effects of shiftwork exposure on DNA methylation using peripheral blood DNA from subjects working in two chemical plants in Northern Italy. The investigation was designed to evaluate (a) DNA methyl- ation changes in Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements as a surrogate of global methylation and (b) promoter methylation of gluco- corticoid receptor (GCR), tumor necrosis factor alpha (TNF-α), and interferon- gamma (IFN-γ). One hundred and fifty white male workers (mean ± SD: 41.0 ± 9 yrs of age) were examined: 100 3 × 8 rotating shiftworkers (40.4 ± 8.7 yrs of age) and 50 day workers (42.2 ± 9.4 yrs of age). The authors used bisulfite-pyrosequencing to esti- mate repetitive elements and gene-specific methylation. Multiple regression analysis, adjusted for age, body mass index (BMI), and job seniority, did not show any signifi- cant association between the five DNA methylation markers and shiftwork. However, job seniority, in all subjects, was significantly associated with Alu (β = −0.019, p = .033) and IFN-γ (β = −0.224, p 15 yrs) was associated with significantly lower Alu (β = −0.86, p = .006) and IFN-γ methylation (β = −6.50, p = .007) after adjust- ment for age, BMI, and morningness/eveningness. In addition, GCR significantly increased with length of shiftwork (β = 3.33, p = .05). The data showed alterations in blood DNA methylation in a group of shiftworkers, including changes in Alu repeti- tive elements methylation and gene-specific methylation of IFN-γ and TNF-α promoters. Further studies are required to determine the role of such alterations in mediating the effects of shiftwork on human health. PMID:20636218

  20. Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

    Science.gov (United States)

    Sun, Kun; Jiang, Peiyong; Chan, K. C. Allen; Wong, John; Cheng, Yvonne K. Y.; Liang, Raymond H. S.; Chan, Wai-kong; Ma, Edmond S. K.; Chan, Stephen L.; Cheng, Suk Hang; Chan, Rebecca W. Y.; Tong, Yu K.; Ng, Simon S. M.; Wong, Raymond S. M.; Hui, David S. C.; Leung, Tse Ngong; Leung, Tak Y.; Lai, Paul B. S.; Chiu, Rossa W. K.; Lo, Yuk Ming Dennis

    2015-01-01

    Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma. PMID:26392541

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

    Science.gov (United States)

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

    2015-02-01

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

  2. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

    2017-03-03

    The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses activity of transposable elements (TEs), affects gene expression, and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of the worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%) in particular, satellite DNA, retrotransposons, and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H=A, C, and T), and CHH sites, whereas the TE pattern differed, depending on the classes 1 (retrotransposons) and 2 (DNA transposons), respectively. Along genes and TEs, the CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing toward a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared to leaves. This article is protected by copyright. All rights reserved.

  3. Profiling DNA Methylation and Hydroxymethylation at Retrotransposable Elements.

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    de la Rica, Lorenzo; Stanley, Jatinder S; Branco, Miguel R

    2016-01-01

    DNA methylation is a key epigenetic modification controlling the transcriptional activity of mammalian retrotransposable elements. Its oxidation to DNA hydroxymethylation has been linked to DNA demethylation and reactivation of retrotransposons. Here we describe in detail protocols for three methods to measure DNA methylation and hydroxymethylation at specific genomic targets: glucMS-qPCR, and two sequencing approaches (pyrosequencing and high-throughput sequencing) for analyzing bisulfite- and oxidative bisulfite-modified DNA. All three techniques provide absolute measurements of methylation and hydroxymethylation levels at single-base resolution. Differences between the methods are discussed, mainly with respect to throughput and target coverage. These constitute the core techniques that are used in our laboratory for accurately surveying the epigenetics of retrotransposable elements.

  4. 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".

  5. 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.

  6. 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.

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

    Science.gov (United States)

    Kim, Jinsil; Pitlick, Mitchell M.; Christine, Paul J.; Schaefer, Amanda R.; Saleme, Cesar; Comas, Belén; Cosentino, Viviana; Gadow, Enrique; Murray, Jeffrey C.

    2013-01-01

    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. PMID:23533356

  8. 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.

  9. From DNA Copy Number to Gene Expression: Local aberrations, Trisomies and Monosomies

    Science.gov (United States)

    Shay, Tal

    The goal of my PhD research was to study the effect of DNA copy number changes on gene expression. DNA copy number aberrations may be local, encompassing several genes, or on the level of an entire chromosome, such as trisomy and monosomy. The main dataset I studied was of Glioblastoma, obtained in the framework of a collaboration, but I worked also with public datasets of cancer and Down's Syndrome. The molecular basis of expression changes in Glioblastoma. Glioblastoma is the most common and aggressive type of primary brain tumors in adults. In collaboration with Prof. Hegi (CHUV, Switzerland), we analyzed a rich Glioblastoma dataset including clinical information, DNA copy number (array CGH) and expression profiles. We explored the correlation between DNA copy number and gene expression at the level of chromosomal arms and local genomic aberrations. We detected known amplification and over expression of oncogenes, as well as deletion and down-regulation of tumor suppressor genes. We exploited that information to map alterations of pathways that are known to be disrupted in Glioblastoma, and tried to characterize samples that have no known alteration in any of the studied pathways. Identifying local DNA aberrations of biological significance. Many types of tumors exhibit chromosomal losses or gains and local amplifications and deletions. A region that is aberrant in many tumors, or whose copy number change is stronger, is more likely to be clinically relevant, and not just a by-product of genetic instability. We developed a novel method that defines and prioritizes aberrations by formalizing these intuitions. The method scores each aberration by the fraction of patients harboring it, its length and its amplitude, and assesses the significance of the score by comparing it to a null distribution obtained by permutations. This approach detects genetic locations that are significantly aberrant, generating a 'genomic aberration profile' for each sample. The 'genomic

  10. Oxidative stress and DNA methylation regulation in the metabolic syndrome.

    Science.gov (United States)

    Yara, Sabrina; Lavoie, Jean-Claude; Levy, Emile

    2015-01-01

    DNA methylation is implicated in tissue-specific gene expression and genomic imprinting. It is modulated by environmental factors, especially nutrition. Modified DNA methylation patterns may contribute to health problems and susceptibility to complex diseases. Current advances have suggested that the metabolic syndrome (MS) is a programmable disease, which is characterized by epigenetic modifications of vital genes when exposed to oxidative stress. Therefore, the main objective of this paper is to critically review the central context of MS while presenting the most recent knowledge related to epigenetic alterations that are promoted by oxidative stress. Potential pro-oxidant mechanisms that orchestrate changes in methylation profiling and are related to obesity, diabetes and hypertension are discussed. It is anticipated that the identification and understanding of the role of DNA methylation marks could be used to uncover early predictors and define drugs or diet-related treatments able to delay or reverse epigenetic changes, thereby combating MS burden.

  11. Analysis of DNA methylation in various swine tissues.

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    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. Methylation matters? Decreased methylation status of genomic DNA in the blood of schizophrenic twins.

    Science.gov (United States)

    Bönsch, Dominikus; Wunschel, Michael; Lenz, Bernd; Janssen, Gesa; Weisbrod, Matthias; Sauer, Heinrich

    2012-08-15

    Studies of schizophrenia inheritance in identical twins show a concordance of about 50%, which supports an epigenetic model. In our present study we investigated methylation of genomic DNA and promoter methylation of Reelin and SOX10 genes in peripheral blood of twins suffering from schizophrenia. Global DNA methylation was reduced (52.3%) in schizophrenic twins if compared with healthy control twins (65.7%). The reduced methylation was significant in males only. We also found a similar hypomethylation in the non-affected twins of discordant pairs and a mixed group of psychiatric controls. In discordant twins there was a relative hypermethylation of the SOX10 promoter. Within-pair-difference of methylation of Reelin promoter was significantly lower in monozygotic twins than in dizygotic twins.

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

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    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.

  14. 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.

  15. [Methods for detection of methylated cytosine residues in DNA].

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V

    2009-01-01

    The article provides analysis of common methods for DNA methylation detection. Advantages and limitations of methods used for different purposes are compared. Clue step for most common methods is bisulfite treatment of DNA samples and its protocol is described in details. Recommendations are formulated for each method best in solving specific problems.

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

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    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.

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

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    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.

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

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    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.

  19. Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

    NARCIS (Netherlands)

    Smulders, M.J.M.; Rus-Kortekaas, W.; Vosman, B.

    1995-01-01

    The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that

  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. Genome-wide DNA methylation profiling of cell-free serum DNA in esophageal adenocarcinoma and Barrett esophagus.

    Science.gov (United States)

    Zhai, Rihong; Zhao, Yang; Su, Li; Cassidy, Lauren; Liu, Geoffrey; Christiani, David C

    2012-01-01

    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.

  2. 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.

  3. Prenatal antiepileptic exposure associates with neonatal DNA methylation differences.

    Science.gov (United States)

    Smith, Alicia K; Conneely, Karen N; Newport, D Jeffrey; Kilaru, Varun; Schroeder, James W; Pennell, Page B; Knight, Bettina T; Cubells, Joseph C; Stowe, Zachary N; Brennan, Patricia A

    2012-05-01

    Antiepileptic drugs (AEDs) are used to treat a variety of neuropsychiatric illnesses commonly encountered in women during their reproductive years, including epilepsy and bipolar disorder. Despite their widespread use, the impact of prenatal exposure on fetal development remains obscure. To evaluate whether AEDs taken by pregnant mothers influence DNA methylation patterns in their neonates, DNA was extracted from the umbilical cord blood of 201 neonates whose mothers were treated for neuropsychiatric illness during pregnancy and interrogated across 27,578 CpG sites using the Illumina HumanMethylation27 BeadChip. The association of each methylation value with the cumulative duration of prenatal AED exposure was examined using a linear mixed model. The average methylation level across all CpG sites was calculated for each subject, and this global methylation measure was evaluated similarly. Neonates with a longer duration of AED exposure in pregnancy showed a decrease in average global methylation (p = 0.0045). Further, DNA methylation of CpG sites in 14 genes significantly decreased with the duration of prenatal AED exposure even after adjusting for multiple comparisons (FDR < 0.05). For a small subset (n = 19) of these neonates, a second tissue, placenta, was available in addition to cord blood. Methylation of 3 of these 14 CpG sites was also significantly decreased in placental tissue. These novel data suggest decreased DNA methylation in neonates of mothers who took AEDs during pregnancy. The long-term stability and potential impact of these changes warrant further attention, and caution may be warranted before prescribing AEDs to pregnant women.

  4. 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.

  5. DNA Methylation Alterations in Breast Cancer

    Science.gov (United States)

    2002-07-01

    EDTA buffer (1-1TE), mary tumor prostate tissues, five matched pairs of normal 5 ViL of 10 x T4 DNA ligase buffer, 1.25 giL each of and primary tumor... DNA ligase were added, and the DNA was incubated breast tissues, was used in the study. The breast and overnight at 16’C. The ligated DNA was...nanograms of Notl primer tion endonuclease Notl and T4 DNA ligase were pur- and 30 ng of Msel primer were used for PCR with chased from Boehringer Mannheim

  6. Salt stress alters DNA methylation levels in alfalfa (Medicago spp).

    Science.gov (United States)

    Al-Lawati, A; Al-Bahry, S; Victor, R; Al-Lawati, A H; Yaish, M W

    2016-02-26

    Modification of DNA methylation status is one of the mechanisms used by plants to adjust gene expression at both the transcriptional and posttranscriptional levels when plants are exposed to suboptimal conditions. Under abiotic stress, different cultivars often show heritable phenotypic variation accompanied by epigenetic polymorphisms at the DNA methylation level. This variation may provide the raw materials for plant breeding programs that aim to enhance abiotic stress tolerance, including salt tolerance. In this study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to assess cytosine methylation levels in alfalfa (Medicago spp) roots exposed to increasing NaCl concentrations (0.0, 8.0, 12.0, and 20.0 dS/m). Eleven indigenous landraces were analyzed, in addition to a salt-tolerant cultivar that was used as a control. There was a slight increase in DNA methylation upon exposure to high levels of soil salinity. Phylogenetic analysis using MSAP showed epigenetic variation within and between the alfalfa landraces when exposed to saline conditions. Based on MSAP and enzyme-linked immunosorbent assay results, we found that salinity increased global DNA methylation status, particularly in plants exposed to the highest level of salinity (20 dS/m). Quantitative reverse transcription-polymerase chain reaction indicated that this might be mediated by the overexpression of methyltransferase homolog genes after exposure to saline conditions. DNA demethylation using 5-azacytidine reduced seedling lengths and dry and fresh weights, indicating a possible decrease in salinity tolerance. These results suggest that salinity affects DNA methylation flexibility.

  7. Dynamic DNA methylation controls glutamate receptor trafficking and synaptic scaling.

    Science.gov (United States)

    Sweatt, J David

    2016-05-01

    Hebbian plasticity, including long-term potentiation and long-term depression, has long been regarded as important for local circuit refinement in the context of memory formation and stabilization. However, circuit development and stabilization additionally relies on non-Hebbian, homeostatic, forms of plasticity such as synaptic scaling. Synaptic scaling is induced by chronic increases or decreases in neuronal activity. Synaptic scaling is associated with cell-wide adjustments in postsynaptic receptor density, and can occur in a multiplicative manner resulting in preservation of relative synaptic strengths across the entire neuron's population of synapses. Both active DNA methylation and demethylation have been validated as crucial regulators of gene transcription during learning, and synaptic scaling is known to be transcriptionally dependent. However, it has been unclear whether homeostatic forms of plasticity such as synaptic scaling are regulated via epigenetic mechanisms. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously largely considered separately: DNA methylation, homeostatic plasticity, and glutamate receptor trafficking. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously considered separately: glutamate receptor trafficking, DNA methylation, and homeostatic plasticity.

  8. 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.

  9. Techniques of DNA methylation analysis with nutritional applications.

    Science.gov (United States)

    Mansego, Maria L; Milagro, Fermín I; Campión, Javier; Martínez, J Alfredo

    2013-01-01

    Epigenetic mechanisms are likely to play an important role in the regulation of metabolism and body weight through gene-nutrient interactions. This review focuses on methods for analyzing one of the most important epigenetic mechanisms, DNA methylation, from single nucleotide to global measurement depending on the study goal and scope. In addition, this study highlights the major principles and methods for DNA methylation analysis with emphasis on nutritional applications. Recent developments concerning epigenetic technologies are showing promising results of DNA methylation levels at a single-base resolution and provide the ability to differentiate between 5-methylcytosine and other nucleotide modifications such as 5-hydroxymethylcytosine. A large number of methods can be used for the analysis of DNA methylation such as pyrosequencing™, primer extension or real-time PCR methods, and genome-wide DNA methylation profile from microarray or sequencing-based methods. Researchers should conduct a preliminary analysis focused on the type of validation and information provided by each technique in order to select the best method fitting for their nutritional research interests.

  10. Chromosomal Aberrations and DNA Repair Gene Variants in a Radon-exposed Population

    Energy Technology Data Exchange (ETDEWEB)

    Kiuru, A.; Lindholm, C.; Koivistoinen, A.; Salomaa, S.

    2004-07-01

    Polymorphisms of XRCC1 (X-ray repair cross-complementing group 1), XRCC3 (X-ray repair cross-complementing group 3), and hOGG1 (the human homologue of the yeast OGG1 gene) DNA repair genes have been associated with altered DNA repair capacity and risk of various cancers. In the present study our goal was to clarify the influence of various DNA repair gene variants on the frequency of chromosomal aberrations (CA) in subjects exposed to residential radon. The study group of 84 non-smoking, healthy individuals exposed to domestic radon were analysed using the fluorescence in-situ hybridization (FISH) technique. No association between radon concentration and CA frequencies was observed. However, a significant increase with age was shown as well as a large variability in translocation frequencies between individuals within the same age group. In order to investigate the role of individual susceptibility to this variation genotypes of DNA repair genes XRCC1 (codons 194, 280 and 399), XRCC3 (codon 241) and hOGG1 (codon 326) were determined from leukocyte DNA using methods based on polymerase chain reaction. Multiple regression analysis was applied to evaluate the effect of the polymorphisms and the other confounding factors (age, exposure to randon etc) to the frequency of CA. The preliminary statistical analyses showed that the different gene appeared not to be related to a pronounced increase in chromosome aberration frequencies observed by FISH painting. However, the analysis indicated that the homozygous variant of XRCC3 codon 241 was associated (P<0.05) with two-ways translocations in conjunction with age. Larger studies, both with regard to the cohort and the number of gene variants are needed to elucidate the influence of other DNA repair variants to the yield of chromosomal aberrations. The results indicate that the chromosomal translocations accumulated by age (spontaneous background) may be partly explained by defects in homologous recombination repair. (Author

  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. DNA methylation is globally disrupted and associated with expression changes in chronic obstructive pulmonary disease small airways.

    Science.gov (United States)

    Vucic, Emily A; Chari, Raj; Thu, Kelsie L; Wilson, Ian M; Cotton, Allison M; Kennett, Jennifer Y; Zhang, May; Lonergan, Kim M; Steiling, Katrina; Brown, Carolyn J; McWilliams, Annette; Ohtani, Keishi; Lenburg, Marc E; Sin, Don D; Spira, Avrum; Macaulay, Calum E; Lam, Stephen; Lam, Wan L

    2014-05-01

    DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illumina's Infinium HM27 and Affymetrix's Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2-related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.

  13. 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...

  14. 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

  15. Genetic variation in DNMT3B and increased global DNA methylation is associated with suicide attempts in psychiatric patients.

    Science.gov (United States)

    Murphy, T M; Mullins, N; Ryan, M; Foster, T; Kelly, C; McClelland, R; O'Grady, J; Corcoran, E; Brady, J; Reilly, M; Jeffers, A; Brown, K; Maher, A; Bannan, N; Casement, A; Lynch, D; Bolger, S; Buckley, A; Quinlivan, L; Daly, L; Kelleher, C; Malone, K M

    2013-02-01

    Recently, a significant epigenetic component in the pathology of suicide has been realized. Here we investigate candidate functional SNPs in epigenetic-regulatory genes, DNMT1 and DNMT3B, for association with suicide attempt (SA) among patients with co-existing psychiatric illness. In addition, global DNA methylation levels [5-methyl cytosine (5-mC%)] between SA and psychiatric controls were quantified using the Methylflash Methylated DNA Quantification Kit. DNA was obtained from blood of 79 suicide attempters and 80 non-attempters, assessed for DSM-IV Axis I disorders. Functional SNPs were selected for each gene (DNMT1; n = 7, DNMT3B; n = 10), and genotyped. A SNP (rs2424932) residing in the 3' UTR of the DNMT3B gene was associated with SA compared with a non-attempter control group (P = 0.001; Chi-squared test, Bonferroni adjusted P value = 0.02). Moreover, haplotype analysis identified a DNMT3B haplotype which differed between cases and controls, however this association did not hold after Bonferroni correction (P = 0.01, Bonferroni adjusted P value = 0.56). Global methylation analysis showed that psychiatric patients with a history of SA had significantly higher levels of global DNA methylation compared with controls (P = 0.018, Student's t-test). In conclusion, this is the first report investigating polymorphisms in DNMT genes and global DNA methylation quantification in SA risk. Preliminary findings suggest that allelic variability in DNMT3B may be relevant to the underlying diathesis for suicidal acts and our findings support the hypothesis that aberrant DNA methylation profiles may contribute to the biology of suicidal acts. Thus, analysis of global DNA hypermethylation in blood may represent a biomarker for increased SA risk in psychiatric patients.

  16. Increased DNA methylation of Dnmt3b targets impairs leukemogenesis.

    Science.gov (United States)

    Schulze, Isabell; Rohde, Christian; Scheller-Wendorff, Marina; Bäumer, Nicole; Krause, Annika; Herbst, Friederike; Riemke, Pia; Hebestreit, Katja; Tschanter, Petra; Lin, Qiong; Linhart, Heinz; Godley, Lucy A; Glimm, Hanno; Dugas, Martin; Wagner, Wolfgang; Berdel, Wolfgang E; Rosenbauer, Frank; Müller-Tidow, Carsten

    2016-03-24

    The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells. Dnmt3b has recently been shown to play a role in genic methylation. To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knock-in mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function. Forced Dnmt3b expression induced widespread DNA hypermethylation inMyc-Bcl2-induced leukemias, preferentially at gene bodies.MLL-AF9-induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Acute myeloid leukemia patients with high expression of Dnmt3b target genes showed inferior survival. Together, these findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function.

  17. 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.

  18. Cord Blood DNA Methylation Biomarkers for Predicting Neurodevelopmental Outcomes

    Directory of Open Access Journals (Sweden)

    Nicolette A. Hodyl

    2016-12-01

    Full Text Available Adverse environmental exposures in pregnancy can significantly alter the development of the fetus resulting in impaired child neurodevelopment. Such exposures can lead to epigenetic alterations like DNA methylation, which may be a marker of poor cognitive, motor and behavioral outcomes in the infant. Here we review studies that have assessed DNA methylation in cord blood following maternal exposures that may impact neurodevelopment of the child. We also highlight some key studies to illustrate the potential for DNA methylation to successfully identify infants at risk for poor outcomes. While the current evidence is limited, in that observations to date are largely correlational, in time and with larger cohorts analyzed and longer term follow-up completed, we may be able to develop epigenetic biomarkers that not only indicate adverse early life exposures but can also be used to identify individuals likely to be at an increased risk of impaired neurodevelopment even in the absence of detailed information regarding prenatal environment.

  19. DNA methylation dynamics in plants and mammals: overview of regulation and dysregulation.

    Science.gov (United States)

    Elhamamsy, Amr Rafat

    2016-07-01

    DNA methylation is a major epigenetic marking mechanism regulating various biological functions in mammals and plant. The crucial role of DNA methylation has been observed in cellular differentiation, embryogenesis, genomic imprinting and X-chromosome inactivation. Furthermore, DNA methylation takes part in disease susceptibility, responses to environmental stimuli and the biodiversity of natural populations. In plant, different types of environmental stress have demonstrated the ability to alter the archetype of DNA methylation through the genome, change gene expression and confer a mechanism of adaptation. DNA methylation dynamics are regulated by three processes de novo DNA methylation, methylation maintenance and DNA demethylation. These processes have their similarities and differences between mammals and plants. Furthermore, the dysregulation of DNA methylation dynamics represents one of the primary molecular mechanisms of developing diseases in mammals. This review discusses the regulation and dysregulation of DNA methylation in plants and mammals. Copyright © 2016 John Wiley & Sons, Ltd.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  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. 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.

  6. Genetic variants of methyl metabolizing enzymes and epigenetic regulators: Associations with promoter CpG island hypermethylation in colorectal cancer

    NARCIS (Netherlands)

    Vogel, S. de; Wouters, K.A.D.; Gottschalk, R.W.H.; Schooten, F.J. van; Goeij, A.F.P.M. de; Bruïne, A.P. de; Goldbohm, R.A.; Brandt, P.A. van den; Weijenberg, M.P.; Engeland, M. van

    2009-01-01

    Aberrant DNA methylation affects carcinogenesis of colorectal cancer. Folate metabolizing enzymes may influence the bioavailability of methyl groups, whereas DNA and histone methyltransferases are involved in epigenetic regulation of gene expression. We studied associations of genetic variants of fo

  7. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

    OpenAIRE

    Lopez de Silanes, I.; Gorospe, M.; Taniguchi, H; Abdelmohsen, K; Srikantan, S.; Alaminos, M.; Berdasco, M.; Urdinguio, R. G.; Fraga, M. F.; Jacinto, F. V.; Esteller, M.

    2009-01-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3???UTR pr...

  8. 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.

  9. DNA methylation by DNMT1 and DNMT3b methyltransferases is driven by the MUC1-C oncoprotein in human carcinoma cells.

    Science.gov (United States)

    Rajabi, H; Tagde, A; Alam, M; Bouillez, A; Pitroda, S; Suzuki, Y; Kufe, D

    2016-12-15

    Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces the expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

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

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    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. Application of the rank-based method to DNA methylation for cancer diagnosis.

    Science.gov (United States)

    Li, Hongdong; Hong, Guini; Xu, Hui; Guo, Zheng

    2015-01-25

    Detecting aberrant DNA methylation as diagnostic or prognostic biomarkers for cancer has been a topic of considerable interest recently. However, current classifiers based on absolute methylation values detected from a cohort of samples are typically difficult to be transferable to other cohorts of samples. Here, focusing on relative methylation levels, we employed a modified rank-based method to extract reversal pairs of CpG sites whose relative methylation level orderings differ between disease samples and normal controls for cancer diagnosis. The reversal pairs identified for five cancer types respectively show excellent prediction performance with the accuracy above 95%. Furthermore, when evaluating the reversal pairs identified for one cancer type in an independent cohorts of samples, we found that they could distinguish different subtypes of this cancer or different malignant stages including early stage of this cancer from normal controls. The identified reversal pairs also appear to be specific to cancer type. In conclusion, the reversal pairs detected by the rank-based method could be used for accurate cancer diagnosis, which are transferable to independent cohorts of samples.

  12. Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay.

    Science.gov (United States)

    Lin, Xiang-Cheng; Zhang, Ting; Liu, Lan; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

    2016-01-19

    Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

  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 defining DNA methylation signature of Floating-Harbor Syndrome.

    Science.gov (United States)

    Hood, Rebecca L; Schenkel, Laila C; Nikkel, Sarah M; Ainsworth, Peter J; Pare, Guillaume; Boycott, Kym M; Bulman, Dennis E; Sadikovic, Bekim

    2016-12-09

    Floating-Harbor syndrome (FHS) is an autosomal dominant genetic condition characterized by short stature, delayed osseous maturation, expressive language impairment, and unique facial dysmorphology. We previously identified mutations in the chromatin remodeling protein SRCAP (SNF2-related CBP Activator Protein) as the cause of FHS. SRCAP has multiple roles in chromatin and transcriptional regulation; however, specific epigenetic consequences of SRCAP mutations remain to be described. Using high resolution genome-wide DNA methylation analysis, we identified a unique and highly specific DNA methylation "epi-signature" in the peripheral blood of individuals with FHS. Both hyper and hypomethylated loci are distributed across the genome, preferentially occurring in CpG islands. Clonal bisulfite sequencing of two hypermethylated (FIGN and STPG2) and two hypomethylated (MYO1F and RASIP1) genes confirmed these findings. The identification of a unique methylation signature in FHS provides further insight into the biological function of SRCAP and provides a unique biomarker for this disorder.

  15. Associations between DNA methylation and schizophrenia-related intermediate phenotypes - a gene set enrichment analysis.

    Science.gov (United States)

    Hass, Johanna; Walton, Esther; Wright, Carrie; Beyer, Andreas; Scholz, Markus; Turner, Jessica; Liu, Jingyu; Smolka, Michael N; Roessner, Veit; Sponheim, Scott R; Gollub, Randy L; Calhoun, Vince D; Ehrlich, Stefan

    2015-06-03

    Multiple genetic approaches have identified microRNAs as key effectors in psychiatric disorders as they post-transcriptionally regulate expression of thousands of target genes. However, their role in specific psychiatric diseases remains poorly understood. In addition, epigenetic mechanisms such as DNA methylation, which affect the expression of both microRNAs and coding genes, are critical for our understanding of molecular mechanisms in schizophrenia. Using clinical, imaging, genetic, and epigenetic data of 103 patients with schizophrenia and 111 healthy controls of the Mind Clinical Imaging Consortium (MCIC) study of schizophrenia, we conducted gene set enrichment analysis to identify markers for schizophrenia-associated intermediate phenotypes. Genes were ranked based on the correlation between DNA methylation patterns and each phenotype, and then searched for enrichment in 221 predicted microRNA target gene sets. We found the predicted hsa-miR-219a-5p target gene set to be significantly enriched for genes (EPHA4, PKNOX1, ESR1, among others) whose methylation status is correlated with hippocampal volume independent of disease status. Our results were strengthened by significant associations between hsa-miR-219a-5p target gene methylation patterns and hippocampus-related neuropsychological variables. IPA pathway analysis of the respective predicted hsa-miR-219a-5p target genes revealed associated network functions in behavior and developmental disorders. Altered methylation patterns of predicted hsa-miR-219a-5p target genes are associated with a structural aberration of the brain that has been proposed as a possible biomarker for schizophrenia. The (dys)regulation of microRNA target genes by epigenetic mechanisms may confer additional risk for developing psychiatric symptoms. Further study is needed to understand possible interactions between microRNAs and epigenetic changes and their impact on risk for brain-based disorders such as schizophrenia.

  16. 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.

  17. DNA methylation in obesity and type 2 diabetes.

    Science.gov (United States)

    de Mello, Vanessa Derenji Ferreira; Pulkkinen, Leena; Lalli, Marianne; Kolehmainen, Marjukka; Pihlajamäki, Jussi; Uusitupa, Matti

    2014-05-01

    To elucidate the mechanisms related to the development of type 2 diabetes (T2D) and other degenerative diseases at a molecular level, a better understanding of the changes in the chromatin structure and the corresponding functional changes in molecular pathways is still needed. For example, persons with low birth weight are at a high risk for development of T2D later in life, suggesting that the intrauterine environment contributes to the disease. One of the hypotheses is that epigenetic regulation, including changes in DNA methylation leading to modifications in chromatin structure, are behind metabolic alterations, e.g. leading to the phenomenon termed metabolic memory. Altered DNA methylation has been shown to affect healthy aging and also to promote age-related health problems. There is suggestive evidence that lifestyle changes including weight loss can have an impact on DNA methylation and consequently gene expression. In this review we provide an overview of human studies investigating DNA methylation in obesity and T2D and associated risk factors behind these diseases.

  18. 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.

  19. 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.

  20. DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development.

    Science.gov (United States)

    Ngernprasirtsiri, J; Kobayashi, H; Akazawa, T

    1988-09-01

    We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.

  1. Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants.

    Science.gov (United States)

    Smulders, M J; Rus-Kortekaas, W; Vosman, B

    1995-12-01

    The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.

  2. DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

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    Alessandra eMaresca

    2015-03-01

    Full Text Available Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is

  3. 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...

  4. Quality evaluation of methyl binding domain based kits for enrichment DNA-methylation sequencing.

    Directory of Open Access Journals (Sweden)

    Tim De Meyer

    Full Text Available DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1 to identify a best performing kit for MBD-based enrichment using independent validation data, and 2 to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3. Reduced representation bisulfite sequencing data (all three cell lines and publicly available Illumina Infinium BeadChip data (DU145 and PC3 were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode's MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications.

  5. No difference in the frequency of locus-specific methylation in the peripheral blood DNA of women diagnosed with breast cancer and age-matched controls

    DEFF Research Database (Denmark)

    Wojdacz, Tomasz K; Thestrup, Britta Boserup; Cold, Søren

    2011-01-01

    , to the pathology of different diseases, remains open. Recently, a number of studies addressed the question of the prevalence of aberrant methylation of cancer-related genes in peripheral blood leukocyte (PBL) DNA and indicated a strong possibility that the presence of constitutional methylation of different genes...... might predispose for cancer development. Here, we have used the methlyation-sensitive high-resolution melting approach to examine the methylation status of the BRCA1, BRCA2, APC, RASSF1A and RARβ2 genes in PBLs of a group of women diagnosed with breast cancer, and an age-matched control group...... with no signs of breast cancer. No significant differences in the frequency of methylation of the above genes were found between cases and controls in our study. Hence, testing for the presence of methylation of cancer-related genes in PBL DNA from women diagnosed with sporadic breast cancer and classified...

  6. Regulators of DNA methylation in mammalian cells

    OpenAIRE

    Termanis, Ausma

    2013-01-01

    Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly t...

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. 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

    2016-07-07

    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.

  11. Histone H1 Limits DNA Methylation in Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Michael Seymour

    2016-07-01

    Full Text Available 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 ΔhH1 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 ΔhH1 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.

  12. DNA methylation programming and reprogramming in primate embryonic stem cells.

    Science.gov (United States)

    Cohen, Netta Mendelson; Dighe, Vikas; Landan, Gilad; Reynisdóttir, Sigrún; Palsson, Arnar; Mitalipov, Shoukhrat; Tanay, Amos

    2009-12-01

    DNA methylation is an important epigenetic mechanism, affecting normal development and playing a key role in reprogramming epigenomes during stem cell derivation. Here we report on DNA methylation patterns in native monkey embryonic stem cells (ESCs), fibroblasts, and ESCs generated through somatic cell nuclear transfer (SCNT), identifying and comparing epigenome programming and reprogramming. We characterize hundreds of regions that are hyper- or hypomethylated in fibroblasts compared to native ESCs and show that these are conserved in human cells and tissues. Remarkably, the vast majority of these regions are reprogrammed in SCNT ESCs, leading to almost perfect correlation between the epigenomic profiles of the native and reprogrammed lines. At least 58% of these changes are correlated in cis to transcription changes, Polycomb Repressive Complex-2 occupancy, or binding by the CTCF insulator. We also show that while epigenomic reprogramming is extensive and globally accurate, the efficiency of adding and stripping DNA methylation during reprogramming is regionally variable. In several cases, this variability results in regions that remain methylated in a fibroblast-like pattern even after reprogramming.

  13. Gene-specific DNA methylation profiles and LINE-1 hypomethylation are associated with myocardial infarction risk

    NARCIS (Netherlands)

    Guarrera, Simonetta; Fiorito, Giovanni; Onland-Moret, N Charlotte; Russo, Alessia; Agnoli, Claudia; Allione, Alessandra; Di Gaetano, Cornelia; Mattiello, Amalia; Ricceri, Fulvio; Chiodini, Paolo; Polidoro, Silvia; Frasca, Graziella; Verschuren, Monique W M; Boer, Jolanda M A; Iacoviello, Licia; van der Schouw, Yvonne T; Tumino, Rosario; Vineis, Paolo; Krogh, Vittorio; Panico, Salvatore; Sacerdote, Carlotta; Matullo, Giuseppe

    2015-01-01

    BACKGROUND: DNA methylation profiles are responsive to environmental stimuli and metabolic shifts. This makes DNA methylation a potential biomarker of environmental-related and lifestyle-driven diseases of adulthood. Therefore, we investigated if white blood cells' (WBCs) DNA methylation profiles ar

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

    Directory of Open Access Journals (Sweden)

    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

  15. 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

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

    Science.gov (United States)

    GUERRERO-PRESTON, RAFAEL; HADAR, TAL; OSTROW, KIMBERLY LASKIE; SOUDRY, ETHAN; ECHENIQUE, MIGUEL; ILI-GANGAS, CARMEN; PÉREZ, GABRIELA; PEREZ, JIMENA; BREBI-MIEVILLE, PRISCILLA; DESCHAMPS, JOSÉ; MORALES, LUISA; BAYONA, MANUEL; SIDRANSKY, DAVID; MATTA, JAIME

    2014-01-01

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

  17. 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.

  18. Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

    Science.gov (United States)

    Salilew-Wondim, Dessie; Fournier, Eric; Hoelker, Michael; Saeed-Zidane, Mohammed; Tholen, Ernst; Looft, Christian; Neuhoff, Christiane; Besenfelder, Urban; Havlicek, Vita; Rings, Franca; Gagné, Dominic; Sirard, Marc-André; Robert, Claude; Shojaei Saadi, Habib A; Gad, Ahmed; Schellander, Karl; Tesfaye, Dawit

    2015-01-01

    Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and

  19. Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

    Directory of Open Access Journals (Sweden)

    Dessie Salilew-Wondim

    Full Text Available Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY, 4-cell (4C or 16-cell (16C were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP. Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic

  20. 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.

  1. 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.

  2. 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.

  3. Analysis of DNA methylation and virus induced DNA methylation change in MD-resistant and –susceptible chickens

    Directory of Open Access Journals (Sweden)

    Juan eLuo

    2012-02-01

    Full Text Available Marek’s disease (MD is a lymphoproliferative disease induced by Marek’s disease virus (MDV infection. With the less efficacy of vaccine in MD prevention, genetically selected MD-resistant chicken becomes more and more important in MD control. To elucidate the mechanism of MD-resistance, lots of researches were focused on the different genetic differences between the resistant and susceptible chickens. However, less is known about the epigenetic features. By using pyrosequencing, the promoter methylations of 18 candidate genes in MD-resistant (L63 and –susceptible (L72 chicken were examined, we found that most of the genes have higher methylation level in L72. MDV infection changed the expressions of all three methyltransferases (DNMT1, DNMT3a and DNMT3b, while the up-regulation of DNMT1 was only shown in L72 and down-regulation of DNMT3b only in L63 at 21dpi. Interestingly, a dynamic change of promoter methylation was observed in most of the genes in both lines during MDV life cycle. Seven genes, including HDAC9, GH, STAT1, CIITA, FABP3, LATS2 and H2Ac, showed different methylation behavior between L63 and L72. In summary, the findings from this study suggested that DNA methylation heterogeneity exists in MD-resistance and –susceptibility chicken, and MDV infection also induces differential methylation alterations, both indicating epigenetic factor may involve in MD.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Hye Youn; Choi, Eun Nam [Department of Biochemistry, School of Medicine, Ewha Womans University, 911-1 Mok-6-dong, Yangcheon-ku, Seoul 158-710 (Korea, Republic of); Ahn Jo, Sangmee [Department of Pharmacy, College of Pharmacy, Dankook University, San 29 Anseo-dong, Dongnam-gu, Cheonan-si, Chungnam 330-714 (Korea, Republic of); Oh, Seikwan [Department of Neuroscience and TIDRC, School of Medicine, Ewha Womans University, 911-1 Mok-6-dong, Yangcheon-ku, Seoul 158-710 (Korea, Republic of); Ahn, Jung-Hyuck, E-mail: ahnj@ewha.ac.kr [Department of Biochemistry, School of Medicine, Ewha Womans University, 911-1 Mok-6-dong, Yangcheon-ku, Seoul 158-710 (Korea, Republic of)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory

  5. Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Rossner, Pavel, E-mail: prossner@biomed.cas.cz; Rossnerova, Andrea; Beskid, Olena; Tabashidze, Nana; Libalova, Helena; Uhlirova, Katerina; Topinka, Jan; Sram, Radim J.

    2014-05-15

    Highlights: • We analyzed the effect of air pollutants on NHEJ and chromosome aberrations. • In HEL12469 cells B[a]P and extractable organic matter induced DSBs. • The compounds induced XRCC4 expression and a weak Ku70/80 response. • We found increased frequency of aberrations of chromosomes 1, 2, 4, 5, 7 and 17. • The tested compounds preferentially affected chromosome 7. - Abstract: In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 μm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24 h with the following concentrations of tested chemicals: B[a]P: 1 μM, 10 μM, 25 μM; EOMs: 1 μg/ml, 10 μg/ml, 25 μg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25 μM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and

  6. Recommendations for a nomenclature system for reporting methylation aberrations in imprinted domains

    DEFF Research Database (Denmark)

    Monk, David; Morales, Joannella; den Dunnen, Johan T;

    2016-01-01

    Disorders we have discussed these issues and designed a nomenclature for naming imprinted DMRs as well as for reporting methylation values. We apply these recommendations for imprinted DMRs that are commonly assayed in clinical laboratories and show how they support standardized database submission....... The recommendations are in line with existing recommendations, most importantly the Human Genome Variation Society nomenclature, and should facilitate accurate reporting and data exchange among laboratories and thereby help to avoid future confusion....

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Genetic differences and aberrant methylation in the apelin system predict the risk of high-altitude pulmonary edema

    Science.gov (United States)

    Mishra, Aastha; Kohli, Samantha; Dua, Sanchi; Thinlas, Tashi; Mohammad, Ghulam; Pasha, M. A. Qadar

    2015-01-01

    Hypoxia-inducible factor stimulates the expression of apelin, a potent vasodilator, in response to reduced blood arterial oxygen saturation. However, aberrations in the apelin system impair pulmonary vascular function, potentially resulting in the development of high-altitude (HA)-related disorders. This study aimed to elucidate the genetic and epigenetic regulation of apelin, apelin receptor (APLNR), and endothelial nitric oxide synthase (NOS3) in HA adaptation and HA pulmonary edema (HAPE). A genome-wide association study and sequencing identified variants of apelin, APLNR, and NOS3 that were validated in a larger sample size of HAPE-patients (HAPE-p), HAPE-free controls (HAPE-f), and healthy highland natives (HLs). Apelin-13 and nitrite levels and apelin and NOS3 expression were down-regulated in HAPE-p (P < 0.001). Among the several studied polymorphisms, apelin rs3761581, rs2235312, and rs3115757; APLNR rs11544374 and rs2282623; and NOS3 4b/4a, rs1799983, and rs7830 were associated with HAPE (P < 0.03). The risk allele rs3761581G was associated with a 58.6% reduction in gene expression (P = 0.017), and the risk alleles rs3761581G and rs2235312T were associated with low levels of apelin-13 and nitrite (P < 0.05). The latter two levels decreased further when both of these risk alleles were present in the patients (P < 0.05). Methylation of the apelin CpG island was significantly higher in HAPE-p at 11.92% than in HAPE-f and HLs at ≤7.1% (P < 0.05). Moreover, the methylation effect was 9% stronger in the 5′ UTR and was associated with decreased apelin expression and apelin-13 levels. The rs3761581 and rs2235312 polymorphisms and methylation of the CpG island influence the expression of apelin in HAPE. PMID:25918383

  12. Chromosomal aberrations, Yq microdeletion, and sperm DNA fragmentation in infertile men opting for assisted reproduction.

    Science.gov (United States)

    Shamsi, Monis B; Kumar, Rajeev; Malhotra, Neena; Singh, Nita; Mittal, Suneeta; Upadhyay, Ashish D; Dada, Rima

    2012-09-01

    Male infertility is a multi-factorial disorder, and identification of its etiology in an individual is critical for treatment. Systematically elucidating the underlying genetic causes (chromosomal and Yq microdeletion) and factors, such as reactive oxygen species (ROS) levels and total antioxidant capacity (TAC), which contribute to sperm DNA damage, may help to reduce the number of men with idiopathic infertility and provide them with the most suitable therapeutics and counseling. This study was done to comprehensively investigate genetic and oxidative stress factors that might be the etiology of a large percentage of men with idiopathic infertility. One hundred twelve infertile men and 76 fertile controls were screened for chromosomal aberrations and Yq microdeletions. ROS, TAC, and sperm DNA damage were assessed in cytogenetically normal, non-azoospermic men with intact Y chromosome (n = 93). ROS was assessed in neat and washed semen by chemiluminescence; seminal TAC with a commercially available kit; and sperm DNA damage by the comet assay. Two men had cytogenetic abnormalities and seven men harbored Yq microdeletions. ROS levels in neat and washed semen of infertile men were significantly higher (P fragmentation in infertile men was significantly higher (P < 0.01) than controls. Genetic factors and oxidative stress cumulatively account for large number of idiopathic infertile cases. Unlike, genetic causes, which cannot be cured, timely identification and management of oxidative stress may help to reverse/reduce the effects on induced DNA damage, and improve the outcomes for infertile males.

  13. DNA methylation profiling of primary neuroblastoma tumors using methyl-CpG-binding domain sequencing.

    Science.gov (United States)

    Decock, Anneleen; Ongenaert, Maté; Van Criekinge, Wim; Speleman, Frank; Vandesompele, Jo

    2016-02-02

    Comprehensive genome-wide DNA methylation studies in neuroblastoma (NB), a childhood tumor that originates from precursor cells of the sympathetic nervous system, are scarce. Recently, we profiled the DNA methylome of 102 well-annotated primary NB tumors by methyl-CpG-binding domain (MBD) sequencing, in order to identify prognostic biomarker candidates. In this data descriptor, we give details on how this data set was generated and which bioinformatics analyses were applied during data processing. Through a series of technical validations, we illustrate that the data are of high quality and that the sequenced fragments represent methylated genomic regions. Furthermore, genes previously described to be methylated in NB are confirmed. As such, these MBD sequencing data are a valuable resource to further study the association of NB risk factors with the NB methylome, and offer the opportunity to integrate methylome data with other -omic data sets on the same tumor samples such as gene copy number and gene expression, also publically available.

  14. Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human

    Science.gov (United States)

    Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

    2017-01-01

    DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation. PMID:28212312

  15. Aberrant Promoter Methylation of the Tumour Suppressor RASSF10 and Its Growth Inhibitory Function in Breast Cancer

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    Antje M. Richter

    2016-02-01

    Full Text Available Breast cancer is the most common cancer in women, with 1.7 million new cases each year. As early diagnosis and prognosis are crucial factors in cancer treatment, we investigated potential DNA methylation biomarkers of the tumour suppressor family Ras-association domain family (RASSF. Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby promotes cancer development and progression. In this study we analysed the tumour suppressors RASSF1A and RASSF10. Our study shows that RASSF10 is expressed in normal breast but inactivated by methylation in breast cancer. We observed a significant inactivating promoter methylation of RASSF10 in primary breast tumours. RASSF10 is inactivated in 63% of primary breast cancer samples but only 4% of normal control breast tissue is methylated (p < 0.005. RASSF1A also shows high promoter methylation levels in breast cancer of 56% vs. 8% of normal tissue (p < 0.005. Interestingly more than 80% of breast cancer samples harboured a hypermethylation of RASSF10 and/or RASSF1A promoter. Matching samples exhibited a strong tumour specific promoter methylation of RASSF10 in comparison to the normal control breast tissue. Demethylation treatment of breast cancer cell lines MCF7 and T47D reversed RASSF10 promoter hypermethylation and re-established RASSF10 expression. In addition, we could show the growth inhibitory potential of RASSF10 in breast cancer cell lines MCF7 and T47D upon exogenous expression of RASSF10 by colony formation. We could further show, that RASSF10 induced apoptotic changes in MCF7 and T47D cells, which was verified by a significant increase in the apoptotic sub G1 fraction by 50% using flow cytometry for MCF7 cells. In summary, our study shows the breast tumour specific inactivation of RASSF10 and RASSF1A due to DNA methylation of their CpG island promoters. Furthermore RASSF10 was characterised by the ability to block growth of breast cancer cell lines by apoptosis

  16. Differential sensitivity to methylated DNA by ETS-family transcription factors is intrinsically encoded in their DNA-binding domains.

    Science.gov (United States)

    Stephens, Dominique C; Poon, Gregory M K

    2016-10-14

    Transactivation by the ETS family of transcription factors, whose members share structurally conserved DNA-binding domains, is variably sensitive to methylation of their target genes. The mechanism by which DNA methylation controls ETS proteins remains poorly understood. Uncertainly also pervades the effects of hemi-methylated DNA, which occurs following DNA replication and in response to hypomethylating agents, on site recognition by ETS proteins. To address these questions, we measured the affinities of two sequence-divergent ETS homologs, PU.1 and Ets-1, to DNA sites harboring a hemi- and fully methylated CpG dinucleotide. While the two proteins bound unmethylated DNA with indistinguishable affinity, their affinities to methylated DNA are markedly heterogeneous and exhibit major energetic coupling between the two CpG methylcytosines. Analysis of simulated DNA and existing co-crystal structures revealed that hemi-methylation induced non-local backbone and groove geometries that were not conserved in the fully methylated state. Indirect readout of these perturbations was differentially achieved by the two ETS homologs, with the distinctive interfacial hydration in PU.1/DNA binding moderating the inhibitory effects of DNA methylation on binding. This data established a biophysical basis for the pioneering properties associated with PU.1, which robustly bound fully methylated DNA, but not Ets-1, which was substantially inhibited.

  17. Epigenomic profiling indicates a role for DNA methylation in early postnatal liver development

    OpenAIRE

    Waterland, Robert A.; Kellermayer, Richard; Rached, Marie-Therese; Tatevian, Nina; Gomes, Marcus V.; Zhang, Jiexin; Li ZHANG; Chakravarty, Abrita; Zhu, Wei; Laritsky, Eleonora; Zhang, Wenjuan; Wang, Xiaodan; Shen, Lanlan

    2009-01-01

    The question of whether DNA methylation contributes to the stabilization of gene expression patterns in differentiated mammalian tissues remains controversial. Using genome-wide methylation profiling, we screened 3757 gene promoters for changes in methylation during postnatal liver development to test the hypothesis that developmental changes in methylation and expression are temporally correlated. We identified 31 genes that gained methylation and 111 that lost methylation from embryonic day...

  18. 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.

  19. 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

  20. 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.

  1. Distinctive Klf4 mutants determine preference for DNA methylation status

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Hideharu; Wang, Dongxue; Steves, Alyse N.; Jin, Peng; Blumenthal, Robert M.; Zhang, Xing; Cheng, Xiaodong

    2016-09-04

    Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay.

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

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    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.

  3. Correlation between aberrant methylation status of ras association domain family 1A and alteration of DNA methyltransferases, hepatitis B virus infection in hepatocellular carcinoma%原发性肝癌组织RASSF1A异常甲基化与DNMTs及HBV的关系

    Institute of Scientific and Technical Information of China (English)

    赵强子; 窦科峰; 祁志芳; 魏万礼; 杨雁灵; 李开宗

    2006-01-01

    目的探讨原发性肝癌ras相关结构域家族1A(ras association domain family 1A,RASSF1A)异常甲基化与肿瘤组织中DNA甲基转移酶(DNA methyltransferases,DNMTs)转录水平变化及HBV感染的关系.方法采用甲基化特异性PCR(methylation specific PCR,MSP)技术检测61例原发性肝癌组织RASSF1A基因异常甲基化情况,RT-PCR法检测其中三种主要DNA甲基转移酶(DNMT1、DNMT3A、DNMT3B)水平的变化.结果61例肝癌标本中RASSF1A基因启动子区异常甲基化45例(73.8%);HBV感染者47例(77.1%),其中MSP阳性32例;无HBV感染者14例,其中MSP阳性13例,RASSF1A基因异常甲基化与HBV感染之间的关系无统计学意义(x2=2.260,P=0.133).肝癌组、肝癌细胞系组及HBV感染组DNMTs表达水平与正常对照组相比均显著升高;组内比较显示肝癌组中DNMT3A、DNMT3B在MSP阳性患者中较阴性患者升高(分别为t=3.494,P<0.01;t=4.258,P<0.01),而DNMT1下降(t=3.221,P<0.05);HBV感染组中,MSP阳性组织其DNMT3B较阴性者升高(t=12.171,P<0.01).结论肝癌组织DNMTs水平变化与RASSF1A异常甲基化有密切关系;HBV感染与DNMT3B转录水平变化相关.

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

    Science.gov (United States)

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

    2015-11-24

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

  5. 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.

  6. 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.

  7. Breast cancer epigenetics: from DNA methylation to microRNAs.

    Science.gov (United States)

    Veeck, Jürgen; Esteller, Manel

    2010-03-01

    Both appropriate DNA methylation and histone modifications play a crucial role in the maintenance of normal cell function and cellular identity. In cancerous cells these "epigenetic belts" become massively perturbed, leading to significant changes in expression profiles which confer advantage to the development of a malignant phenotype. DNA (cytosine-5)-methyltransferase 1 (Dnmt1), Dnmt3a and Dnmt3b are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells. Intriguingly, DNMTs were found to be overexpressed in cancerous cells, which is believed to partly explain the hypermethylation phenomenon commonly observed in tumors. However, several lines of evidence indicate that further layers of gene regulation are critical coordinators of DNMT expression, catalytic activity and target specificity. Splice variants of DNMT transcripts have been detected which seem to modulate methyltransferase activity. Also, the DNMT mRNA 3'UTR as well as the coding sequence harbors multiple binding sites for trans-acting factors guiding post-transcriptional regulation and transcript stabilization. Moreover, microRNAs targeting DNMT transcripts have recently been discovered in normal cells, yet expression of these microRNAs was found to be diminished in breast cancer tissues. In this review we summarize the current knowledge on mechanisms which potentially lead to the establishment of a DNA hypermethylome in cancer cells.

  8. Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island.

    Directory of Open Access Journals (Sweden)

    Heather M O'Hagan

    2008-08-01

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

  9. 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

  10. 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.

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

    Science.gov (United States)

    Jiang, Yan; Chen, Jiahong; Tong, Jian; Chen, Tao

    2014-01-01

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

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

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    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.

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

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    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.

  14. DNA methylation patterns provide insight into epigenetic regulation in the Pacific oyster (Crassostrea gigas

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    Gavery Mackenzie R

    2010-08-01

    Full Text Available Abstract Background DNA methylation is an epigenetic mechanism with important regulatory functions in animals. While the mechanism itself is evolutionarily ancient, the distribution and function of DNA methylation is diverse both within and among phylogenetic groups. Although DNA methylation has been well studied in mammals, there are limited data on invertebrates, particularly molluscs. Here we characterize the distribution and investigate potential functions of DNA methylation in the Pacific oyster (Crassostrea gigas. Results Methylation sensitive PCR and bisulfite sequencing PCR approaches were used to identify CpG methylation in C. gigas genes and demonstrated that this species possesses intragenic methylation. In silico analysis of CpGo/e ratios in publicly available sequence data suggests that DNA methylation is a common feature of the C. gigas genome, and that specific functional categories of genes have significantly different levels of methylation. Conclusions The Pacific oyster genome displays intragenic DNA methylation and contains genes necessary for DNA methylation in animals. Results of this investigation suggest that DNA methylation has regulatory functions in Crassostrea gigas, particularly in gene families that have inducible expression, including those involved in stress and environmental responses.

  15. Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

    Science.gov (United States)

    TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocyti...

  16. 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

  17. 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.

  18. 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.

  19. Identification of DNA methylation biomarkers from Infinium arrays

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    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.

  20. 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.

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

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    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

  2. 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.

  3. Chromosome aberrations induced by zebularine in triticale.

    Science.gov (United States)

    Ma, Xuhui; Wang, Qing; Wang, Yanzhi; Ma, Jieyun; Wu, Nan; Ni, Shuang; Luo, Tengxiao; Zhuang, Lifang; Chu, Chenggen; Cho, Seong-Woo; Tsujimoto, Hisashi; Qi, Zengjun

    2016-07-01

    Chromosome engineering is an important approach for generating wheat germplasm. Efficient development of chromosome aberrations will facilitate the introgression and application of alien genes in wheat. In this study, zebularine, a DNA methylation transferase inhibitor, was successfully used to induce chromosome aberrations in the octoploid triticale cultivar Jinghui#1. Dry seeds were soaked in zebularine solutions (250, 500, and 750 μmol/L) for 24 h, and the 500 μmol/L treatment was tested in three additional treatment times, i.e., 12, 36, and 48 h. All treatments induced aberrations involving wheat and rye chromosomes. Of the 920 cells observed in 67 M1 plants, 340 (37.0%) carried 817 aberrations with an average of 0.89 aberrations per cell (range: 0-12). The aberrations included probable deletions, telosomes and acentric fragments (49.0%), large segmental translocations (28.9%), small segmental translocations (17.1%), intercalary translocations (2.6%), long chromosomes that could carry more than one centromere (2.0%), and ring chromosomes (0.5%). Of 510 M2 plants analyzed, 110 (21.6%) were found to carry stable aberrations. Such aberrations included 79 with varied rye chromosome numbers, 7 with wheat and rye chromosome translocations, 15 with possible rye telosomes/deletions, and 9 with complex aberrations involving variation in rye chromosome number and wheat-rye translocations. These indicated that aberrations induced by zebularine can be steadily transmitted, suggesting that zebularine is a new efficient agent for chromosome manipulation.

  4. DNA methylation and gene expression of HIF3A

    DEFF Research Database (Denmark)

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

    2016-01-01

    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....... Interestingly, HIF3A expression in SAT, but not in muscle, associated negatively with BMI and whole-body insulin resistance. We found a significant effect of familiality on HIF3A methylation levels in the blood and HIF3A expression levels in skeletal muscle. CONCLUSIONS: Our findings are in line...... 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...

  5. A methylation-stimulated DNA machine: an autonomous isothermal route to methyltransferase activity and inhibition analysis.

    Science.gov (United States)

    Zhu, Changfeng; Wen, Yanqin; Peng, Hongzhen; Long, Yitao; He, Yao; Huang, Qing; Li, Di; Fan, Chunhai

    2011-04-01

    The operation of DNA nanomachines is generally triggered by either conformational changes of DNA nanostructure or external environmental stimuli. In the present study, we demonstrate an alternative driving force, DNA methylation, to stimulate DNA machine operation. DNA methylation changes neither DNA sequence and conformation nor external environment, however, blocks its cleavage by corresponding methylation-sensitive restriction endonuclease. We thus designed a strand displacement amplification DNA machine, which could be stimulated upon DNA methylation and then autonomously generates accumulated amounts of peroxidase-mimicking DNAzyme signaling machine products in an isothermal manner. The machine product DNAzyme could catalyze the H(2)O(2)-mediated oxidation of 2,2'-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS(2-)) to a colored product ABTS(·-). This methylation-stimulated DNA machine was further used as a colorimetric assay for analysis of methyltransferases activities and screening of methylation inhibitors. As compared with classical methylation assay, this facile isothermal DNA machine avoids the introduction of methylation-specific polymerase chain reaction and radioactive labels, which might be employed as an effective tool for DNA methylation analysis.

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

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    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.

  7. The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation

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    Agnieszka Kaufman-Szymczyk

    2015-12-01

    Full Text Available Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i post-translation histone modification (i.e., deacetylation and methylation; (ii DNA global hypomethylation; (iii promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review.

  8. Exploring the utility of human DNA methylation arrays for profiling mouse genomic DNA.

    Science.gov (United States)

    Wong, Nicholas C; Ng, Jane; Hall, Nathan E; Lunke, Sebastian; Salmanidis, Marika; Brumatti, Gabriela; Ekert, Paul G; Craig, Jeffrey M; Saffery, Richard

    2013-07-01

    Illumina Infinium Human Methylation (HM) BeadChips are widely used for measuring genome-scale DNA methylation, particularly in relation to epigenome-wide association studies (EWAS) studies. The methylation profile of human samples can be assessed accurately and reproducibly using the HM27 BeadChip (27,578 CpG sites) or its successor, the HM450 BeadChip (482,421 CpG sites). To date no mouse equivalent has been developed, greatly hindering the application of this methodology to the wide range of valuable murine models of disease and development currently in existence. We found 1308 and 13,715 probes from HM27 and HM450 BeadChip respectively, uniquely matched the bisulfite converted reference mouse genome (mm9). We demonstrate reproducible measurements of DNA methylation at these probes in a range of mouse tissue samples and in a murine cell line model of acute myeloid leukaemia. In the absence of a mouse counterpart, the Infinium Human Methylation BeadChip arrays have utility for methylation profiling in non-human species.

  9. 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 contai...

  10. Methylated DNA is over-represented in whole-genome bisulfite sequencing data

    Directory of Open Access Journals (Sweden)

    Lexiang eJi

    2014-10-01

    Full Text Available The development of whole-genome bisulfite sequencing (WGBS has led to a number of exciting discoveries about the role of DNA methylation leading to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently advanced to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of reads from methylated DNA in WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. It is especially critical for studies that seek to accurately quantify DNA methylation levels in populations that may segregate for allelic DNA methylation states.

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

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    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.

  12. 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.

  13. Adjusting for Cell Type Composition in DNA Methylation Data Using a Regression-Based Approach.

    Science.gov (United States)

    Jones, Meaghan J; Islam, Sumaiya A; Edgar, Rachel D; Kobor, Michael S

    2017-01-01

    Analysis of DNA methylation in a population context has the potential to uncover novel gene and environment interactions as well as markers of health and disease. In order to find such associations it is important to control for factors which may mask or alter DNA methylation signatures. Since tissue of origin and coinciding cell type composition are major contributors to DNA methylation patterns, and can easily confound important findings, it is vital to adjust DNA methylation data for such differences across individuals. Here we describe the use of a regression method to adjust for cell type composition in DNA methylation data. We specifically discuss what information is required to adjust for cell type composition and then provide detailed instructions on how to perform cell type adjustment on high dimensional DNA methylation data. This method has been applied mainly to Illumina 450K data, but can also be adapted to pyrosequencing or genome-wide bisulfite sequencing data.

  14. Psychosocial stress experience and DNA methylation in humans - implications for stress-adaptation and -resilience

    OpenAIRE

    Unternaehrer, Eva

    2014-01-01

    Abstract: Background: Psychosocial stress, especially early in life, is a risk factor for mental disorders. Recent evidence suggests that stress-related changes in epigenetic patterns, including DNA methylation, could mediate this association. Aim: to examine a potential association between psychosocial stress exposure and DNA methylation of two stress-related genes: the oxytocin receptor (OXTR) and the brain-derived neurotrophic factor (BDNF). Methods: We investigated DNA methylation i...

  15. 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.

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

    OpenAIRE

    Hellman Asaf; Chess Andrew

    2010-01-01

    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 int...

  17. 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.

  18. Analysis of DNA methylation change induced by Dnmt3b in mouse hepatocytes.

    Science.gov (United States)

    Takahashi, Mayumi; Kamei, Yasutomi; Ehara, Tatsuya; Yuan, Xunmei; Suganami, Takayoshi; Takai-Igarashi, Takako; Hatada, Izuho; Ogawa, Yoshihiro

    2013-05-17

    DNA methylation is a key epigenetic contributor to gene regulation in mammals. We have recently found that in the mouse liver, the promoter region of glycerol-3-phosphate acyltransferase 1, a rate-limiting enzyme of de novo lipogenesis, is regulated by DNA methylation, which is mediated by Dnmt3b, an enzyme required for the initiation of de novo methylation. In this study, using primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3b, we characterized Dnmt3b-dependent DNA methylation on a genome-wide basis. A genome-wide DNA methylation analysis, called microarray-based integrated analysis of methylation by isoschizomers, identified 108 genes with Dnmt3b dependent DNA methylation. In DNA expression array analysis, expression of some genes with Dnmt3b-dependent DNA methylation was suppressed. Studies with primary mouse hepatocytes overexpressing Dnmt3b or Dnmt3a revealed that many genes with Dnmt3b-dependent methylation are not methylated by Dnmt3a, whereas those methylated by Dnmt3a are mostly methylated by Dnmt3b. Bioinformatic analysis showed that the CANAGCTG and CCGGWNCSC (N denotes A, T, G, or C; W denotes A or T; and S denotes C or G) sequences are enriched in genes methylated by overexpression of Dnmt3b and Dnmt3a, respectively. We also observed a large number of genes with Dnmt3b-dependent DNA methylation in primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3, suggesting that Dnmt3b is an important DNA methyltransferase in primary mouse hepatocytes, targets specific genes, and potentially plays a role in vivo.

  19. 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.

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

    Science.gov (United States)

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

    2016-09-01

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

  1. DNA methylation is crucial for the early development in the Oyster C. gigas.

    Science.gov (United States)

    Riviere, Guillaume; Wu, Guan-Chung; Fellous, Alexandre; Goux, Didier; Sourdaine, Pascal; Favrel, Pascal

    2013-12-01

    In vertebrates, epigenetic modifications influence gene transcription, and an appropriate DNA methylation is critical in development. Indeed, a precise temporal and spatial pattern of early gene expression is mandatory for a normal embryogenesis. However, such a regulation and its underlying mechanisms remain poorly understood in more distant organisms such as Lophotrochozoa. Thus, despite DNA in the oyster genome being methylated, the role of DNA methylation in development is unknown. To clarify this point, oyster genomic DNA was examined during early embryogenesis and found differentially methylated. Reverse transcriptase quantitative polymerase chain reaction indicated stage-specific levels of transcripts encoding DNA-methyltransferase (DNMT) and methyl-binding domain proteins. In addition, as highlighted by electronic microscopy and immunohistochemistry, the DNMT inhibitor 5-aza-cytidine induced alterations in the quantity and the localisation of methylated DNA and severe dose-dependent development alterations and was lethal after zygotic genome reinitiation. Furthermore, methyl-DNA-immunoprecipitation-quantitative polymerase chain reaction revealed that the transcription level of most of the homeobox gene orthologues examined, but not of the other early genes investigated, was inversely correlated with their specific DNA methylation. Altogether, our results demonstrate that DNA methylation influences gene expression in Crassostrea gigas and is critical for oyster development, possibly by specifically controlling the transcription level of homeobox orthologues. These findings provide evidence for the importance of epigenetic regulation of development in Lophotrochozoans and bring new insights into the early life of C. gigas, one of the most important aquaculture resources worldwide.

  2. 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...

  3. Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation.

    Science.gov (United States)

    Weisenberger, Daniel J; Velicescu, Mihaela; Cheng, Jonathan C; Gonzales, Felicidad A; Liang, Gangning; Jones, Peter A

    2004-01-01

    Several alternatively spliced variants of DNA methyltransferase (DNMT) 3b have been described. Here, we identified new murine Dnmt3b mRNA isoforms and found that mouse embryonic stem (ES) cells expressed only Dnmt3b transcripts that contained exons 10 and 11, whereas the Dnmt3b transcripts in somatic cells lacked these exons, suggesting that this region is important for embryonic development. DNMT3b2 and 3b3 were the major isoforms expressed in human cell lines and the mRNA levels of these isoforms closely correlated with their protein levels. Although DNMT3b3 may be catalytically inactive, it still may be biologically important because D4Z4 and satellites 2 and 3 repeat sequences, all known DNMT3b target sequences, were methylated in cells that predominantly expressed DNMT3b3. Treatment of cells with the mechanism-based inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) caused a complete depletion of DNMT1, 3a, 3b1, and 3b2 proteins. Human DNMT3b3 and the murine Dnmt3b3-like isoform, Dnmt3b6, were also depleted although less efficiently, suggesting that DNMT3b3 also may be capable of DNA binding. Moreover, de novo methylation of D4Z4 in T24 cancer cells after 5-Aza-CdR treatment only occurred when DNMT3b3 was expressed, reinforcing its role as a contributing factor of DNA methylation. The expression of either DNMT3b2 or 3b3, however, was not sufficient to explain the abnormal methylation of DNMT3b target sequences in human cancers, which may therefore be dependent on factors that affect DNMT3b targeting. Methylation analyses of immunodeficiency, chromosomal instabilities, and facial abnormalities cells revealed that an Alu repeat sequence was highly methylated, suggesting that Alu sequences are not DNMT3b targets.

  4. Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

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    Chang Su

    2014-12-01

    Full Text Available DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees.

  5. DNA methylation analysis of chromosome 21 gene promoters at single base pair and single allele resolution.

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    Yingying Zhang

    2009-03-01

    Full Text Available Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii methylation levels of individual cells in one tissue are very similar, and iii methylation patterns follow a relaxed site-specific distribution. Furthermore, iv we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA.

    Science.gov (United States)

    López de Silanes, Isabel; Gorospe, Myriam; Taniguchi, Hiroaki; Abdelmohsen, Kotb; Srikantan, Subramanya; Alaminos, Miguel; Berdasco, María; Urdinguio, Rocío G; Fraga, Mario F; Jacinto, Filipe V; Esteller, Manel

    2009-05-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

  11. 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.

  12. 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.

  13. DNA methylation patterns of protein-coding genes and long non-coding RNAs in males with schizophrenia.

    Science.gov (United States)

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

    2015-11-01

    Schizophrenia (SCZ) is one of the most complex mental illnesses affecting ~1% of the population worldwide. SCZ pathogenesis is considered to be a result of genetic as well as epigenetic alterations. Previous studies have aimed to identify the causative genes of SCZ. However, DNA methylation of long non-coding RNAs (lncRNAs) involved in SCZ has not been fully elucidated. In the present study, a comprehensive genome-wide analysis of DNA methylation was conducted using samples from two male patients with paranoid and undifferentiated SCZ, respectively. Methyl-CpG binding domain protein-enriched genome sequencing was used. In the two patients with paranoid and undifferentiated SCZ, 1,397 and 1,437 peaks were identified, respectively. Bioinformatic analysis demonstrated that peaks were enriched in protein-coding genes, which exhibited nervous system and brain functions. A number of these peaks in gene promoter regions may affect gene expression and, therefore, influence SCZ-associated pathways. Furthermore, 7 and 20 lncRNAs, respectively, in the Refseq database were hypermethylated. According to the lncRNA dataset in the NONCODE database, ~30% of intergenic peaks overlapped with novel lncRNA loci. The results of the present study demonstrated that aberrant hypermethylation of lncRNA genes may be an important epigenetic factor associated with SCZ. However, further studies using larger sample sizes are required.

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

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    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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. GWAS of DNA Methylation Variation Within Imprinting Control Regions Suggests Parent-of-Origin Association

    NARCIS (Netherlands)

    Renteria, M.E.; Coolen, M.W.; Statham, A.L.; Choi, R.S.; Qu, W.; Campbell, M.J.; Smith, S.; Henders, A.K.; Montgomery, G.W.; Clark, S. J.; Martin, N.G.; Medland, S.E.

    2013-01-01

    Imprinting control regions (ICRs) play a fundamental role in establishing and maintaining the non-random monoallelic expression of certain genes, via common regulatory elements such as non-coding RNAs and differentially methylated regions (DMRs) of DNA. We recently surveyed DNA methylation levels wi

  1. 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.

  2. The interplay between environmental factors and DNA methylation in psychotic disorders : Environmental orchestration of the epigenome

    NARCIS (Netherlands)

    Houtepen, LC

    2016-01-01

    Introduction: Environmental exposures during early- life increase the risk of developing a psychotic disorder, but it remains unclear how early life events can have such persistent later life consequences. DNA methylation is the addition of a methyl group to a DNA base and is part of a group of epig

  3. Tobacco smoking is associated with DNA methylation of diabetes susceptibility genes

    NARCIS (Netherlands)

    S. Ligthart (Symen); R.V. Steenaard (Rebecca V.); M.J. Peters (Marjolein); J.B.J. van Meurs (Joyce); E.J.G. Sijbrands (Eric); A.G. Uitterlinden (André); M.J. Bonder (Marc Jan); A. Hofman (Albert); O.H. Franco (Oscar); A. Dehghan (Abbas); B.T. Heijman (Bastiaan); P.A.C. 't Hoen (Peter); J.B.J. van Meurs (Joyce); A.J. Isaacs (Aaron); R. Jansen; L. Franke (Lude); D.I. Boomsma (Dorret); R. Pool (Reńe); J. van Dongen (Jenny); J.J. Hottenga (Jouke Jan); M.M. van Greevenbroek; C.D.A. Stehouwer (Coen D.A.); C.J. van der Kallen; C.G. Schalkwijk (Casper); C. Wijmenga (Cisca); A. Zhernakova (Alexandra); E.F. Tigchelaar (Ettje F.); P. Eline Slagboom; M. Beekman (Marian); J. Deelen (Joris); D. van Heemst (Diana); J.H. Veldink (Jan); L.H. van den Berg (Leonard); C.M. van Duijn (Cock); A. Hofman (Albert); A.G. Uitterlinden (Andre G.); P. Mila Jhamai; M.M.P.J. Verbiest (Michael); H. Eka; D. Suchiman; M. Verkerk (Marijn); R. van der Breggen (Ruud); J. van Rooij (Jeroen); N. Lakenberg (Nico); H. Mei (Hailiang); M. van Iterson; M. Van Galen (Michiel); J.J. Bot (Jan); P. van 't Hof (Peter); P. Deelen (Patrick); I. Nooren (Irene); H. Moed (Heleen); M. Vermaat (Martijn); D.V. Zhernakova (Dasha V.); R. Luijk (René); M.J. Bonder (Marc Jan); F. van Dijk (F.); W. Arindrarto (Wibowo); S.M. Kielbasa (Szymon M.); M. Swertz (Morris); E.W. van Zwet (Erik)

    2016-01-01

    textabstractAims/hypothesis: Tobacco smoking, a risk factor for diabetes, is an established modifier of DNA methylation. We hypothesised that tobacco smoking modifies DNA methylation of genes previously identified for diabetes. Methods: We annotated CpG sites available on the Illumina Human Methylat

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

    Science.gov (United States)

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

  5. Delineation of Methyl-DNA Binding Protein Interactions in the Prostate Cancer Genome (PC110091)

    Science.gov (United States)

    2014-03-01

    DNA Binding Protein Interactions in the Prostate Cancer Genome (PC110091) PRINCIPAL INVESTIGATOR: Roderick T Hori, PhD...13. SUPPLEMENTARY NOTES Prostate Cancer, Methylated DNA, Methyl- CpG Binding Domain, Chromatin Immunoprecipitation 14. ABSTRACT The purpose...of this study is to generate a genome-wide association profile of Methyl- CpG Domain-containing (MBD) proteins, such as MeCP2, MBD1, MBD2 and MBD4, in

  6. The dynamics of DNA methylation and hydroxymethylation during amelogenesis.

    Science.gov (United States)

    Yoshioka, Hirotaka; Minamizaki, Tomoko; Yoshiko, Yuji

    2015-11-01

    Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear. Here, we have uncovered the spatial distributions of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) to determine epigenetic events in the mandibular incisors of mice. Immunohistochemistry and dot blotting showed that 5-hmC in ameloblasts increased from the secretory stage to the later maturation stage. We also demonstrated the distribution of 5-mC-positive ameloblasts with punctate nuclear labeling from sometime after the initiation of the secretory stage to the later maturation stage; however, dot blotting failed to detect this change. No obvious alteration of 5-mC/5-hmC staining in odontoblasts and dental pulp cells was observed. Concomitant with quantitative expression data, immunohistochemistry showed that maintenance DNA methyltransferase DNMT1 was highly expressed in immature dental epithelial cells and subsequently decreased at later stages of development. Meanwhile, de novo DNA methyltransferase Dnmt3a and Dnmt3b and DNA demethylase Tet family genes were universally expressed, except Tet1 that was highly expressed in immature dental epithelial cells. Thus, DNMT1 may sustain the undifferentiated status of dental epithelial cells through the maintenance of DNA methylation, while the hydroxylation of 5-mC may occur through the whole differentiation process by TET activity. Taken together, these data indicate that the dynamic changes of 5-mC and 5-hmC may be critical for the regulation of amelogenesis.

  7. 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.

  8. Comparisons of Non-Gaussian Statistical Models in DNA Methylation Analysis

    Directory of Open Access Journals (Sweden)

    Zhanyu Ma

    2014-06-01

    Full Text Available As a key regulatory mechanism of gene expression, DNA methylation patterns are widely altered in many complex genetic diseases, including cancer. DNA methylation is naturally quantified by bounded support data; therefore, it is non-Gaussian distributed. In order to capture such properties, we introduce some non-Gaussian statistical models to perform dimension reduction on DNA methylation data. Afterwards, non-Gaussian statistical model-based unsupervised clustering strategies are applied to cluster the data. Comparisons and analysis of different dimension reduction strategies and unsupervised clustering methods are presented. Experimental results show that the non-Gaussian statistical model-based methods are superior to the conventional Gaussian distribution-based method. They are meaningful tools for DNA methylation analysis. Moreover, among several non-Gaussian methods, the one that captures the bounded nature of DNA methylation data reveals the best clustering performance.

  9. 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...

  10. Genome-wide analysis of DNA methylation in five tissues of Zhikong scallop, Chlamys farreri.

    Directory of Open Access Journals (Sweden)

    Yan Sun

    Full Text Available DNA methylation plays a vital role in tissue development and differentiation in eukaryotes. Epigenetic studies have been seldom conducted in the extremely diverse and evolutionarily highly successful bilaterian lineage Mollusca. In the present study, we conducted the genome-wide profiling of DNA methylation for five tissues of a bivalve mollusc, Chlamys farreri using the methylation-sensitive amplification polymorphism (MSAP technique. The methylation levels were quite similar among tissues, ranging from 20.9% to 21.7%. CG methylation was the dominant type (14.9%-16.5% in the C. farreri genome, but CHG methylation also accounted for a substantial fraction of total methylation (5.1%-6.3%. Relatively high methylation diversity was observed within tissues. Methylation differentiation between tissues was evaluated and 460 tissue-specific epiloci were identified. Kidney differs from the other tissues in DNA methylation profiles. Our study presents the first look at the tissue-specific DNA methylation patterns in a bivalve mollusc and represents an initial step towards understanding of epigenetic regulatory mechanism underlying tissue development and differentiation in bivalves.

  11. Methylation-sensitive restriction enzyme nested real time PCR, a potential approach for sperm DNA identification.

    Science.gov (United States)

    Bai, Lijuan; Yan, Peng; Cao, Ximei; Jia, Linna; Zhang, Ce; Guo, Dawei

    2015-08-01

    Mammal H19 gene is an imprinting gene in which the paternal allele is silenced. On H19 imprinting control region (ICR), one of the mechanisms regulating the paternal allelic specific silence is DNA methylation in somatic cells throughout the individual's whole life. Nevertheless, this pattern of DNA methylation is erased and re-established in germline. As results, in mature sperm H19 ICR shows biallelic methylation instead of paternal specific methylation in somatic cells. Although the data were mainly from experiments on mice the same mechanisms are believed existing in human germline. We designed an experiment to probe the sperm DNA by methylation sensitive restriction enzyme based nested qPCR (MSRE-nested-qPCR). The genomic DNA digested/undigested by HhaI was amplified by outer primers encompassing four HhaI sites on H19 ICR. These PCR products were used as templates for second round real-time PCR to quantify the DNA methylation level. The results showed that DNA methylation level at H19 ICR were 55.27 ± 8.36% in 32 blood samples and 101.94 ± 11.66% in 31 semen samples. Based on our data sperm DNA could be identified if H19 ICR methylation level is over 78.62%.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Influence of tissue, age, and environmental quality on DNA methylation in Alligator mississippiensis.

    Science.gov (United States)

    Parrott, Benjamin B; Bowden, John A; Kohno, Satomi; Cloy-McCoy, Jessica A; Hale, Matthew D; Bangma, Jacqueline T; Rainwater, Thomas R; Wilkinson, Phillip M; Kucklick, John R; Guillette, Louis J

    2014-01-01

    Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography-tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2'-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.

  14. 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

  15. Characterization of a new aberration of the human Y chromosome by banding methods and DNA restriction endonuclease analysis.

    Science.gov (United States)

    Schmid, M; Gall, H; Schempp, W; Weber, L; Schmidtke, J

    1981-01-01

    Comparative cytogenetic analyses were performed with ten different banding methods on a previously undescribed, inherited structural aberration of a Y chromosome, and the results compared with those of normal Y chromosomes occurring in the same family. The value of the individual staining techniques in investigations of Y chromosomal aberrations is emphasized. The aberrant Y chromosome analyzed can be formally derived from an isodicentric Y chromosome for the short arm with a very terminal long-arm breakpoint, in which the centromere, an entire short arm, and the proximal region on one long arm was lost. This interpretation was confirmed by determining the amount of the two Y-specific DNA sequences (2.1 and 3.4 kb in length) by means of Hae III restriction endonuclease analysis. The karyotype-phenotype correlations in the men with this aberrant Y chromosome, especially the fertility dysfunctions (oligoasthenoteratozoospermia, cryptozoospermia), are discussed. The possibility of the existence of fertility factors involved in the control of spermatogenesis within the quinacrine-bright heterochromatic region of the Y long arm is presented.

  16. Genome-wide DNA methylation modified by soy phytoestrogens: role for epigenetic therapeutics in prostate cancer?

    Science.gov (United States)

    Karsli-Ceppioglu, Seher; Ngollo, Marjolaine; Adjakly, Mawussi; Dagdemir, Aslihan; Judes, Gaëlle; Lebert, André; Boiteux, Jean-Paul; Penault-LLorca, Frédérique; Bignon, Yves-Jean; Guy, Laurent; Bernard-Gallon, Dominique

    2015-04-01

    In prostate cancer, DNA methylation is significantly associated with tumor initiation, progression, and metastasis. Previous studies have suggested that soy phytoestrogens might regulate DNA methylation at individual candidate gene loci and that they play a crucial role as potential therapeutic agents for prostate cancer. The purpose of our study was to examine the modulation effects of phytoestrogens on a genome-wide scale in regards to DNA methylation in prostate cancer. Prostate cancer cell lines DU-145 and LNCaP were treated with 40 μM of genistein and 110 μM of daidzein. DNMT inhibitor 5-azacytidine (2 μM) and the methylating agent budesonide (2 μM) were used to compare their demethylation/methylation effects with phytoestrogens. The regulatory effects of phytoestrogens on DNA methylation were analyzed by using a methyl-DNA immunoprecipitation method coupled with Human DNA Methylation Microarrays (MeDIP-chip). We observed that the methylation profiles of 58 genes were altered by genistein and daidzein treatments in DU-145 and LNCaP prostate cancer cells. In addition, the methylation frequencies of the MAD1L1, TRAF7, KDM4B, and hTERT genes were remarkably modified by genistein treatment. Our results suggest that the modulation effects of phytoestrogens on DNA methylation essentially lead to inhibition of cell growth and induction of apoptosis. Genome-wide methylation profiling reported here suggests that epigenetic regulation mechanisms and, by extension, epigenetics-driven novel therapeutic candidates warrant further consideration in future "omics" studies of prostate cancer.

  17. Insulin and Glucose Alter Death-Associated Protein Kinase 3 (DAPK3) DNA Methylation in Human Skeletal Muscle

    DEFF Research Database (Denmark)

    Mudry, Jonathan M; Lassiter, David G; Nylén, Carolina

    2016-01-01

    DNA methylation is altered by environmental factors. We hypothesized DNA methylation is altered in skeletal muscle in response to either insulin or glucose exposure. We performed a genome-wide DNA methylation analysis in muscle from healthy men before and after insulin exposure. DNA methylation...... of selected genes was determined in muscle from healthy and type 2 diabetic men before and after a glucose tolerance test. Insulin altered DNA methylation in the 3'UTR of the calcium pump ATP2A3 gene. Insulin increased DNA methylation in the gene body of DAPK3, a gene involved in cell proliferation, apoptosis...... glucose incorporation to glycogen was unaltered by siRNA against DAPK3, palmitate oxidation was increased. In conclusion, insulin and glucose exposure acutely alter the DNA methylation profile of skeletal muscle, indicating DNA methylation constitutes a rapidly and adaptive epigenetic mark. Furthermore...

  18. Broad DNA methylation changes of spermatogenesis, inflammation and immune response-related genes in a subgroup of sperm samples for assisted reproduction.

    Science.gov (United States)

    Schütte, B; El Hajj, N; Kuhtz, J; Nanda, I; Gromoll, J; Hahn, T; Dittrich, M; Schorsch, M; Müller, T; Haaf, T

    2013-11-01

    Aberrant sperm DNA methylation patterns, mainly in imprinted genes, have been associated with male subfertility and oligospermia. Here, we performed a genome-wide methylation analysis in sperm samples representing a wide range of semen parameters. Sperm DNA samples of 38 males attending a fertility centre were analysed with Illumina HumanMethylation27 BeadChips, which quantify methylation of >27 000 CpG sites in cis-regulatory regions of almost 15 000 genes. In an unsupervised analysis of methylation of all analysed sites, the patient samples clustered into a major and a minor group. The major group clustered with samples from normozoospermic healthy volunteers and, thus, may more closely resemble the normal situation. When correlating the clusters with semen and clinical parameters, the sperm counts were significantly different between groups with the minor group exhibiting sperm counts in the low normal range. A linear model identified almost 3000 CpGs with significant methylation differences between groups. Functional analysis revealed a broad gain of methylation in spermatogenesis-related genes and a loss of methylation in inflammation- and immune response-related genes. Quantitative bisulfite pyrosequencing validated differential methylation in three of five significant candidate genes on the array. Collectively, we identified a subgroup of sperm samples for assisted reproduction with sperm counts in the low normal range and broad methylation changes (affecting approximately 10% of analysed CpG sites) in specific pathways, most importantly spermatogenesis-related genes. We propose that epigenetic analysis can supplement traditional semen parameters and has the potential to provide new insights into the aetiology of male subfertility.

  19. MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes.

    Science.gov (United States)

    Wang, Shi; Lv, Jia; Zhang, Lingling; Dou, Jinzhuang; Sun, Yan; Li, Xue; Fu, Xiaoteng; Dou, Huaiqian; Mao, Junxia; Hu, Xiaoli; Bao, Zhenmin

    2015-11-01

    Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the 'perfect solution', and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost.

  20. Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.

    Science.gov (United States)

    Baubec, Tuncay; Colombo, Daniele F; Wirbelauer, Christiane; Schmidt, Juliane; Burger, Lukas; Krebs, Arnaud R; Akalin, Altuna; Schübeler, Dirk

    2015-04-09

    DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.

  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. 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.

  3. Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA.

    Science.gov (United States)

    Choi, Ajin; Shin, Kyoung-Jin; Yang, Woo Ick; Lee, Hwan Young

    2014-01-01

    Identification of body fluids found at crime scenes provides important information that can support a link between sample donors and actual criminal acts. Previous studies have reported that DNA methylation analysis at several tissue-specific differentially methylated regions (tDMRs) enables successful identification of semen, and the detection of certain bacterial DNA can allow for identification of saliva and vaginal fluid. In the present study, a method for detecting bacterial DNA was integrated into a previously reported multiplex methylation-sensitive restriction enzyme-polymerase chain reaction. The developed multiplex PCR was modified by the addition of a new semen-specific marker and by including amplicons for the 16S ribosomal RNA gene of saliva- and vaginal fluid-specific bacteria to improve the efficacy to detect a specific type of body fluid. Using the developed multiplex system, semen was distinguishable by unmethylation at the USP49, DACT1, and PFN3 tDMRs and by hypermethylation at L81528, and saliva could be identified by detection of saliva-specific bacteria, Veillonella atypica and/or Streptococcus salivarius. Additionally, vaginal fluid and menstrual blood were differentiated from other body fluids by hypomethylation at the PFN3 tDMR and the presence of vaginal fluid-specific bacteria, Lactobacillus crispatus and/or Lactobacillus gasseri. Because the developed multiplex system uses the same biological source of DNA for individual identification profiling and simultaneously analyses various types of body fluid in one PCR reaction, this method will facilitate more efficient body fluid identification in forensic casework.

  4. Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation.

    Directory of Open Access Journals (Sweden)

    Andrea Fuso

    Full Text Available Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs, having degenerated bases (G/A to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

  5. 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.

  6. Genome-wide association between DNA methylation and alternative splicing in an invertebrate

    Directory of Open Access Journals (Sweden)

    Flores Kevin

    2012-09-01

    Full Text Available Abstract Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee and Nasonia vitripennis (jewel wasp analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice

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

    Directory of Open Access Journals (Sweden)

    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.

  8. A DNA methylation-based definition of biologically distinct breast cancer subtypes.

    Science.gov (United States)

    Stefansson, Olafur A; Moran, Sebastian; Gomez, Antonio; Sayols, Sergi; Arribas-Jorba, Carlos; Sandoval, Juan; Hilmarsdottir, Holmfridur; Olafsdottir, Elinborg; Tryggvadottir, Laufey; Jonasson, Jon G; Eyfjord, Jorunn; Esteller, Manel

    2015-03-01

    In cancer, epigenetic states are deregulated and thought to be of significance in cancer development and progression. We explored DNA methylation-based signatures in association with breast cancer subtypes to assess their impact on clinical presentation and patient prognosis. DNA methylation was analyzed using Infinium 450K arrays in 40 tumors and 17 normal breast samples, together with DNA copy number changes and subtype-specific markers by tissue microarrays. The identified methylation signatures were validated against a cohort of 212 tumors annotated for breast cancer subtypes by the PAM50 method (The Cancer Genome Atlas). Selected markers were pyrosequenced in an independent validation cohort of 310 tumors and analyzed with respect to survival, clinical stage and grade. The results demonstrate that DNA methylation patterns linked to the luminal-B subtype are characterized by CpG island promoter methylation events. In contrast, a large fraction of basal-like tumors are characterized by hypomethylation events occurring within the gene body. Based on these hallmark signatures, we defined two DNA methylation-based subtypes, Epi-LumB and Epi-Basal, and show that they are associated with unfavorable clinical parameters and reduced survival. Our data show that distinct mechanisms leading to changes in CpG methylation states are operative in different breast cancer subtypes. Importantly, we show that a few selected proxy markers can be used to detect the distinct DNA methylation-based subtypes thereby providing valuable information on disease prognosis.

  9. 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.

  10. Detection of DNA methylation changes during seed germination in rapeseed (Brassica napus)

    Institute of Scientific and Technical Information of China (English)

    LU Guangyuan; WU Xiaoming; CHEN Biyun; GAO Guizhen; XU Kun; LI Xiangzhi

    2006-01-01

    DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. In this study, we assessed the extent and pattern of cytosine methylation during rapeseed (Brassica napus L.) seed germination, and compared the methylation level of various tissues in seedling, using the techniques of methylation-sensitive amplified polymorphism (MSAP) and HPLC separation and quantification of nucleosides. In all, 484 bands, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified by 12 pairs of selective primers in DNA obtained from dry seeds. A total of 76 sites were found to be differentially digested by the isoschizomers, indicating that approximately 15.7% of 5(-CCGG-3( sites in the genome were cytosine methylated. Four classes of patterns were observed in a comparative assay of cytosine methylation in the dry and germinating seeds; a small number of hypermethylation events occurred at 5(-CCGG-3( sites in germinating seeds compared with dry seeds, while many more hypomethylation events were detected after seed germination. Differences in DNA methylation level in various tissues were also detected; radicel was less methylated than hypocotyl and cotyledon. These observations were further confirmed by HPLC analysis. In addition, sequencing of eleven differentially methylated fragments and the subsequent blast search revealed that cytosine methylated 5(-CCGG- 3( sequences were equally distributed between coding and non-coding regions. These results clearly demonstrate the power of MSAP technique for large-scale DNA methylation detection in rapeseed genome, and the complexity of DNA methylation change during seed germination. DNA Hypomethylation going with seed germination appears to be a necessary step toward transcriptional activation in gene expression, and 5 well contribute to the developmental gene regulation.

  11. 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....

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

    Directory of Open Access Journals (Sweden)

    Stéphanie Cornen

    Full Text Available Breast cancers (BCs of the luminal B subtype are estrogen receptor-positive (ER+, highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs, DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15 and UTRN (6q24, were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.

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

    Science.gov (United States)

    Cornen, Stéphanie; Guille, Arnaud; Adélaïde, José; Addou-Klouche, Lynda; Finetti, Pascal; Saade, Marie-Rose; Manai, Marwa; Carbuccia, Nadine; Bekhouche, Ismahane; Letessier, Anne; Raynaud, Stéphane; Charafe-Jauffret, Emmanuelle; Jacquemier, Jocelyne; Spicuglia, Salvatore; de The, Hugues; Viens, Patrice; Bertucci, François; Birnbaum, Daniel; Chaffanet, Max

    2014-01-01

    Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.

  14. Kaempferol Modulates DNA Methylation and Downregulates DNMT3B in Bladder Cancer

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    Wei Qiu

    2017-03-01

    Full Text Available Background: Genomic DNA methylation plays an important role in both the occurrence and development of bladder cancer. Kaempferol (Kae, a natural flavonoid that is present in many fruits and vegetables, exhibits potent anti-cancer effects in bladder cancer. Similar to other flavonoids, Kae possesses a flavan nucleus in its structure. This structure was reported to inhibit DNA methylation by suppressing DNA methyltransferases (DNMTs. However, whether Kae can inhibit DNA methylation remains unclear. Methods: Nude mice bearing bladder cancer were treated with Kae for 31 days. The genomic DNA was extracted from xenografts and the methylation changes was determined using an Illumina Infinium HumanMethylation 450 BeadChip Array. The ubiquitination was detected using immuno-precipitation assay. Results: Our data indicated that Kae modulated DNA methylation in bladder cancer, inducing 103 differential DNA methylation positions (dDMPs associated with genes (50 hyper-methylated and 53 hypo-methylated. DNA methylation is mostly relied on the levels of DNMTs. We observed that Kae specifically inhibited the protein levels of DNMT3B without altering the expression of DNMT1 or DNMT3A. However, Kae did not downregulate the transcription of DNMT3B. Interestingly, we observed that Kae induced a premature degradation of DNMT3B by inhibiting protein synthesis with cycloheximide (CHX. By blocking proteasome with MG132, we observed that Kae induced an increased ubiquitination of DNMT3B. These results suggested that Kae could induce the degradation of DNMT3B through ubiquitin-proteasome pathway. Conclusion: Our data indicated that Kae is a novel DNMT3B inhibitor, which may promote the degradation of DNMT3B in bladder cancer.

  15. DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Gao, Fei; Li, Shengting; Lin, Lin;

    2011-01-01

    in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls......To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...

  16. 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.

  17. Comparative DNA Methylation Profiling Reveals an Immunoepigenetic Signature of HIV-related Cognitive Impairment.

    Science.gov (United States)

    Corley, Michael J; Dye, Christian; D'Antoni, Michelle L; Byron, Mary Margaret; Yo, Kaahukane Leite-Ah; Lum-Jones, Annette; Nakamoto, Beau; Valcour, Victor; SahBandar, Ivo; Shikuma, Cecilia M; Ndhlovu, Lishomwa C; Maunakea, Alika K

    2016-09-15

    Monocytes/macrophages contribute to the neuropathogenesis of HIV-related cognitive impairment (CI); however, considerable gaps in our understanding of the precise mechanisms driving this relationship remain. Furthermore, whether a distinct biological profile associated with HIV-related CI resides in immune cell populations remains unknown. Here, we profiled DNA methylomes and transcriptomes of monocytes derived from HIV-infected individuals with and without CI using genome-wide DNA methylation and gene expression profiling. We identified 1,032 CI-associated differentially methylated loci in monocytes. These loci related to gene networks linked to the central nervous system (CNS) and interactions with HIV. Most (70.6%) of these loci exhibited higher DNA methylation states in the CI group and were preferentially distributed over gene bodies and intergenic regions of the genome. CI-associated DNA methylation states at 12 CpG sites associated with neuropsychological testing performance scores. CI-associated DNA methylation also associated with gene expression differences including CNS genes CSRNP1 (P = 0.017), DISC1 (P = 0.012), and NR4A2 (P = 0.005); and a gene known to relate to HIV viremia, THBS1 (P = 0.003). This discovery cohort data unveils cell type-specific DNA methylation patterns related to HIV-associated CI and provide an immunoepigenetic DNA methylation "signature" potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against CI.

  18. 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.

  19. 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.

  20. Air pollution and DNA methylation alterations in lung cancer: A systematic and comparative study

    Science.gov (United States)

    Jiang, Cheng-Lan; He, Shui-Wang; Zhang, Yun-Dong; Duan, He-Xian; Huang, Tao; Huang, Yun-Chao; Li, Gao-Feng; Wang, Ping; Ma, Li-Ju; Zhou, Guang-Biao; Cao, Yi

    2017-01-01

    The lung cancer incidence in the Xuanwei and neighboring region, Yunnan, China, is among the highest in China and is attributed to severe air pollution with high benzo(a)pyrene levels. We systematically and comparatively analyzed DNA methylation alterations at genome and gene levels in Xuanwei lung cancer tissues and cell lines, as well as benzo(a)pyrene-treated cells and mouse samples. We obtained a comprehensive dataset of genome-wide cytosine-phosphate-guanine island methylation in air pollution-related lung cancer samples. Benzo(a)pyrene exposure induced multiple alterations in DNA methylation and in mRNA expressions of DNA methyltransferases and ten-11 translocation proteins; these alterations partially occurred in Xuanwei lung cancer. Furthermore, benzo(a)pyrene-induced DKK2 and EN1 promoter hypermethylation and LPAR2 promoter hypomethylation led to down-regulation and up-regulation of the genes, respectively; the down-regulation of DKK2 and EN1 promoted the cellular proliferation. Thus, DNA methylation alterations induced by benzo(a)pyrene contribute partially to abnormal DNA methylation in air pollution-related lung cancer, and these DNA methylation alterations may affect the development and progression of lung cancer. Additionally, vitamin C and B6 can reduce benzo(a)pyrene-induced DNA methylation alterations and may be used as chemopreventive agents for air pollution-related lung cancer. PMID:27901495

  1. 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.

  2. 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.

  3. Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling.

    Science.gov (United States)

    Nones, Katia; Waddell, Nic; Song, Sarah; Patch, Ann-Marie; Miller, David; Johns, Amber; Wu, Jianmin; Kassahn, Karin S; Wood, David; Bailey, Peter; Fink, Lynn; Manning, Suzanne; Christ, Angelika N; Nourse, Craig; Kazakoff, Stephen; Taylor, Darrin; Leonard, Conrad; Chang, David K; Jones, Marc D; Thomas, Michelle; Watson, Clare; Pinese, Mark; Cowley, Mark; Rooman, Ilse; Pajic, Marina; Butturini, Giovanni; Malpaga, Anna; Corbo, Vincenzo; Crippa, Stefano; Falconi, Massimo; Zamboni, Giuseppe; Castelli, Paola; Lawlor, Rita T; Gill, Anthony J; Scarpa, Aldo; Pearson, John V; Biankin, Andrew V; Grimmond, Sean M

    2014-09-01

    The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

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

    Directory of Open Access Journals (Sweden)

    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

  5. 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

    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-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...... and insulin-stimulated SAT from LBW and matched normal birth weight (NBW) subjects during control and high-fat overfeeding. MATERIALS/METHODS: Nineteen young healthy men with LBW and 26 NBW controls were studied after both a 5-day high-fat overfeeding and a control diet in a randomized crossover setting. DNA...

  6. Comparative epigenomics: a powerful tool to understand the evolution of DNA methylation.

    Science.gov (United States)

    Zhong, Xuehua

    2016-04-01

    Understanding how developmental and functional complexity of organisms evolves is a longstanding challenge in biology. Genetic mutation has long been thought to be the cause of biological complexity. However, increasing evidence indicates that epigenetic variation provides a parallel path for the evolution of biological complexity. Cytosine DNA methylation, the addition of a chemical mark on DNA, is a conserved and essential gene regulatory mechanism. Recent studies have greatly advanced our understanding of the DNA methylation landscapes and key regulatory components across many species. In this review, I summarize recent advances in understanding DNA methylation from an evolutionary perspective. Using comparative approaches, I highlight the conservation and divergence of DNA methylation patterns and regulatory machinery in plants and other eukaryotic organisms.

  7. MicroRNA expression profiling and DNA methylation signature for deregulated microRNA in cutaneous T-cell lymphoma.

    Science.gov (United States)

    Sandoval, Juan; Díaz-Lagares, Angel; Salgado, Rocío; Servitje, Octavio; Climent, Fina; Ortiz-Romero, Pablo L; Pérez-Ferriols, Amparo; Garcia-Muret, Maria P; Estrach, Teresa; Garcia, Mar; Nonell, Lara; Esteller, Manel; Pujol, Ramon M; Espinet, Blanca; Gallardo, Fernando

    2015-04-01

    MicroRNAs usually regulate gene expression negatively, and aberrant expression has been involved in the development of several types of cancers. Microarray profiling of microRNA expression was performed to define a microRNA signature in a series of mycosis fungoides tumor stage (MFt, n=21) and CD30+ primary cutaneous anaplastic large cell lymphoma (CD30+ cALCL, n=11) samples in comparison with inflammatory dermatoses (ID, n=5). Supervised clustering confirmed a distinctive microRNA profile for cutaneous T-cell lymphoma (CTCL) with respect to ID. A 40 microRNA signature was found in MFt including upregulated onco-microRNAs (miR-146a, miR-142-3p/5p, miR-21, miR-181a/b, and miR-155) and downregulated tumor-suppressor microRNAs (miR-200ab/429 cluster, miR-10b, miR-193b, miR-141/200c, and miR-23b/27b). Regarding CD30+ cALCL, 39 differentially expressed microRNAs were identified. Particularly, overexpression of miR-155, miR-21, or miR-142-3p/5p and downregulation of the miR-141/200c clusters were observed. DNA methylation in microRNA gene promoters, as expression regulatory mechanism for deregulated microRNAs, was analyzed using Infinium 450K array and approximately one-third of the differentially expressed microRNAs showed significant DNA methylation differences. Two different microRNA methylation signatures for MFt and CD30+ cALCL were found. Correlation analysis showed an inverse relationship for microRNA promoter methylation and microRNA expression. These results reveal a subgroup-specific epigenetically regulated microRNA signatures for MFt and CD30+ cALCL patients.

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

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    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.

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

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    Andrew D. King

    2016-09-01

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

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

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    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...

  11. Comparison of methods for quantification of global DNA methylation in human cells and tissues.

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    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.

  12. DNA Methylation and Chromatin Remodeling: The Blueprint of Cancer Epigenetics

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    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.

  13. 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

  14. Enhanced one-carbon flux towards DNA methylation: Effect of dietary methyl supplements against gamma-radiation-induced epigenetic modifications.

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    Batra, Vipen; Sridhar, Swathi; Devasagayam, Thomas Paul Asir

    2010-02-12

    Radiation exposure poses a major risk for workers in the nuclear power plants and other radiation related industry. In this context, we demonstrate that gamma-radiation is an efficient DNA demethylating agent and its injurious effect can be minimized by dietary methyl supplements (folate, choline and vitamin B12). To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on normal control diet (NCD) and methyl-supplemented diet (MSD). After 2 weeks of NCD and MSD dietary regimen, we exposed the animals to gamma-radiation (2, 4 and 6Gy) and investigated the profile of downstream metabolites and activity levels of one-carbon (C(1)) flux generating enzymes. In MSD fed and irradiated animals, hepatic folate levels increased (Pmaintenance of genomic DNA methylation under gamma-radiation stress might be a very dynamic, progressive diet dependent process that could involve increased one-carbon flux through various C(1) metabolites.

  15. Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

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    Yuan, Xiao-Long; Gao, Ning; Xing, Yan; Zhang, Hai-Bin; Zhang, Ai-Ling; Liu, Jing; He, Jin-Long; Xu, Yuan; Lin, Wen-Mian; Chen, Zan-Mou; Zhang, Hao; Zhang, Zhe; Li, Jia-Qi

    2016-02-25

    Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

  16. Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer

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    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.

  17. 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

  18. Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

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    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.

  19. EGCG Prevents High Fat Diet-Induced Changes in Gut Microbiota, Decreases of DNA Strand Breaks, and Changes in Expression and DNA Methylation of Dnmt1 and MLH1 in C57BL/6J Male Mice

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    Marlene Remely

    2017-01-01

    Full Text Available Obesity as a multifactorial disorder involves low-grade inflammation, increased reactive oxygen species incidence, gut microbiota aberrations, and epigenetic consequences. Thus, prevention and therapies with epigenetic active antioxidants, (--Epigallocatechin-3-gallate (EGCG, are of increasing interest. DNA damage, DNA methylation and gene expression of DNA methyltransferase 1, interleukin 6, and MutL homologue 1 were analyzed in C57BL/6J male mice fed a high-fat diet (HFD or a control diet (CD with and without EGCG supplementation. Gut microbiota was analyzed with quantitative real-time polymerase chain reaction. An induction of DNA damage was observed, as a consequence of HFD-feeding, whereas EGCG supplementation decreased DNA damage. HFD-feeding induced a higher inflammatory status. Supplementation reversed these effects, resulting in tissue specific gene expression and methylation patterns of DNA methyltransferase 1 and MutL homologue 1. HFD feeding caused a significant lower bacterial abundance. The Firmicutes/Bacteroidetes ratio is significantly lower in HFD + EGCG but higher in CD + EGCG compared to control groups. The results demonstrate the impact of EGCG on the one hand on gut microbiota which together with dietary components affects host health. On the other hand effects may derive from antioxidative activities as well as epigenetic modifications observed on CpG methylation but also likely to include other epigenetic elements.

  20. EGCG Prevents High Fat Diet-Induced Changes in Gut Microbiota, Decreases of DNA Strand Breaks, and Changes in Expression and DNA Methylation of Dnmt1 and MLH1 in C57BL/6J Male Mice

    Science.gov (United States)

    Remely, Marlene; Ferk, Franziska; Sterneder, Sonja; Setayesh, Tahereh; Roth, Sylvia; Kepcija, Tatjana; Noorizadeh, Rahil; Rebhan, Irene; Greunz, Martina; Beckmann, Johanna; Wagner, Karl-Heinz; Knasmüller, Siegfried

    2017-01-01

    Obesity as a multifactorial disorder involves low-grade inflammation, increased reactive oxygen species incidence, gut microbiota aberrations, and epigenetic consequences. Thus, prevention and therapies with epigenetic active antioxidants, (−)-Epigallocatechin-3-gallate (EGCG), are of increasing interest. DNA damage, DNA methylation and gene expression of DNA methyltransferase 1, interleukin 6, and MutL homologue 1 were analyzed in C57BL/6J male mice fed a high-fat diet (HFD) or a control diet (CD) with and without EGCG supplementation. Gut microbiota was analyzed with quantitative real-time polymerase chain reaction. An induction of DNA damage was observed, as a consequence of HFD-feeding, whereas EGCG supplementation decreased DNA damage. HFD-feeding induced a higher inflammatory status. Supplementation reversed these effects, resulting in tissue specific gene expression and methylation patterns of DNA methyltransferase 1 and MutL homologue 1. HFD feeding caused a significant lower bacterial abundance. The Firmicutes/Bacteroidetes ratio is significantly lower in HFD + EGCG but higher in CD + EGCG compared to control groups. The results demonstrate the impact of EGCG on the one hand on gut microbiota which together with dietary components affects host health. On the other hand effects may derive from antioxidative activities as well as epigenetic modifications observed on CpG methylation but also likely to include other epigenetic elements. PMID:28133504

  1. Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing

    Science.gov (United States)

    Chan, K. C. Allen; Jiang, Peiyong; Chan, Carol W. M.; Sun, Kun; Wong, John; Hui, Edwin P.; Chan, Stephen L.; Chan, Wing Cheong; Hui, David S. C.; Ng, Simon S. M.; Chan, Henry L. Y.; Wong, Cesar S. C.; Ma, Brigette B. Y.; Chan, Anthony T. C.; Lai, Paul B. S.; Sun, Hao; Chiu, Rossa W. K.; Lo, Y. M. Dennis

    2013-01-01

    We explored the detection of genome-wide hypomethylation in plasma using shotgun massively parallel bisulfite sequencing as a marker for cancer. Tumor-associated copy number aberrations (CNAs) could also be observed from the bisulfite DNA sequencing data. Hypomethylation and CNAs were detected in the plasma DNA of patients with hepatocellular carcinoma, breast cancer, lung cancer, nasopharyngeal cancer, smooth muscle sarcoma, and neuroendocrine tumor. For the detection of nonmetastatic cancer cases, plasma hypomethylation gave a sensitivity and specificity of 74% and 94%, respectively, when a mean of 93 million reads per case were obtained. Reducing the sequencing depth to 10 million reads per case was found to have no adverse effect on the sensitivity and specificity for cancer detection, giving respective figures of 68% and 94%. This characteristic thus indicates that analysis of plasma hypomethylation by this sequencing-based method may be a relatively cost-effective approach for cancer detection. We also demonstrated that plasma hypomethylation had utility for monitoring hepatocellular carcinoma patients following tumor resection and for detecting residual disease. Plasma hypomethylation can be combined with plasma CNA analysis for further enhancement of the detection sensitivity or specificity using different diagnostic algorithms. Using the detection of at least one type of aberration to define an abnormality, a sensitivity of 87% could be achieved with a specificity of 88%. These developments have thus expanded the applications of plasma DNA analysis for cancer detection and monitoring. PMID:24191000

  2. Influence of DNMT genotype on global and site specific DNA methylation patterns in neonates and pregnant women.

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    Catherine Potter

    Full Text Available This study examines the relationship between common genetic variation within DNA methyltransferase genes and inter-individual variation in DNA methylation. Eleven polymorphisms spanning DNMT1 and DNMT3B were genotyped. Global and gene specific (IGF2, IGFBP3, ZNT5 DNA methylation was quantified by LUMA and bisulfite Pyrosequencing assays, respectively, in neonatal cord blood and in maternal peripheral blood. Associations between maternal genotype and maternal methylation (n (≈ 333, neonatal genotype and neonatal methylation (n (≈ 454, and maternal genotype and neonatal methylation (n (≈ 137 were assessed. The findings of this study provide some support to the hypothesis that genetic variation in DNA methylating enzymes influence DNA methylation at global and gene-specific levels; however observations were not robust to correction for multiple testing. More comprehensive analysis of the influence of genetic variation on global and site specific DNA methylation is warranted.

  3. A review of dietary factors and its influence on DNA methylation in colorectal carcinogenesis.

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    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.

  4. Genome-wide screen for differential DNA methylation associated with neural cell differentiation in mouse.

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    Rene Cortese

    Full Text Available Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs in undifferentiated embryonic stem cells (ESCs, in in-vitro induced neural stem cells (NSCs and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p1.96 enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.

  5. Divergence of gene body DNA methylation and evolution of plant duplicate genes.

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    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.

  6. DNA methylation profiles of polycystic ovarian syndrome in Chinese women: A case-control study

    DEFF Research Database (Denmark)

    Li, Shuxia; Duan, Hongmei; Zhu, D;

    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 using Illumina’s HumanMethylation450 BeadChip array. We identifi ed a substantial number of genomic sites diff...... erentially methylated in the whole blood of 30 PCOS patients and 30 healthy controls (52 sites, false discovery rate

  7. HIF3A DNA Methylation Is Associated with Childhood Obesity and ALT

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    Shuo Wang; Jieyun Song; Yide Yang; Yining Zhang; Haijun Wang; Jun Ma

    2015-01-01

    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 as...

  8. The role of DNA methylation on Octopus vulgaris development and their perspectives

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    Eva eDíaz-Freije

    2014-02-01

    Full Text Available DNA methylation is a common regulator of gene expression and development in mammalian and other vertebrate genomes. DNA methylation has been studied so far in a few bivalve mollusk species, finding a wide spectrum of levels. We focused our study in the common octopus, Octopus vulgaris, an important organism for neuroscience, physiology and ethology research as well as for human consumption. We aim to confirm the existence of DNA methylation in O. vulgaris and ultimately, if methylation plays a role in gene regulation during octopus development. We used a genome-wide approach, methylation-sensitive amplified polymorphism (MSAP, firstly in four different tissues from the same specimens from adult benthonic individuals to test whether gene expression is regulated by methylation. Secondly, we tested the hypothesis that methylation underlies development by assessing MSAP patters from paralarvae to adult developmental stages. Our data indicate that octopus genome is widely methylated since clear differences can be observed, and the methylation pattern change with the development. The statistical analyses showed significant differences in methylation pattern between paralarvae, where higher internal cytosine methylation is observed, and the three other post-hatching stages. This suggests an important role of cytosine methylation during the first step of development, when major morphological changes take place. However, methylation seems to have little effect on gene expression during the benthonic phase, since any significant effect was revealed in the AMOVA performed. Our observations highlight the importance of epigenetic mechanism in the first developmental steps of the common octopus and open new perspectives to overcome high mortality rate during paralarvae growth. Thus, better understanding the molecular regulation patterns could lead to new approaches that increase the efficiency of husbandry of this emergent species for aquaculture.

  9. Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

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    Diane I Schroeder

    2015-08-01

    Full Text Available Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs and highly methylated domains (HMDs with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo.

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

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    Jian-Hong Xu

    Full Text Available An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types, z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control, normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25-30% appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pat