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Sample records for cpg methylation loss

  1. Methylation of a CpG Island within the Uroplakin Ib Promoter: A Possible Mechanism for Loss of Uroplakin lb Expression in Bladder Carcinoma

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    Andrea E. Varga

    2004-03-01

    Full Text Available Uroplakin Ib is a structural protein on the surface of urothelial cells. Expression of uroplakin Ib mRNA is reduced or absent in many transitional cell carcinomas (TCCs but molecular mechanisms underlying loss of expression remain to be determined. Analysis of the uroplakin Ib promoter identified a weak CpG island spanning the proximal promoter, exon 1, and the beginning of intron 1. This study examined the hypothesis that methylation of this CpG island regulates uroplakin Ib expression. Uroplakin Ib mRNA levels were determined by reverse transcription polymerase chain reaction and CpG methylation was assessed by bisulfite modification of DNA, PCR, and sequencing. A correlation was demonstrated in 15 TCC lines between uroplakin Ib mRNA expression and lack of CpG methylation. In support of a regulatory role for methylation, incubating uroplakin Ib-negative lines with 5-aza-2′ -deoxycytidine reactivated uroplakin Ib mRNA expression. A trend between uroplakin Ib mRNA expression and CpG methylation was also observed in normal urothelium and bladder carcinomas. In particular, loss of uroplakin Ib expression correlated with methylation of a putative Spi/NFκB binding motif. The data are consistent with the hypothesis that methylation of specific sites within the uroplakin Ib promoter may be an important factor in the loss of uroplakin Ib expression in TCCs.

  2. High CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of tetralogy of Fallot

    Indian Academy of Sciences (India)

    SI-JU GAO; GUI-FANG ZHANG; RONG-PENG ZHANG

    2016-12-01

    We examined CpG island methylation in p16 gene and its effect on p16 protein expression in tetralogy of Fallot (ToF) patients to explore its potential implications in the development and progression of ToF. The study subjects consisted of 75 healthy controls and 63 ToF patients recruited at Linyi People’s Hospital between January 2012 and June 2014. The 4 mL of peripheral venous blood of each subject was obtained and saved in ethylene diamine tetraacetic acid (EDTA) tubes. Methylation-specific polymerase chain reaction (MSP) was employed to detect CpG island methylation in p16 promoter region andWestern blotting was used to detect p16 expression of all subjects. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was performed to test p16 mRNA expression. The results showed that p16-methylation rates in ToF group were significantly higher than the control group (ToF group, 58.73%; control group, 13.33%; P < 0.001). Remarkably, Western blotting and FQ-PCR results derived from RVOT revealed that p16 protein expression was significantly lower in ToF group compared to the control group (0.76± 0.21 versus 2.31 ± 0.35; P < 0.001), and p16 gene expression was also markedly decreased in ToF group (1.212 ± 0.152 versus 1.346 ± 0.191, P< 0.001). Additionally, our analysis suggested that CpG island methylation in p16 promoters in ToF patients was negatively correlated with p16 protein and gene expression (both P < 0.05). Our study reports that high CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of ToF, which may have significant therapeutic applications for ToF.

  3. High CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of tetralogy of Fallot.

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    Gao, Si-Ju; Zhang, Gui-Fang; Zhang, Rong-Peng

    2016-12-01

    We examined CpG island methylation in p16 gene and its effect on p16 protein expression in tetralogy of Fallot (ToF) patients to explore its potential implications in the development and progression of ToF. The study subjects consisted of 75 healthy controls and 63 ToF patients recruited at Linyi People's Hospital between January 2012 and June 2014. The 4 mL of peripheral venous blood of each subject was obtained and saved in ethylene diamine tetraacetic acid (EDTA) tubes. Methylation-specific polymerase chain reaction (MSP) was employed to detect CpG island methylation in p16 promoter region andWestern blotting was used to detect p16 expression of all subjects. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was performed to test p16 mRNA expression. The results showed that p16-methylation rates in ToF group were significantly higher than the control group (ToF group, 58.73%; control group, 13.33%; P p16 protein expression was significantly lower in ToF group compared tothe control group (0.76 ± 0.21 versus 2.31 ± 0.35; P p16 gene expression was also markedly decreased in ToF group (1.212 ± 0.152 versus 1.346 ± 0.191, P p16 promoters in ToF patients was negatively correlated with p16 protein and gene expression (both P p16 gene and loss of p16 protein expression associate with the development and progression of ToF, which may have significant therapeutic applications for ToF.

  4. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

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    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James; Vivekanandan, Perumal

    2013-12-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

  5. Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin

    DEFF Research Database (Denmark)

    Tariq, M.; Saze, H.; Probst, A.;

    2003-01-01

    DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation...... in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation....

  6. Nucleosome Positions and Differential Methylation Status of Various Regions within MLH1 CpG Island

    Institute of Scientific and Technical Information of China (English)

    BAI Hua; ZHOU Jing; DENG Da-jun

    2008-01-01

    Objective:To determine the relationship between nucleosome positions and formation of differential methylation of the reported region A,B,C,and D within the MLH1 CpG island. Methods:Methylation of the MLH1 promoter was analyzed by combined of bisulfite restriction assay.Chromatin of RKO and MGC803 cells were extracted and digested by Mnase.Mononucleosomal DNA fragment was isolated and used as templates for detection of nucleosomal distribution by a battery of quantitative PCRs covering the full MLH1 promoter region. Results:The MLH1 was methylated in RKO and unmethylated in MGC803.At the region B,where methylation of CpG sites did not correlated with transcription of this gene well,qPCR product of the M-3(-599nt~-475nt)fragment was amplified in both RKO and MGC803 cells.However,at the region C and D within the core promoter,where methylation of CpG sites correlated with loss of MLH1 transcription well,the M-7(-257nt~-153nt)and M-8(-189nt~-71nt)fragments were amplified remarkably only in RKO cells. Conclusion:Nucleosome may be the basic unit for both CpG methylation and methylation-related regulation of gene transcription.Methylation status of CpG sites within the same nucleosome may be homogeneous;between different nucleosomes,homogeneous or heterogeneous.

  7. Collaborations between CpG sites in DNA methylation

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    Song, You; Ren, Honglei; Lei, Jinzhi

    2017-08-01

    DNA methylation patterns have profound impacts on genome stability, gene expression and development. The molecular base of DNA methylation patterns has long been focused at single CpG sites level. Here, we construct a kinetic model of DNA methylation with collaborations between CpG sites, from which a correlation function was established based on experimental data. The function consists of three parts that suggest three possible sources of the correlation: movement of enzymes along DNA, collaboration between DNA methylation and nucleosome modification, and global enzyme concentrations within a cell. Moreover, the collaboration strength between DNA methylation and nucleosome modification is universal for mouse early embryo cells. The obtained correlation function provides insightful understanding for the mechanisms of inheritance of DNA methylation patterns.

  8. CpG distribution and methylation pattern in porcine parvovirus.

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    Renáta Tóth

    Full Text Available Based on GC content and the observed/expected CpG ratio (oCpGr, we found three major groups among the members of subfamily Parvovirinae: Group I parvoviruses with low GC content and low oCpGr values, Group II with low GC content and high oCpGr values and Group III with high GC content and high oCpGr values. Porcine parvovirus belongs to Group I and it features an ascendant CpG distribution by position in its coding regions similarly to the majority of the parvoviruses. The entire PPV genome remains hypomethylated during the viral lifecycle independently from the tissue of origin. In vitro CpG methylation of the genome has a modest inhibitory effect on PPV replication. The in vitro hypermethylation disappears from the replicating PPV genome suggesting that beside the maintenance DNMT1 the de novo DNMT3a and DNMT3b DNA methyltransferases can't methylate replicating PPV DNA effectively either, despite that the PPV infection does not seem to influence the expression, translation or localization of the DNA methylases. SNP analysis revealed high mutability of the CpG sites in the PPV genome, while introduction of 29 extra CpG sites into the genome has no significant biological effects on PPV replication in vitro. These experiments raise the possibility that beyond natural selection mutational pressure may also significantly contribute to the low level of the CpG sites in the PPV genome.

  9. 18q loss of heterozygosity in microsatellite stable colorectal cancer is correlated with CpG island methylator phenotype-negative (CIMP-0 and inversely with CIMP-low and CIMP-high

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    Kirkner Gregory J

    2007-05-01

    Full Text Available Abstract Background: The CpG island methylator phenotype (CIMP with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high and BRAF mutations. 18q loss of heterozygosity (LOH commonly present in colorectal cancer with chromosomal instability (CIN is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and MLH1 methylation in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation and CIMP-0 (CIMP-negative, determined by quantitative DNA methylation analysis. Methods: Utilizing MethyLight technology (real-time PCR, we quantified DNA methylation in 8 CIMP-specific promoters {CACNA1G, CDKN2A (p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1} in 758 non-MSI-high colorectal cancers obtained from two large prospective cohorts. Using four 18q microsatellite markers (D18S55, D18S56, D18S67 and D18S487 and stringent criteria for 18q LOH, we selected 374 tumors (236 LOH-positive tumors with ≥ 2 markers showing LOH; and 138 LOH-negative tumors with ≥ 3 informative markers and no LOH. Results: CIMP-0 (0/8 methylated promoters was significantly more common in 18q LOH-positive tumors (59% = 139/236, p = 0.002 than 18q LOH-negative tumors (44% = 61/138, while CIMP-low/high (1/8–8/8 methylated promoters was significantly more common (56% in 18q LOH-negative tumors than 18q LOH-positive tumors (41%. These relations persisted after stratification by sex, location, or the status of MSI, p53 expression (by immunohistochemistry, or KRAS/BRAF mutation. Conclusion: 18q LOH is correlated positively with CIMP-0 and inversely with CIMP-low and CIMP-high. Our findings provide supporting evidence for relationship between CIMP-0 and 18q LOH as well as a molecular difference between CIMP-0 and CIMP-low in

  10. Methylation Linear Discriminant Analysis (MLDA for identifying differentially methylated CpG islands

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    Vass J Keith

    2008-08-01

    Full Text Available Abstract Background Hypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA. Results MLDA was programmed in R (version 2.7.0 and the package is available at CRAN 1. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing. Conclusion MLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of

  11. Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

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    Li, Lili; Li, Chen; Mao, Haitao; Du, Zhenfang; Chan, Wai Yee; Murray, Paul; Luo, Bing; Chan, Anthony TC; Mok, Tony SK; Chan, Francis KL; Ambinder, Richard F; Tao, Qian

    2016-01-01

    Promoter CpG methylation is a fundamental regulatory process of gene expression. TET proteins are active CpG demethylases converting 5-methylcytosine to 5-hydroxymethylcytosine, with loss of 5 hmC as an epigenetic hallmark of cancers, indicating critical roles of TET proteins in epigenetic tumorigenesis. Through analysis of tumor methylomes, we discovered TET1 as a methylated target, and further confirmed its frequent downregulation/methylation in cell lines and primary tumors of multiple carcinomas and lymphomas, including nasopharyngeal, esophageal, gastric, colorectal, renal, breast and cervical carcinomas, as well as non-Hodgkin, Hodgkin and nasal natural killer/T-cell lymphomas, although all three TET family genes are ubiquitously expressed in normal tissues. Ectopic expression of TET1 catalytic domain suppressed colony formation and induced apoptosis of tumor cells of multiple tissue types, supporting its role as a broad bona fide tumor suppressor. Furthermore, TET1 catalytic domain possessed demethylase activity in cancer cells, being able to inhibit the CpG methylation of tumor suppressor gene (TSG) promoters and reactivate their expression, such as SLIT2, ZNF382 and HOXA9. As only infrequent mutations of TET1 have been reported, compared to TET2, epigenetic silencing therefore appears to be the dominant mechanism for TET1 inactivation in cancers, which also forms a feedback loop of CpG methylation during tumorigenesis. PMID:27225590

  12. Regulation of DNA transposition by CpG methylation and chromatin structure in human cells.

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    Jursch, Tobias; Miskey, Csaba; Izsvák, Zsuzsanna; Ivics, Zoltán

    2013-05-15

    The activity of transposable elements can be regulated by different means. DNA CpG methylation is known to decrease or inhibit transpositional activity of diverse transposons. However, very surprisingly, it was previously shown that CpG methylation of the Sleeping Beauty (SB) transposon significantly enhanced transposition in mouse embryonic stem cells. In order to investigate the unexpected response of SB transposition to CpG methylation, related transposons from the Tc1/mariner superfamily, that is, Tc1, Himar1, Hsmar1, Frog Prince (FP) and Minos were tested to see how transposition was affected by CpG methylation. A significant increase of >20-fold in transposition of SB, FP and Minos was seen, whereas Tc1, Himar1 and Hsmar1 showed no difference in transposition upon CpG-methylation. The terminal inverted repeats (TIRs) of the SB, FP and Minos elements share a common structure, in which each TIR contains two functionally important binding sites for the transposase (termed the IR/DR structure). The group of IR/DR elements showed increased excision after CpG methylation compared to untreated transposon donor plasmids. We found that de novo CpG methylation is not required for transposition. A mutated FP donor plasmid with depleted CpG sites in both TIRs was as efficient in transposition as the wild-type transposon, indicating that CpG sites inside the TIRs are not responsible for altered binding of factors potentially modulating transposition. By using an in vivo one-hybrid DNA-binding assay in cultured human cells we found that CpG methylation had no appreciable effect on the affinity of SB transposase to its binding sites. However, chromatin immunoprecipitation indicated that CpG-methylated transposon donor plasmids are associated with a condensed chromatin structure characterized by trimethylated histone H3K9. Finally, DNA compaction by protamine was found to enhance SB transposition. We have shown that DNA CpG methylation upregulates transposition of IR

  13. Deletions of a differentially methylated CpG island at SNRPN define a putative imprinting control region

    Energy Technology Data Exchange (ETDEWEB)

    Sutcliffe, J.S.,; Nakao, M.; Beaudet, A.L. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies, respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy, or other mutations. Four transcripts designated PAR-5, PAR-7, PAR-1 and PAR-4 were isolated and localized to a region within 300 kb telomeric to the gene encoding small nuclear ribonucleoprotein-associated polypeptide N (SNRPN). Analysis of the transcripts in cultured fibroblasts and lymphoblasts from deletion patients demonstrated that SNRPN, PAR-5 and PAR-1 are expressed exclusively from the paternal chromosome, defining an imprinted domain that spans at least 200 kb. All three imprinted transcripts were absent in cells from three PWS patients (one pair of sibs and one sporadic case) with small deletions that involve a differentially methylated CpG island containing a previously undescribed 5{prime} untranslated exon ({alpha}) of SNRPN. Methylation of the CpG island is specific for the maternal chromosome consistent with paternal expression of the imprinted domain. One deletion, which is benign when maternally transmitted, extends upstream <30 kb from the CpG island, and is associated with altered methylation centromeric to SNRPN, and loss of transcription telomeric to SNRPN, implying the presence of an imprinting control region around the CpG island containing exon {alpha}.

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

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    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.

  15. A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray

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    Qiao Yingjuan

    2008-01-01

    Full Text Available Abstract Background DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing. Results We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray, provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients. Conclusion This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

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

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

    1999-01-01

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

  17. CpG Island Methylation in Human Lymphocytes Is Highly Correlated with DNA Sequence, Repeats, and Predicted DNA Structure

    OpenAIRE

    Christoph Bock; Martina Paulsen; Sascha Tierling; Thomas Mikeska; Thomas Lengauer; Jörn Walter

    2006-01-01

    CpG island methylation plays an important role in epigenetic gene control during mammalian development and is frequently altered in disease situations such as cancer. The majority of CpG islands is normally unmethylated, but a sizeable fraction is prone to become methylated in various cell types and pathological situations. The goal of this study is to show that a computational epigenetics approach can discriminate between CpG islands that are prone to methylation from...

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

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

    2015-01-01

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

  19. Methylation of the estrogen receptor CpG island distinguishes spontaneous and plutonium-induced tumors from nitrosamine-induced lung tumors

    Energy Technology Data Exchange (ETDEWEB)

    Belinsky, S.A.; Baylin, S.B.; Issa, J.J. [Johns Hopkins Univ., Baltimore, MD (United States)

    1995-12-01

    CpG islands located in the promoter region of genes constitute one mechanism for regulating transcription. These islands are normally free of methylation, regardless of the expression state of the gene. Hypermethylation of CpG islands, the addition of a methyl group to the internal cytosine within CpG dinucleotides, can cause silencing of a gene. Hypermethylation has been detected as an early event at specific chromosome loci during the development of colon cancer and represents one mechanism used by neoplatic cells to inactivate tumor suppressor genes. Recent studies have demonstrated this mechanism in inactivation of the VHL tumor suppressor gene in 19% of sporadic renal tumors and the p16 {sup INK4a} tumor suppressor gene in 30% of non-small cell lung cancers. A recent report indicates that the estrogen receptor gene could also be inactivated through methylation. In addition, estrogen receptor CpG island methylation arises as a direct function of age in normal colonic mucosa and is present in virtually all colonic tumors. In cultured colon cancer cells, methylation-associated loss of expression of the estrogen receptor gene results in deregulated growth, suggesting a role for the estrogen receptor in colon cancer development. These results provide further evidence that gene silencing through methylation could be a predominant epigenetic mechanism underlying the development of many different types of cancer. The purpose of the current investigation was to determine whether estrogen receptor CpG island methylation is involved in the development of lung cancer. The frequency for methylation of the estrogen receptor CpG island in rodent lung tumors is summarized.

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

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

    2017-01-01

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

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

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    Guadalupe S. López-Álvarez

    2017-01-01

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

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

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

    2015-01-01

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

  3. Genome-wide methylation analysis identifies differentially methylated CpG loci associated with severe obesity in childhood.

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    Huang, R C; Garratt, E S; Pan, H; Wu, Y; Davis, E A; Barton, S J; Burdge, G C; Godfrey, K M; Holbrook, J D; Lillycrop, K A

    2015-01-01

    Childhood obesity is a major public health issue. Here we investigated whether differential DNA methylation was associated with childhood obesity. We studied DNA methylation profiles in whole blood from 78 obese children (mean BMI Z-score: 2.6) and 71 age- and sex-matched controls (mean BMI Z-score: 0.1). DNA samples from obese and control groups were pooled and analyzed using the Infinium HumanMethylation450 BeadChip array. Comparison of the methylation profiles between obese and control subjects revealed 129 differentially methylated CpG (DMCpG) loci associated with 80 unique genes that had a greater than 10% difference in methylation (P-value obesity were validated using sodium bisulfite pyrosequencing across loci within the FYN, PIWIL4, and TAOK3 genes in individual subjects. Three CpG loci within FYN were hypermethylated in obese individuals (all P obesity was associated with lower methylation of CpG loci within PIWIL4 (P = 0.003) and TAOK3 (P = 0.001). After building logistic regression models, we determined that a 1% increase in methylation in TAOK3, multiplicatively decreased the odds of being obese by 0.91 (95% CI: 0.86 - 0.97), and an increase of 1% methylation in FYN CpG3, multiplicatively increased the odds of being obese by 1.03 (95% CI: 0.99 - 1.07). In conclusion, these findings provide evidence that childhood obesity is associated with specific DNA methylation changes in whole blood, which may have utility as biomarkers of obesity risk.

  4. Analysis of CpG methylation sites and CGI among human papillomavirus DNA genomes

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    Galván Silvia C

    2011-11-01

    Full Text Available Abstract Background The Human Papillomavirus (HPV genome is divided into early and late coding sequences, including 8 open reading frames (ORFs and a regulatory region (LCR. Viral gene expression may be regulated through epigenetic mechanisms, including cytosine methylation at CpG dinucleotides. We have analyzed the distribution of CpG sites and CpG islands/clusters (CGI among 92 different HPV genomes grouped in function of their preferential tropism: cutaneous or mucosal. We calculated the proportion of CpG sites (PCS for each ORF and calculated the expected CpG values for each viral type. Results CpGs are underrepresented in viral genomes. We found a positive correlation between CpG observed and expected values, with mucosal high-risk (HR virus types showing the smallest O/E ratios. The ranges of the PCS were similar for most genomic regions except E4, where the majority of CpGs are found within islands/clusters. At least one CGI belongs to each E2/E4 region. We found positive correlations between PCS for each viral ORF when compared with the others, except for the LCR against four ORFs and E6 against three other ORFs. The distribution of CpG islands/clusters among HPV groups is heterogeneous and mucosal HR-HPV types exhibit both lower number and shorter island sizes compared to cutaneous and mucosal Low-risk (LR HPVs (all of them significantly different. Conclusions There is a difference between viral and cellular CpG underrepresentation. There are significant correlations between complete genome PCS and a lack of correlations between several genomic region pairs, especially those involving LCR and E6. L2 and L1 ORF behavior is opposite to that of oncogenes E6 and E7. The first pair possesses relatively low numbers of CpG sites clustered in CGIs while the oncogenes possess a relatively high number of CpG sites not associated to CGIs. In all HPVs, E2/E4 is the only region with at least one CGI and shows a higher content of CpG sites in every

  5. Levels of DNA Methylation Vary at CpG Sites across the BRCA1 Promoter, and Differ According to Triple Negative and "BRCA-Like" Status, in Both Blood and Tumour DNA.

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    Sarah L Daniels

    Full Text Available Triple negative breast cancer is typically an aggressive and difficult to treat subtype. It is often associated with loss of function of the BRCA1 gene, either through mutation, loss of heterozygosity or methylation. This study aimed to measure methylation of the BRCA1 gene promoter at individual CpG sites in blood, tumour and normal breast tissue, to assess whether levels were correlated between different tissues, and with triple negative receptor status, histopathological scoring for BRCA-like features and BRCA1 protein expression. Blood DNA methylation levels were significantly correlated with tumour methylation at 9 of 11 CpG sites examined (p<0.0007. The levels of tumour DNA methylation were significantly higher in triple negative tumours, and in tumours with high BRCA-like histopathological scores (10 of 11 CpG sites; p<0.01 and p<0.007 respectively. Similar results were observed in blood DNA (6 of 11 CpG sites; p<0.03 and 7 of 11 CpG sites; p<0.02 respectively. This study provides insight into the pattern of CpG methylation across the BRCA1 promoter, and supports previous studies suggesting that tumours with BRCA1 promoter methylation have similar features to those with BRCA1 mutations, and therefore may be suitable for the same targeted therapies.

  6. DELETION AND 5'CPG ISLAND METHYLATION OF p15 GENE IN BRAIN GLIOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the abnormality of p15 gene in brain glioma and the correlation of it with occurrence or malignant progression of brain glioma. Methods: Deletion and 5'CPG island methylation of p15 gene were detected by the methods of PCR and PCR-based methylation in 56 cases of brain glioma. Results: Out of 43 cases of high grade glioma, 14 cases were found to have homozygous deletion of p15E1, while none of the 13 cases of low grade glioma was found to have deletion of p15E1 (P<0.05). Methylation of 5'CPG Island of p15 gene was found only in four cases of glioma. Conclusion: Abnormality of p15 gene may involved in the occurrence and malignant progression of brain glioma. Homozygous deletion of gene is the major mechanism of inactivation for p15 gene in brain glioma.

  7. 5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases.

    Science.gov (United States)

    Valton, Julien; Daboussi, Fayza; Leduc, Sophie; Molina, Rafael; Redondo, Pilar; Macmaster, Rachel; Montoya, Guillermo; Duchateau, Philippe

    2012-08-31

    In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.

  8. A novel CpG island set identifies tissue-specific methylation at developmental gene loci.

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    Robert Illingworth

    2008-01-01

    Full Text Available CpG islands (CGIs are dense clusters of CpG sequences that punctuate the CpG-deficient human genome and associate with many gene promoters. As CGIs also differ from bulk chromosomal DNA by their frequent lack of cytosine methylation, we devised a CGI enrichment method based on nonmethylated CpG affinity chromatography. The resulting library was sequenced to define a novel human blood CGI set that includes many that are not detected by current algorithms. Approximately half of CGIs were associated with annotated gene transcription start sites, the remainder being intra- or intergenic. Using an array representing over 17,000 CGIs, we established that 6%-8% of CGIs are methylated in genomic DNA of human blood, brain, muscle, and spleen. Inter- and intragenic CGIs are preferentially susceptible to methylation. CGIs showing tissue-specific methylation were overrepresented at numerous genetic loci that are essential for development, including HOX and PAX family members. The findings enable a comprehensive analysis of the roles played by CGI methylation in normal and diseased human tissues.

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

    Science.gov (United States)

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

    2006-11-15

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

  10. Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

    Science.gov (United States)

    Camoriano, Marta; Morey Kinney, Shannon R.; Moser, Michael T.; Foster, Barbara A.; Mohler, James L.; Trump, Donald L.; Karpf, Adam R.; Smiraglia, Dominic J.

    2010-01-01

    Aberrant DNA methylation plays a significant role in nearly all human cancers and may contribute to disease progression to advanced phenotypes. Study of advanced prostate cancer phenotypes in the human disease is hampered by limited availability of tissues. We therefore took advantage of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model to study whether three different phenotypes of TRAMP tumors (PRIM, late-stage primary tumors; AIP, androgen-independent primary tumors; and MET, metastases) displayed specific patterns of CpG island hypermethylation using Restriction Landmark Genomic Scanning. Each tumor phenotype displayed numerous hypermethylation events, with the most homogeneous methylation pattern in AIP and the most heterogeneous pattern in MET. Several loci displayed a phenotype-specific methylation pattern; the most striking pattern being loci methylated at high frequency in PRIM and AIP but rarely in MET. Examination of the mRNA expression of three genes, BC058385, Goosecoid, and Neurexin 2, which exhibited nonpromoter methylation, revealed increased expression associated with downstream methylation. Only methylated samples showed mRNA expression, in which tumor phenotype was a key factor determining the level of expression. The CpG island in the human orthologue of BC058385 was methylated in human AIP but not in primary androgen-stimulated prostate cancer or benign prostate. The clinical data show a proof-of-principle that the TRAMP model can be used to identify targets of aberrant CpG island methylation relevant to human disease. In conclusion, phenotype-specific hypermethylation events were associated with the overexpression of different genes and may provide new markers of prostate tumorigenesis. PMID:18519676

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

    Science.gov (United States)

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

    2012-01-01

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

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

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    Delphine Fradin

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

  13. Significance of CpG methylation for solar UV-induced mutagenesis and carcinogenesis in skin.

    Science.gov (United States)

    Ikehata, Hironobu; Ono, Tetsuya

    2007-01-01

    Mutations detected in the p53 gene in human nonmelanoma skin cancers show a highly UV-specific mutation pattern, a dominance of C --> T base substitutions at dipyrimidine sites plus frequent CC --> TT tandem substitutions, indicating a major involvement of solar UV in the skin carcinogenesis. These mutations also have another important characteristic of frequent occurrences at CpG dinucleotide sites, some of which actually show prominent hotspots in the p53 gene. Although mammalian solar UV-induced mutation spectra were studied intensively in the aprt gene using rodent cultured cells and the UV-specific mutation pattern was confirmed, the second characteristic of the p53 mutations in human skin cancers had not been reproduced. However, studies with transgenic mouse systems developed thereafter for mutation research, which harbor methyl CpG-abundant transgenes as mutation markers, yielded complete reproductions of the situation of the human skin cancer mutations in terms of both the UV-specific pattern and the frequent occurrence at CpG sites. In this review, we evaluate the significance of the CpG methylation for solar UV mutagenesis in the mammalian genome, which would lead to skin carcinogenesis. We propose that the UV-specific mutations at methylated CpG sites, C --> T transitions at methyl CpG-associated dipyrimidine sites, are a solar UV-specific mutation signature, and have estimated the wavelength range effective for the solar-UV-specific mutation as 310-340 nm. We also recommend the use of methyl CpG-enriched sequences as mutational targets for studies on solar-UV genotoxicity for human, rather than conventional mammalian mutational marker genes such as the aprt and hprt genes.

  14. Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

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    Farrar William L

    2010-10-01

    Full Text Available Abstract Background Recently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation. Results Differentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1. Conclusions Using this

  15. Methylation-Specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences

    OpenAIRE

    Nygren, Anders O.H.; Ameziane, Najim; Duarte, Helena M. B.; Vijzelaar, Raymon N. C. P.; Waisfisz, Quinten; Hess, Corine J.; Jan. P. Schouten; Errami, Abdellatif

    2005-01-01

    Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of ...

  16. CpG island methylator phenotype and its relationship with prognosis in adult acute leukemia patients.

    Science.gov (United States)

    Fu, Hai-Ying; Wu, Dan-Sen; Zhou, Hua-Rong; Shen, Jian-Zhen

    2014-09-01

    To investigated the relationship between CpG island methylator phenotype (CIMP) and prognosis in adults with acute leukemia. Bone marrow samples from 53 acute myeloid leukemia and 50 acute lymphoblastic leukemia patients were collected. The methylation status of 18 tumor suppressor genes was determined using methylation-specific polymerase chain reaction. Greater than 30% of acute leukemia patients had methylated p15, p16, CDH1, CDH13, RUNX3, sFRP1, ID4, and DLC-1 genes; methylation of ≥4 were defined as CIMP positive. Age, type of leukemia, white blood cell count, and CIMP status were significantly associated with recurrence-free survival (RFS) and overall survival (OS) (P acute leukemia.

  17. Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants.

    Science.gov (United States)

    How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg

    2015-07-01

    Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

  18. CpG island methylator phenotype of multigene in serum of sporadic breast carcinoma.

    Science.gov (United States)

    Jing, Feng; Yuping, Wang; Yong, Chen; Jie, Luo; Jun, Lu; Xuanbing, Tang; Lihua, Hu

    2010-08-01

    CpG island methylator phenotype (CIMP) involves methylation targeted toward the promoters of multiple genes. We determined a methylation profile of tumor-related genes in serum of sporadic breast cancer (SBC). The multigene methylation was examined by methylation-specific polymerase chain reaction assay in serum of 50 SBCs and 50 paired nontumors, and CIMP+ was defined as having three genes that are concordantly methylated. The methylation frequency of ten genes in serum of 50 SBCs varied from 10% in FHIT to 74% in RASSF1A. The methylation status of RASSF1A, BRCA1, p16, CDH1, ER, RARbeta2, APC, and DAPK was significantly correlated with SBC and nontumor serum (P < 0.05). Methylation of at least one gene was found in 92% SBC; CIMP was more frequent in SBC than nontumor serum (P < 0.001). There was a significant association between CIMP and methylation of RASSF1A, BRCA1, p16, CDH1, ER, RARbeta2, APC, and DAPK (P < 0.05); the methylation link profile of CDH1, RASSF1A, BRCA1, and RARbeta2 as breast cancer marker may contribute high sensitivity (90%) and specificity (88%). ER and RARbeta2 methylation was associated with elevated serum CA153 levels in 39 SBC samples with CIMP+ (P < 0.05). Multivariate analysis showed that living area of patients was found to provide independent prognostic information associated with a relative risk of tumor recurrence of 5.3. Multigene-specific methylation profile in serum was association with the recurrence risk of rural SBC, and positive correlation of CIMP can serve as a promising molecular marker of SBC.

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

  20. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks.

    Science.gov (United States)

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-22

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named "DeepMethyl" to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

  1. Nanopores suggest a negligible influence of CpG methylation on nucleosome packaging and stability.

    Science.gov (United States)

    Langecker, Martin; Ivankin, Andrey; Carson, Spencer; Kinney, Shannon R M; Simmel, Friedrich C; Wanunu, Meni

    2015-01-14

    Nucleosomes are the fundamental repeating units of chromatin, and dynamic regulation of their positioning along DNA governs gene accessibility in eukaryotes. Although epigenetic factors have been shown to influence nucleosome structure and dynamics, the impact of DNA methylation on nucleosome packaging remains controversial. Further, all measurements to date have been carried out under zero-force conditions. In this paper, we present the first automated force measurements that probe the impact of CpG DNA methylation on nucleosome stability. In solid-state nanopore force spectroscopy, a nucleosomal DNA tail is captured into a pore and pulled on with a time-varying electrophoretic force until unraveling is detected. This is automatically repeated for hundreds of nucleosomes, yielding statistics of nucleosome lifetime vs electrophoretic force. The force geometry, which is similar to displacement forces exerted by DNA polymerases and helicases, reveals that nucleosome stability is sensitive to DNA sequence yet insensitive to CpG methylation. Our label-free method provides high-throughput data that favorably compares with other force spectroscopy experiments and is suitable for studying a variety of DNA-protein complexes.

  2. Developmentally programmed 3' CpG island methylation confers tissue- and cell-type-specific transcriptional activation

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    During development, a small but significant number of CpG islands (CGIs) becomes methylated. The timing of developmentally programmed CGI methylation and associated mechanisms of transcriptional regulation during cellular differentiation, however, remain poorly characterized. Here we used genome-wid...

  3. IGFBP3 Promoter Methylation in Colorectal Cancer: Relationship with Microsatellite Instability, CpG Island Methylator Phenotype, p53

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    Takako Kawasaki

    2007-12-01

    Full Text Available Insulin-like growth factor binding protein 3 (IGFBP3, which is induced by wild-type p53, regulates IGF and interacts with the TGF-β pathway. IGFBP3 promoter methylation may occur in colorectal cancer with or without the CpG island methylator phenotype (CIMP, which is associated with microsatellite instability (MSI and TGFBR2 mutation. We examined the relationship between IGFBP3 methylation, p53 expression, CIMP and MSI in 902 population-based colorectal cancers. Utilizing real-time PCR (MethyLight, we quantified promoter methylation in IGFBP3 and eight other CIMP-high-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1. IGFBP3 methylation was far more frequent in non-MSI-high CIMP-high tumors (85% = 35/41 than in MSI-high CIMPhigh (49% = 44/90, P < .0001, MSI-high non-CIMP-high (17% = 6/36, P < .0001, non-MSI-high non-CIMP-high tumors (22% = 152/680, P < .0001. Among CIMPhigh tumors, the inverse relationship between MSI and IGFBP3 methylation persisted in p53-negative tumors (P < .0001, but not in p53-positive tumors. IGFBP3 methylation was associated inversely with TGFBR2 mutation in MSI-high non-CIMP-high tumors (P = .02. In conclusion, IGFBP3 methylation is inversely associated with MSI in CIMP-high colorectal cancers, this relationship is limited to p53-negative tumors. Our data suggest complex relationship between global genomic/epigenomic phenomena (such as MSI/ CIMP, single molecular events (e.g., IGFBP3 methylation, TP53 mutation, TGFBR2 mutation, the related pathways.

  4. The 5'-end transitional CpGs between the CpG islands and retroelements are hypomethylated in association with loss of heterozygosity in gastric cancers

    Directory of Open Access Journals (Sweden)

    Seo Eun-Joo

    2006-07-01

    Full Text Available Abstract Background A loss of heterozygosity (LOH represents a unilateral chromosomal loss that reduces the dose of highly repetitive Alu, L1, and LTR retroelements. The aim of this study was to determine if the LOH events can affect the spread of retroelement methylation in the 5'-end transitional area between the CpG islands and their nearest retroelements. Methods The 5'-transitional area of all human genes (22,297 was measured according to the nearest retroelements to the transcription start sites. For 50 gastric cancer specimens, the level of LOH events on eight cancer-associated chromosomes was estimated using the microsatellite markers, and the 5'-transitional CpGs of 20 selected genes were examined by methylation analysis using the bisulfite-modified DNA. Results The extent of the transitional area was significantly shorter with the nearest Alu elements than with the nearest L1 and LTR elements, as well as in the extragenic regions containing a higher density of retroelements than in the intragenic regions. The CpG islands neighbouring a high density of Alu elements were consistently hypomethylated in both normal and tumor tissues. The 5'-transitional methylated CpG sites bordered by a low density of Alu elements or the L1 and LTR elements were hypomethylated more frequently in the high-level LOH cases than in the low-level LOH cases. Conclusion The 5'-transitional methylated CpG sites not completely protected by the Alu elements were hypomethylated in association with LOH events in gastric cancers. This suggests that an irreversible unbalanced decrease in the genomic dose reduces the spread of L1 methylation in the 5'-end regions of genes.

  5. Prognostic significance of CpG island methylator phenotype in surgically resected small cell lung carcinoma.

    Science.gov (United States)

    Saito, Yuichi; Nagae, Genta; Motoi, Noriko; Miyauchi, Eisaku; Ninomiya, Hironori; Uehara, Hirofumi; Mun, Mingyon; Okumura, Sakae; Ohyanagi, Fumiyoshi; Nishio, Makoto; Satoh, Yukitoshi; Aburatani, Hiroyuki; Ishikawa, Yuichi

    2016-03-01

    Methylation is closely involved in the development of various carcinomas. However, few datasets are available for small cell lung cancer (SCLC) due to the scarcity of fresh tumor samples. The aim of the present study is to clarify relationships between clinicopathological features and results of the comprehensive genome-wide methylation profile of SCLC. We investigated the genome-wide DNA methylation status of 28 tumor and 13 normal lung tissues, and gene expression profiling of 25 SCLC tissues. Following unsupervised hierarchical clustering and non-negative matrix factorization, gene ontology analysis was performed. Clustering of SCLC led to the important identification of a CpG island methylator phenotype (CIMP) of the tumor, with a significantly poorer prognosis (P = 0.002). Multivariate analyses revealed that postoperative chemotherapy and non-CIMP were significantly good prognostic factors. Ontology analyses suggested that the extrinsic apoptosis pathway was suppressed, including TNFRSF1A, TNFRSF10A and TRADD in CIMP tumors. Here we revealed that CIMP was an important prognostic factor for resected SCLC. Delineation of this phenotype may also be useful for the development of novel apoptosis-related chemotherapeutic agents for treatment of the aggressive tumor.

  6. Incorrect DNA methylation of the DAZL promoter CpG island associates with defective human sperm†

    Science.gov (United States)

    Navarro-Costa, Paulo; Nogueira, Paulo; Carvalho, Marta; Leal, Fernanda; Cordeiro, Isabel; Calhaz-Jorge, Carlos; Gonçalves, João; Plancha, Carlos E.

    2010-01-01

    BACKGROUND Successful gametogenesis requires the establishment of an appropriate epigenetic state in developing germ cells. Nevertheless, an association between abnormal spermatogenesis and epigenetic disturbances in germline-specific genes remains to be demonstrated. METHODS In this study, the DNA methylation pattern of the promoter CpG island (CGI) of two germline regulator genes—DAZL and DAZ, was characterized by bisulphite genomic sequencing in quality-fractioned ejaculated sperm populations from normozoospermic (NZ) and oligoasthenoteratozoospermic (OAT) men. RESULTS OAT patients display increased methylation defects in the DAZL promoter CGI when compared with NZ controls. Such differences are recorded when analyzing sperm fractions enriched either in normal or defective germ cells (P< 0.001 in both cases). Significant differences in DNA methylation profiles are also observable when comparing the qualitatively distinct germ cell fractions inside the NZ and OAT groups (P= 0.003 and P= 0.007, respectively). Contrastingly, the unmethylation pattern of the DAZ promoter CGI remains correctly established in all experimental groups. CONCLUSIONS An association between disrupted DNA methylation of a key spermatogenesis gene and abnormal human sperm is described here for the first time. These results suggest that incorrect epigenetic marks in germline genes may be correlated with male gametogenic defects. PMID:20685756

  7. Three SRA-domain methylcytosine-binding proteins cooperate to maintain global CpG methylation and epigenetic silencing in Arabidopsis.

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    Hye Ryun Woo

    2008-08-01

    Full Text Available Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1 encodes an SRA (SET- and RING-associated domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5' region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing.

  8. Three SRA-domain methylcytosine-binding proteins cooperate to maintain global CpG methylation and epigenetic silencing in Arabidopsis.

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    Hye Ryun Woo

    Full Text Available Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1 encodes an SRA (SET- and RING-associated domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5' region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing.

  9. Comparative Genomic Study Reveals a Transition from TA Richness in Invertebrates to GC Richness in Vertebrates at CpG Flanking Sites: An Indication for Context-Dependent Mutagenicity of Methylated CpG Sites

    Institute of Scientific and Technical Information of China (English)

    Yong Wang; Frederick C.C. Leung

    2008-01-01

    Vertebrate genomes are characterized with CpG deficiency, particularly for GC-poor regions. The GC content-related CpG deficiency is probably caused by context-dependent deamination of methylated CpG sites. This hypothesis was examined in this study by comparing nucleotide frequencies at CpG flanking positions among invertebrate and vertebrate genomes. The finding is a transition of nucleotide preference of 5' T to 5' A at the invertebrate-vertebrate boundary, indicating that a large number of CpG sites with 5' Ts were depleted because of global DNA methylation developed in vertebrates. At genome level, we investigated CpG observed/expected (obs/exp) values in 500 bp fragments, and found that higher CpG obs/exp value is shown in GC-poor regions of invertebrate genomes (except sea urchin) but in GC-rich sequences of vertebrate genomes. We next compared GC content at CpG flanking positions with genomic average, showing that the GC content is lower than the average in invertebrate genomes, but higher than that in vertebrate genomes. These results indicate that although 5' T and 5' A are different in inducing deamination of methylated CpG sites, GC content is even more important in affecting the deamination rate. In all the tests, the results of sea urchin are similar to vertebrates perhaps due to its fractional DNA methylation.CpG deficiency is therefore suggested to be mainly a result of high mutation rates of methylated CpG sites in GC-poor regions.

  10. CpG island methylator phenotype association with upregulated telomerase activity in hepatocellular carcinoma.

    Science.gov (United States)

    Zhang, Changsong; Guo, Xianling; Jiang, Guocheng; Zhang, Li; Yang, Yang; Shen, Feng; Wu, Mengchao; Wei, Lixin

    2008-09-01

    CpG island methylator phenotype (CIMP) involves the targeting of multiple genes by promoter hypermethylation. Telomerase plays an important role in the development of cellular immortality and oncogenesis. To gain insight into the role of epigenetic aberration of telomerase-related genes in hepatocarcinogenesis, we determined a hypermethylation profile in HCC. We examined the promoter methylation status of 9 genes associated with telomerase activity in 120 HCC, 120 cirrhosis tissues and 10 normal liver tissues by methylation-specific PCR. Assay of telomerase activity was by TRAP-ELISA. The frequency of promoter methylation of each gene was P21 63.3%, P15 42.5%, P16 62.5%, P53 14.2%, RB 32.5%, P27 48.3%, WTI 54.2%, E2F-1 70.8% and P300 65.8% of 120 HCC. Methylation status of P21, P15, P16, WTI and E2F-1 was significantly associated with HCC and nontumor tissues (p < 0.05). CIMP+ was detected in 61.7% (74/120) HCC and 15% (18/120) cirrhosis tissues, no CIMP+ was present in normal liver tissues (p < 0.001). A significant difference between CIMP status and metastasis was been found in HCC (p < 0.001). Results showed that 94.6% (70/74) HCC and 55.6% (10/18) cirrhosis patients with CIMP+ show expression of high telomerase activity than 45.5% (10/22) HCC and 6.25% (1/16) cirrhosis patients with CIMP- (p < 0.001). CIMP lead to high levels of expression of telomerase activity through the simultaneous inactivation of multiple genes associated with telomerase activity by concordant methylation.

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

    Science.gov (United States)

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

    2016-08-01

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

  12. In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases.

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    Julia Arand

    2012-06-01

    Full Text Available The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites. The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM to calculate the relative contribution of DNA methyltransferases (Dnmts for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.

  13. High resolution detection and analysis of CpG dinucleotides methylation using MBD-Seq technology.

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    Xun Lan

    Full Text Available Methyl-CpG binding domain protein sequencing (MBD-seq is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ∼100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution.

  14. DNA methylation-based age prediction from saliva: High age predictability by combination of 7 CpG markers.

    Science.gov (United States)

    Hong, Sae Rom; Jung, Sang-Eun; Lee, Eun Hee; Shin, Kyoung-Jin; Yang, Woo Ick; Lee, Hwan Young

    2017-07-01

    DNA methylation is currently one of the most promising age-predictive biomarkers. Many studies have reported DNA methylation-based age predictive models, but most of these are based on DNA methylation patterns from blood. Only a few studies have examined age-predictive DNA patterns in saliva, which is one of the most frequently-encountered body fluids at crime scenes. In this study, we generated genome-wide DNA methylation profiles of saliva from 54 individuals and identified CpG markers that showed a high correlation between methylation and age. Because the age-associated marker candidates from saliva differed from those of blood, we investigated DNA methylation patterns of 6 age-associated CpG marker candidates (cg00481951, cg19671120, cg14361627, cg08928145, cg12757011, and cg07547549 of the SST, CNGA3, KLF14, TSSK6, TBR1, and SLC12A5 genes, respectively) in addition to a cell type-specific CpG marker (cg18384097 of the PTPN7 gene) in an independent set of saliva samples obtained from 226 individuals aged 18 to 65 years. Multiplex methylation SNaPshot reactions were used to generate the data. We then generated a linear regression model with age information and the methylation profile from the 113 training samples. The model exhibited a 94.5% correlation between predicted and chronological age with a mean absolute deviation (MAD) from chronological age of 3.13 years. In subsequent validation using 113 test samples, we also observed a high correlation between predicted and chronological age (Spearman's rho=0.952, MAD from chronological age=3.15years). The model composed of 7 selected CpG sites enabled age prediction in saliva with high accuracy, which will be useful in saliva analysis for investigative leads. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients.

    Science.gov (United States)

    Mur, Pilar; Rodríguez de Lope, Ángel; Díaz-Crespo, Francisco Javier; Hernández-Iglesias, Teresa; Ribalta, Teresa; Fiaño, Concepción; García, Juan Fernando; Rey, Juan Antonio; Mollejo, Manuela; Meléndez, Bárbara

    2015-05-01

    Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP+) and promoter methylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) gene. Among these markers, a predictive value was reported in glioblastomas (GBM) for MGMT promoter methylation, in particular in elderly GBM patients. In this study, methylation data from 46 glioma samples with the Illumina 450K platform were obtained and extended using external data to include a total of 247 glioma samples. Methylation analysis of the whole MGMT gene with this platform revealed two strongly survival-associated CpG regions within the promoter and the gene body, which were confirmed in a reported dataset of high grade-gliomas. Methylation at the promoter (CpG 25, cg12981137 and the prognostic model MGMT-STP27) and at the gene body CpG 165 (cg07933035), were significantly associated with better overall survival, and strongly correlated with G-CIMP+ status. In this series, the prognostic value of MGMT methylation at the promoter was not observed in G-CIMP- cases, although around 50 % of them were MGMT-methylated. These results were also obtained in an homogeneously-treated series of chemoradiated G-CIMP- GBMs analyzed by MSP and qMSP, and confirmed in a reported pyrosequencing-analyzed series of gliomas. Interestingly, in contrast to the MGMT promoter, gene body methylation was of prognostic value in G-CIMP-patients older than 65 years. Our study highlights the relevance of the prognostic value of the different regions of methylation throughout the MGMT gene that could be affected by specific G-CIMP profiles and age groups.

  16. Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis

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    Gong Zhiyuan

    2011-01-01

    Full Text Available Abstract Background DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis. Results A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(aanthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation. Conclusion The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver.

  17. CpG Island Methylator Phenotype and Prognosis of Colorectal Cancer in Northeast China

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    Xia Li

    2014-01-01

    Full Text Available Purpose. To investigate the association between CpG island methylator phenotype (CIMP and the overall survival of sporadic colorectal cancer (CRC in Northeast China. Methods. 282 sporadic CRC patients were recruited in this study. We selected MLH1, MGMT, p16, APC, MINT1, MINT31, and RUNX3 as the CIMP panel markers. The promoter methylation was assessed by methylation sensitive high resolution melting (MS-HRM. Proportional hazards-regression models were fitted with computing hazard ratios (HR and the corresponding 95% confidence intervals (95% CI. Results. 12.77% (36/282 of patients were CIMP-0, 74.1% (209/282 of patients were CIMP-L, and 13.12% (37/282 of patients were CIMP-H. The five-year survival of the 282 CRC patients was 58%. There was significant association between APC gene promoter methylation and CRC overall survival (HR = 1.61; 95% CI: 1.05–2.46; P=0.03. CIMP-H was significantly associated with worse prognosis compared to CIMP-0 (HR = 3.06; 95% CI: 1.19–7.89; P=0.02 and CIMP-L (HR = 1.97; 95% CI: 1.11–3.48; P=0.02, respectively. While comparing with the combine of CIMP-L and CIMP-0 (CIMP-L/0, CIMP-H also presented a worse prognosis (HR = 2.31; 95% CI: 1.02–5.24; P=0.04. Conclusion. CIMP-H may be a predictor of a poor prognosis of CRC in Northeast China patients.

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

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    Snijders Peter JF

    2010-06-01

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

  19. A Panel of CpG Methylation Sites Distinguishes Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

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    Kevin Huang

    2014-01-01

    Full Text Available Whether human induced pluripotent stem cells (hiPSCs are epigenetically identical to human embryonic stem cells (hESCs has been debated in the stem cell field. In this study, we analyzed DNA methylation patterns in a large number of hiPSCs (n = 114 and hESCs (n = 155, and identified a panel of 82 CpG methylation sites that can distinguish hiPSCs from hESCs with high accuracy. We show that 12 out of the 82 CpG sites were subject to hypermethylation in part by DNMT3B. Notably, DNMT3B contributes directly to aberrant hypermethylation and silencing of the signature gene, TCERG1L. Overall, we conclude that DNMT3B is involved in a wave of de novo methylation during reprogramming, a portion of which contributes to the unique hiPSC methylation signature. These 82 CpG methylation sites may be useful as biomarkers to distinguish between hiPSCs and hESCs.

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

    Science.gov (United States)

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

    2016-05-01

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

  1. Microarray-based method for detecting methylation changes of p16Ink4a gene 5'-CpG islands in gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Peng Hou; Jia-Yao Shen; Mei-Ju Ji; Nong-Yue He; Zu-Hong Lu

    2004-01-01

    AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of highthroughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer.METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status.Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP).RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover,it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP.CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.

  2. CpG sites with continuously increasing or decreasing methylation from early to late human fetal brain development.

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    Schneider, Eberhard; Dittrich, Marcus; Böck, Julia; Nanda, Indrajit; Müller, Tobias; Seidmann, Larissa; Tralau, Tim; Galetzka, Danuta; El Hajj, Nady; Haaf, Thomas

    2016-10-30

    Normal human brain development is dependent on highly dynamic epigenetic processes for spatial and temporal gene regulation. Recent work identified wide-spread changes in DNA methylation during fetal brain development. We profiled CpG methylation in frontal cortex of 27 fetuses from gestational weeks 12-42, using Illumina 450K methylation arrays. Sites showing genome-wide significant correlation with gestational age were compared to a publicly available data set from gestational weeks 3-26. Altogether, we identified 2016 matching developmentally regulated differentially methylated positions (m-dDMPs): 1767m-dDMPs were hypermethylated and 1149 hypomethylated during fetal development. M-dDMPs are underrepresented in CpG islands and gene promoters, and enriched in gene bodies. They appear to cluster in certain chromosome regions. M-dDMPs are significantly enriched in autism-associated genes and CpGs. Our results promote the idea that reduced methylation dynamics during fetal brain development may predispose to autism. In addition, m-dDMPs are enriched in genes with human-specific brain expression patterns and/or histone modifications. Collectively, we defined a subset of dDMPs exhibiting constant methylation changes from early to late pregnancy. The same epigenetic mechanisms involving methylation changes in cis-regulatory regions may have been adopted for human brain evolution and ontogeny. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells

    KAUST Repository

    Takahashi, Yuta

    2017-05-05

    CpG islands (CGIs) are primarily promoter-associated genomic regions and are mostly unmethylated within highly methylated mammalian genomes. The mechanisms by which CGIs are protected from de novo methylation remain elusive. Here we show that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation. By targeting the DNA mismatch repair gene MLH1 CGI, we could generate a PSC model of a cancer-related epimutation. Furthermore, we successfully corrected aberrant imprinting in induced PSCs derived from an Angelman syndrome patient. Our results provide insights into how CpG-free DNA induces de novo CGI methylation and broaden the application of targeted epigenome editing for a better understanding of human development and disease.

  4. CpG island methylator phenotype in plasma is associated with hepatocellular carcinoma prognosis

    Institute of Scientific and Technical Information of China (English)

    Ji-Bin Liu; Yi-Xin Zhang; Shu-Hui Zhou; Min-Xin Shi; Jin Cai; Yan Liu; Ke-Ping Chen; Fu-Lin Qiang

    2011-01-01

    AIM: To evaluate the clinical significance of CpG island methylator phenotype (CIMP) in plasma and its asso ciation with hepatocellular carcinoma (HCC) progress.METHODS: CIMP status of 108 HCC patients was analyzed using a methylation marker panel in tumor tissues and plasma with methylation-specific poly merase chain reaction. Fifteen samples of non-neo plastic liver tissues and 60 of plasma from healthy persons were examined simultaneously. Examined genes included APC, WIF-1, RUNX-3, DLC-1, SFRP-1, DKK and E-cad.RESULTS: The frequencies of high-level methyla tion in HCC tissue and plasma were at least 15% for the seven genes: APC, 48/108, 44.44% in tissue and 26/108, 24.07% in plasma; WIF-1, 53/108, 49.07% in tissue and 35/108, 32.41% in plasma; RUNX-3, 52/108, 48.14% in tissue and 42/108, 38.89% in plasma; DLC-1, 38/108, 35.18% in tissue and 23/108, 21.30% in plasma; SFRP-1, 40/108, 37.04% in tissue and 31/108, 28.7% in plasma; DKK, 39/108, 36.1% in tis sue and 25/108, 23.14% in plasma; and E-cad, 37/108, 34.3% in tissue and 18/108, 16.67% in plasma. CIMP+ (S 3 methylated genes) was detected in 68 (60.2%) tumor tissue samples and 62 (57.4%) plasma samples. CIMP was not detected in non-neoplastic liver tissues or plasma of healthy persons. CIMP status in tumor tissues differed significantly in gender, hepatitis B surface antigen, alpha-fetoprotein, and tumor-node-metastasis stage (P < 0.05). Similar results were ob tained with plasma samples (P < 0.05). There was no difference in CIMP status in age, presence of hepatitis C virus antibody, cirrhosis, number of nodes, number of tumors, tumor size, or Edmondson-Steiner stage. A one-year follow-up found that the metastatic rate and recurrence rate in the CIMP+ group were significantly higher than in the CIMP- group as assessed with plas ma samples (P < 0.05).CONCLUSION: Plasma DNA can be a reliable sample source for CIMP analysis. CIMP in plasma may serve as a molecular marker of late-stage and poor-prognosis HCC.

  5. PRIMEGENS-v2: genome-wide primer design for analyzing DNA methylation patterns of CpG islands.

    Science.gov (United States)

    Srivastava, Gyan P; Guo, Juyuan; Shi, Huidong; Xu, Dong

    2008-09-01

    DNA methylation plays important roles in biological processes and human diseases, especially cancers. High-throughput bisulfite genomic sequencing based on new generation of sequencers, such as the 454-sequencing system provides an efficient method for analyzing DNA methylation patterns. The successful implementation of this approach depends on the use of primer design software capable of performing genome-wide scan for optimal primers from in silico bisulfite-treated genome sequences. We have developed a method, which fulfills this requirement and conduct primer design for sequences including regions of given promoter CpG islands. The developed method has been implemented using the C and JAVA programming languages. The primer design results were tested in the PCR experiments of 96 selected human DNA sequences containing CpG islands in the promoter regions. The results indicate that this method is efficient and reliable for designing sequence-specific primers. The sequence-specific primer design for DNA meth-ylated sequences including CpG islands has been integrated into the second version of PRIMEGENS as one of the primer design features. The software is freely available for academic use at http://digbio.missouri.edu/primegens/.

  6. Tissue specific DNA methylation of CpG islands in normal human adult somatic tissues distinguishes neural from non-neural tissues.

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    Ghosh, Srimoyee; Yates, Allan J; Frühwald, Michael C; Miecznikowski, Jeffrey C; Plass, Christoph; Smiraglia, Dominic

    2010-08-16

    Although most CpG islands are generally thought to remain unmethylated in all adult somatic tissues, recent genome-wide approaches have found that some CpG islands have distinct methylation patterns in various tissues, with most differences being seen between germ cells and somatic tissues. Few studies have addressed this among human somatic tissues and fewer still have studied the same sets of tissues from multiple individuals. In the current study, we used Restriction Landmark Genomic Scanning to study tissue specific methylation patterns in a set of twelve human tissues collected from multiple individuals. We identified 34 differentially methylated CpG islands among these tissues, many of which showed consistent patterns in multiple individuals. Of particular interest were striking differences in CpG island methylation, not only among brain regions, but also between white and grey matter of the same region. These findings were confirmed for selected loci by quantitative bisulfite sequencing. Cluster analysis of the RLGS data indicated that several tissues clustered together, but the strongest clustering was in brain. Tissues from different brain regions clustered together, and, as a group, brain tissues were distinct from either mesoderm or endoderm derived tissues which demonstrated limited clustering. These data demonstrate consistent tissue specific methylation for certain CpG islands, with clear differences between white and grey matter of the brain. Furthermore, there was an overall pattern of tissue specifically methylated CpG islands that distinguished neural tissues from non-neural.

  7. CpG dinucleotide positioning patterns determine the binding affinity of methyl-binding domain to nucleosomes.

    Science.gov (United States)

    Mendonca, Agnes; Sanchez, Oscar F; Liu, Wenjie; Li, Zhe; Yuan, Chongli

    2017-06-01

    The methyl-binding domain of MBD1 is a common methyl CpG binding motif and has been linked to transcriptional repression. Understanding the dynamics of MBD1 binding to nucleosomal DNA is crucial, but the molecular interactions between MBD1 and chromatin remain elusive. In this study, we found the binding of MBD1 to nucleosomes demonstrates sequence preferences depending on the position of the methyl groups on the nucleosome. Specifically, binding was favored at (me)CpG sites in the dyad proximal region and facing towards the histone octamers. At locations where the (me)CpG sites face away from the histone octamer, the binding affinity was significantly lower. Interestingly, the binding of ΔMBD1 at methylated CpG sites facing away from histone octamers induces conformational changes of nucleosomes, resulting in a more "open" conformation. The biological implication of DNA methylation is thus likely to be synergistically regulated via DNA sequences contents and their nucleosome-positioning patterns based on our in vitro findings. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. A distinct group of CpG islands shows differential DNA methylation between replicas of the same cell line in vitro.

    Science.gov (United States)

    Cocozza, Sergio; Scala, Giovanni; Miele, Gennaro; Castaldo, Imma; Monticelli, Antonella

    2013-10-10

    CpG dinucleotide-rich genomic DNA regions, known as CpG islands (CGIs), can be methylated at their cytosine residues as an epigenetic mark that is stably inherited during cell mitosis. Differentially methylated regions (DMRs) are genomic regions showing different degrees of DNA methylation in multiple samples. In this study, we focused our attention on CGIs showing different DNA methylation between two culture replicas of the same cell line. We used methylation data of 35 cell lines from the Encyclopedia of DNA Elements (ENCODE) consortium to identify CpG islands that were differentially methylated between replicas of the same cell line and denoted them Inter Replicas Differentially Methylated CpG islands (IRDM-CGIs). We identified a group of IRDM-CGIs that was consistently shared by different cell lines, and denoted it common IRDM-CGIs. X chromosome CGIs were overrepresented among common IRDM-CGIs. Autosomal IRDM-CGIs were preferentially located in gene bodies and intergenic regions had a lower G + C content, a smaller mean length, and a reduced CpG percentage. Functional analysis of the genes associated with autosomal IRDM-CGIs showed that many of them are involved in DNA binding and development. Our results show that several specific functional and structural features characterize common IRDM-CGIs. They may represent a specific subset of CGIs that are more prone to being differentially methylated for their intrinsic characteristics.

  9. CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy.

    Science.gov (United States)

    Barbé, Lise; Lanni, Stella; López-Castel, Arturo; Franck, Silvie; Spits, Claudia; Keymolen, Kathelijn; Seneca, Sara; Tomé, Stephanie; Miron, Ioana; Letourneau, Julie; Liang, Minggao; Choufani, Sanaa; Weksberg, Rosanna; Wilson, Michael D; Sedlacek, Zdenek; Gagnon, Cynthia; Musova, Zuzana; Chitayat, David; Shannon, Patrick; Mathieu, Jean; Sermon, Karen; Pearson, Christopher E

    2017-03-02

    CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10(-12)). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5, thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator.

  10. Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

    Science.gov (United States)

    Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

    2016-01-01

    Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested

  11. The application of methylation specific electrophoresis (MSE to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

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    Yokoyama Seiya

    2012-02-01

    Full Text Available Abstract Background Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. Methods Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE. The MSE method is comprised of the following steps: (a bisulfite treatment of genomic DNA, (b amplification of the target DNA by a nested PCR approach and (c applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. Result The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is Conclusions The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

  12. Inheritance of an epigenetic mark: the CpG DNA methyltransferase 1 is required for de novo establishment of a complex pattern of non-CpG methylation.

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    Valérie Grandjean

    Full Text Available Site-specific methylation of cytosines is a key epigenetic mark of vertebrate DNA. While a majority of the methylated residues are in the symmetrical (meCpG:Gp(meC configuration, a smaller, but significant fraction is found in the CpA, CpT and CpC asymmetric (non-CpG dinucleotides. CpG methylation is reproducibly maintained by the activity of the DNA methyltransferase 1 (Dnmt1 on the newly replicated hemimethylated substrates (meCpG:GpC. On the other hand, establishment and hereditary maintenance of non-CpG methylation patterns have not been analyzed in detail. We previously reported the occurrence of site- and allele-specific methylation at both CpG and non-CpG sites. Here we characterize a hereditary complex of non-CpG methylation, with the transgenerational maintenance of three distinct profiles in a constant ratio, associated with extensive CpG methylation. These observations raised the question of the signal leading to the maintenance of the pattern of asymmetric methylation. The complete non-CpG pattern was reinstated at each generation in spite of the fact that the majority of the sperm genomes contained either none or only one methylated non-CpG site. This observation led us to the hypothesis that the stable CpG patterns might act as blueprints for the maintenance of non-CpG DNA methylation. As predicted, non-CpG DNA methylation profiles were abrogated in a mutant lacking Dnmt1, the enzymes responsible for CpG methylation, but not in mutants defective for either Dnmt3a or Dnmt2.

  13. Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.

    Science.gov (United States)

    Nygren, Anders O H; Ameziane, Najim; Duarte, Helena M B; Vijzelaar, Raymon N C P; Waisfisz, Quinten; Hess, Corine J; Schouten, Jan P; Errami, Abdellatif

    2005-08-16

    Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA-probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA-probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader-Willy syndrome, Angelman syndrome or acute myeloid leukemia.

  14. Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.

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    Zhifu Sun

    Full Text Available We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+ and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A, and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

  15. Methylation and sequence analysis around EagI sites: identification of 28 new CpG islands in XQ24-XQ28.

    Science.gov (United States)

    Tribioli, C; Tamanini, F; Patrosso, C; Milanesi, L; Villa, A; Pergolizzi, R; Maestrini, E; Rivella, S; Bione, S; Mancini, M

    1992-02-25

    Thirty-two probes for CpG islands of the distal long arm of the human X chromosome have been identified. From a genomic library of DNA of the hamster-human cell hybrid X3000.1 digested with the rare cutter restriction enzyme EagI, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5' end of genes of the X chromosome has been used to distinguish between EagI sites in CpG islands versus isolated EagI sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5' end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated EagI site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylation of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.

  16. CpG Island Methylation, Microsatellite Instability, and BRAF Mutations and Their Clinical Application in the Treatment of Colon Cancer

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    Christina Wu

    2012-01-01

    Full Text Available There have been significant developments in colon cancer research over the last few years, enabling us to better characterize tumors individually and classifying them according to certain molecular or genetic features. Currently, we are able to use KRAS mutational status as a guide to therapy with anti-epidermal growth factor receptor antibodies. Other molecular features under research include BRAF mutation, microsatellite instability, and CpG island methylation. These three molecular features are often associated with tumors that have overlapping phenotypes and can be present simultaneously in the same tumor. However, they carry different prognostic and predictive qualities, making analysis of their interaction relatively complex. Much research thus far has examined the clinical relevance of microsatellite instability in helping determine prognosis and the predictive value of adjuvant 5-fluorouracil chemotherapy in stages II and III colon cancers. BRAF mutation appears to be a biomarker for poor prognosis. CpG island methylation is tightly associated with microsatellite instable tumors and BRAF mutation, but its clinical utility remains uncertain. Hereby, we examine preclinical and clinical data that supports the utilization of all three phenotypes in future research applied to clinical practice.

  17. Epigenetic silencing of BTB and CNC homology 2 and concerted promoter CpG methylation in gastric cancer.

    Science.gov (United States)

    Haam, Keeok; Kim, Hee-Jin; Lee, Kyung-Tae; Kim, Jeong-Hwan; Kim, Mirang; Kim, Seon-Young; Noh, Seung-Moo; Song, Kyu-Sang; Kim, Yong Sung

    2014-09-01

    BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription factor with a prominent role in B-cell development. Genetic polymorphisms within a single locus encoding BACH2 are associated with various autoimmune diseases and allergies. In this study, restriction landmark genomic scanning revealed methylation at a NotI site in a CpG island covering the BACH2 promoter in gastric cancer cell lines and primary gastric tumors. Increased methylation of the BACH2 promoter was observed in 52% (43/83) of primary gastric tumors, and BACH2 hypermethylation was significantly associated with decreased gene expression. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin. A restored BACH2 expression in BACH2-silenced gastric cancer cell lines, and knockdown of BACH2 using short hairpin RNA (i.e. RNA interference) increased cell proliferation in gastric cancer cells. Clinicopathologic data showed that decreased BACH2 expression occurred significantly more frequently in intestinal-type (27/44, 61%) compared with diffuse-type (13/50, 26%) gastric cancers (P<0.001). Furthermore, BACH2 promoter methylation paralleled that of previously identified targets, such as LRRC3B, LIMS2, PRKD1 and POPDC3, in a given set of gastric tumors. We propose that concerted methylation in many promoters plays a role in accelerating gastric tumor formation and that methylated promoter loci may be targets for therapeutic treatment, such as the recently introduced technique of epigenetic editing.

  18. Functional Characterization and Drug Response of Freshly Established Patient-Derived Tumor Models with CpG Island Methylator Phenotype.

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    Claudia Maletzki

    Full Text Available Patient-individual tumor models constitute a powerful platform for basic and translational analyses both in vitro and in vivo. However, due to the labor-intensive and highly time-consuming process, only few well-characterized patient-derived cell lines and/or corresponding xenografts exist. In this study, we describe successful generation and functional analysis of novel tumor models from patients with sporadic primary colorectal carcinomas (CRC showing CpG island methylator phenotype (CIMP. Initial DNA fingerprint analysis confirmed identity with the patient in all four cases. These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated. Cell lines were of epithelial origin (EpCAM+ with distinct morphology and growth kinetics. Response to chemotherapeutics was quite individual between cells, with stage I-derived cell line HROC60 being most susceptible towards standard clinically approved chemotherapeutics (e.g. 5-FU, Irinotecan. Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib. This comprehensive analysis of tumor biology, genetic alterations and assessment of chemosensitivity towards a broad range of (chemo- therapeutics helps bringing forward the concept of personalized tumor therapy.

  19. In silico enhanced restriction enzyme based methylation analysis of the human glioblastoma genome using Agilent 244K CpG Island microarrays

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    Anh Tran

    2010-01-01

    Full Text Available Genome wide methylation profiling of gliomas is likely to provide important clues to improving treatment outcomes. Restriction enzyme based approaches have been widely utilized for methylation profiling of cancer genomes and will continue to have importance in combination with higher density microarrays. With the availability of the human genome sequence and microarray probe sequences, these approaches can be readily characterized and optimized via in silico modeling. We adapted the previously described HpaII/MspI based Methylation Sensitive Restriction Enzyme (MSRE assay for use with two-color Agilent 244K CpG island microarrays. In this assay, fragmented genomic DNA is digested in separate reactions with isoschizomeric HpaII (methylation-sensitive and MspI (methylation-insensitive restriction enzymes. Using in silico hybridization, we found that genomic fragmentation with BfaI was superior to MseI, providing a maximum effective coverage of 22,362 CpG islands in the human genome. In addition, we confirmed the presence of an internal control group of fragments lacking HpaII/MspI sites which enable separation of methylated and unmethylated fragments. We used this method on genomic DNA isolated from normal brain, U87MG cells, and a glioblastoma patient tumor sample and confirmed selected differentially methylated CpG islands using bisulfite sequencing. Along with additional validation points, we performed a receiver operating characteristics (ROC analysis to determine the optimal threshold (p ≤ 0.001. Based on this threshold, we identified ~2400 CpG islands common to all three samples and 145 CpG islands unique to glioblastoma. These data provide more general guidance to individuals seeking to maximize effective coverage using restriction enzyme based methylation profiling approaches.

  20. Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation

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    Koeffler Phillip

    2010-02-01

    Full Text Available Abstract Background Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer. Methods Methylation specific PCR (MSP and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8 and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay. Results The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines. Conclusions CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer.

  1. Weight loss after gastric bypass surgery in human obesity remodels promoter methylation

    DEFF Research Database (Denmark)

    Barres, Romain; Kirchner, Henriette; Rasmussen, Morten

    2013-01-01

    observed in the normal-weight, healthy subjects. Using bisulfite sequencing, we show that promoter methylation of PGC-1a and PDK4 is altered with obesity and restored to nonobese levels after RYGB-induced weight loss. A genome-wide DNA methylation analysis of skeletal muscle revealed that obesity...... is associated with hypermethylation at CpG shores and exonic regions close to transcription start sites. Our results provide evidence that obesity and RYGB-induced weight loss have a dynamic effect on the epigenome....... of genes enriched in metabolic process and mitochondrial function. After weight loss, the expression of the majority of the identified genes was normalized to levels observed in normal-weight, healthy controls. Among the 14 metabolic genes analyzed, promoter methylation of 11 genes was normalized to levels...

  2. Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation.

    Science.gov (United States)

    Wang, Xiaotong; Li, Qiye; Lian, Jinmin; Li, Li; Jin, Lijun; Cai, Huimin; Xu, Fei; Qi, Haigang; Zhang, Linlin; Wu, Fucun; Meng, Jie; Que, Huayong; Fang, Xiaodong; Guo, Ximing; Zhang, Guofan

    2014-12-16

    Studies of DNA methylomes in a wide range of eukaryotes have revealed both conserved and divergent characteristics of DNA methylation among phylogenetic groups. However, data on invertebrates particularly molluscs are limited, which hinders our understanding of the evolution of DNA methylation in metazoa. The sequencing of the Pacific oyster Crassostrea gigas genome provides an opportunity for genome-wide profiling of DNA methylation in this model mollusc. Homologous searches against the C. gigas genome identified functional orthologs for key genes involved in DNA methylation: DNMT1, DNMT2, DNMT3, MBD2/3 and UHRF1. Whole-genome bisulfite sequencing (BS-seq) of the oyster's mantle tissues revealed that more than 99% methylation modification was restricted to cytosines in CpG context and methylated CpGs accumulated in the bodies of genes that were moderately expressed. Young repeat elements were another major targets of CpG methylation in oysters. Comparison with other invertebrate methylomes suggested that the 5'-end bias of gene body methylation and the negative correlation between gene body methylation and gene length were the derived features probably limited to the insect lineage. Interestingly, phylostratigraphic analysis showed that CpG methylation preferentially targeted genes originating in the common ancestor of eukaryotes rather than the oldest genes originating in the common ancestor of cellular organisms. Comparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these 'relatively young' genes was critical for the origin and radiation of eukaryotes.

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

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    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.

  4. Lead exposure suppressed ALAD transcription by increasing methylation level of the promoter CpG islands.

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    Li, Chunping; Xu, Ming; Wang, Sumeng; Yang, Xiaolin; Zhou, Shourong; Zhang, Jingping; Liu, Qizhan; Sun, Yujie

    2011-05-30

    DNA methylation provides a plausible link between the environment and alterations in gene expression that may lead to disease phenotypes. Lead exposure can change DNA methylation status. Here, we hypothesized that the methylation of the ALAD gene promoter may play an important role in lead toxicity. To determine whether the methylation level of the ALAD promoter is associated with the risk of lead poisoning, we conducted a case-control study of 103 workers from a battery plant and 103 healthy volunteers with matching age and gender distribution. We employed real-time PCR and methylation-specific PCR (MSP) in cell models to determine the relationship between ALAD methylation level and transcription level. We found lead exposure to increase the ALAD gene methylation level and down-regulate ALAD transcription. The difference in methylation frequencies between exposures and controls was statistically significant (p=0.002), and individuals with methylated ALAD gene showed an increased risk of lead poisoning (adjusted OR=3.57, 95% CI, 1.55-8.18). This study suggests that the lead-exposure-induced increases in ALAD methylation may be involved in the mechanism of lead toxicity. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Association between GSTP1 CpG methylation and the early phase of lead exposure.

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    Li, Chunping; Yang, Xiaolin; Xu, Ming; Zhang, Jinlong; Sun, Na

    2014-02-01

    GSTP1 is induced by lead, and thus serves as a biomarker of lead exposure. Lead exposure changes DNA methylation status. We attempted to prove that the methylation of the GSTP1 promoter plays an important role in lead toxicity. We conducted a case-control study of 53 workers from a battery plant and 53 age and sex matched healthy volunteers to determine whether the methylation level of the GSTP1 promoter is associated with the risk of lead poisoning. We employed methylation-specific PCR (MSP) in cell models to determine the relationship between the GSTP1 methylation level and lead exposure. We found no association between GSTP1 methylation and lead exposure. The difference in methylation frequencies between the exposure group and the controls was not statistically significant (p = 0.401), and individuals with the methylated GSTP1 gene was not associated with the risk of lead poisoning (adjusted OR = 1.36, 95% CI, 0.22-8.24). This study suggests that GSTP1 methylation is not involved in the early phase of lead toxicity. Further studies should be performed to detect the association between GSTP1 methylation and the risk of lead poisoning in later phases.

  6. Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape.

    Science.gov (United States)

    Day, Kenneth; Waite, Lindsay L; Thalacker-Mercer, Anna; West, Andrew; Bamman, Marcas M; Brooks, James D; Myers, Richard M; Absher, Devin

    2013-01-01

    DNA methylation is an epigenetic modification that changes with age in human tissues, although the mechanisms and specificity of this process are still poorly understood. We compared CpG methylation changes with age across 283 human blood, brain, kidney, and skeletal muscle samples using methylation arrays to identify tissue-specific age effects. We found age-associated CpGs (ageCGs) that are both tissue-specific and common across tissues. Tissue-specific age CGs are frequently located outside CpG islands with decreased methylation, and common ageCGs show the opposite trend. AgeCGs are significantly associated with poorly expressed genes, but those with decreasing methylation are linked with higher tissue-specific expression levels compared with increasing methylation. Therefore, tissue-specific gene expression may protect against common age-dependent methylation. Distinguished from other tissues, skeletal muscle age CGs are more associated with expression, enriched near genes related to myofiber contraction, and closer to muscle-specific CTCF binding sites. Kidney-specific ageCGs are more increasingly methylated compared to other tissues as measured by affiliation with kidney-specific expressed genes. Underlying chromatin features also mark common and tissue-specific age effects reflective of poised and active chromatin states, respectively. In contrast with decreasingly methylated ageCGs, increasingly methylated ageCGs are also generally further from CTCF binding sites and enriched within lamina associated domains. Our data identified common and tissue-specific DNA methylation changes with age that are reflective of CpG landscape and suggests both common and unique alterations within human tissues. Our findings also indicate that a simple epigenetic drift model is insufficient to explain all age-related changes in DNA methylation.

  7. Nucleosomes correlate with in vivo progression pattern of de novo methylation of p16 CpG islands in human gastric carcinogenesis.

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    Zhe-Ming Lu

    Full Text Available BACKGROUND: The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the nucleosome position within the p16 CpG island and established a seeding methylation-specific PCR (sMSP assay based on bisulfite modification to enrich the p16 alleles containing methylated-CpG at the methylation "seeding" sites within its intron-1 in gastric carcinogenesis. The sMSP-positive rate in primary gastric carcinoma (GC samples (36/40 was significantly higher than that observed in gastritis (19/45 or normal samples (7/13 (P<0.01. Extensive clone sequencing of these sMSP products showed that the density of methylated-CpGs in p16 CpG islands increased gradually along with the severity of pathological changes in gastric tissues. In gastritis lesions the methylation was frequently observed in the region corresponding to the exon-1 coding-nucleosome and the 5'UTR-nucleosome; the methylation was further extended to the region corresponding to the promoter-nucleosome in GC samples. Only few methylated-CpG sites were randomly detected within p16 CpG islands in normal tissues. The significantly inversed relationship between the p16 exon-1 methylation and its transcription was observed in GC samples. An exact p16 promoter-specific 83 bp-MSP assay confirms the result of sMSP (33/55 vs. 1/6, P<0.01. In addition, p16 methylation in chronic gastritis lesions significantly correlated with H. pylori infection; however, such correlation was not observed in GC specimens. CONCLUSIONS/SIGNIFICANCE: It was determined that de novo methylation was initiated in the coding region of p16 exon-1 in gastritis, then progressed to its 5'UTR, and ultimately to the proximal promoter in GCs. Nucleosomes may function as the basic extension/progression unit of de novo methylation of p16 CpG islands in vivo.

  8. Breed-specific expression of GR exon 1 mRNA variants and profile of GR promoter CpG methylation in the hippocampus of newborn piglets.

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    Sun, Q; Jia, Y; Li, R; Li, X; Yang, X; Zhao, R

    2014-11-01

    Glucocorticoid receptor (GR) transcription is driven by alternative promoters to produce different exon 1 mRNA variants. CpG methylation on GR promoters profoundly affects GR transcription. GR in hippocampus is critical for energy homeostasis and stress responses, yet it remains unclear whether hippocampal expression of GR exon 1 mRNA variants and the methylation status of GR promoters differ between Large White (LW) and Erhualian (EHL) pigs showing distinct metabolic and stress-coping characteristics. EHL pigs had higher hippocampus weight relative to BW (PGR did not differ between breeds, yet GR exon 1 to 11 mRNA was significantly higher (PGR protein content. No significant breed difference was detected for the methylation status across the whole region of the proximal GR promoter, while CpG334 and CpG266.267 were differentially methylated, in a reversed manner, between breeds. The methylation status of CpGs 248, 259, 260, 268 and 271 was negatively correlated (PGR exon 1 to 11 mRNA abundance. Our results provide fundamental information on the breed-specific characteristics of GR and its mRNA variants expression and the status of DNA methylation on the proximal GR promoter in the pig hippocampus.

  9. INACTIVATION OF THE CDKN2/pl6 GENE INDUCED BY METHYLATION AT 5'-CpG ISLAND AND ITS RELATION TO LUNG CANCER

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objectives: To investigate the methylation status of the CDKN2/pl6 gene 5'-CpG island, and study the relationship between the CDKN2/pl6 gene inacti-vation by methylation and lung cancer. Methods: Genomic DNA extracted from the specimens of lung cancer and normal lung tissue was digested with methylation-sensitive endonucleases, and Southern blotting was used to analyze the methylation status of the CDKN2/pl6 gene in 89 cases of lung cancer and 10 cases of normal lung tissue. Results: In 89 cases of lung cancer studied, the CDKN2/pl6 gene was shown to be methylated in 21 cases with the total methylation rate of 23.6% (21/89), in which there were 15 cases (16.9%) methylated at SmaI sites, 12 cases (13.5%) at SacII sites and 6 cases at both SmaI and SacII sites. The methylation of the CDKN2/pl6 gene occurred in 17 among 42 of pl6 protein negative cases of lung cancer with a rate of 40.5% (17/42), and in 3 among 47 of pl6 protein positive cases with a Conclusion: The aberrant methylation of the CDKN2/pl6 gene 5'-CpG island is probably an important mechanism of the gene inactivation, it may be involved in the genesis and progress of lung cancer.

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

    Science.gov (United States)

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

    2009-11-01

    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 folate metabolizing enzymes (MTHFR, MTR, and MTRR), DNA methyltransferase DNMT3b, and histone methyltransferases (EHMT1, EHMT2, and PRDM2), with colorectal cancers, with or without the CpG island methylator phenotype (CIMP), MLH1 hypermethylation, or microsatellite instability. Incidence rate ratios were calculated in case-cohort analyses, with common homozygotes as reference, among 659 cases and 1,736 subcohort members of the Netherlands Cohort Study on diet and cancer (n = 120,852). Men with the MTHFR 677TT genotype were at decreased colorectal cancer risk (incidence rate ratio, 0.49; P = 0.01), but the T allele was associated with increased risk in women (incidence rate ratio, 1.39; P = 0.02). The MTR 2756GG genotype was associated with increased colorectal cancer risk (incidence rate ratio, 1.58; P = 0.04), and inverse associations were observed among women carrying DNMT3b C-->T (rs406193; incidence rate ratio, 0.72; P = 0.04) or EHMT2 G-->A (rs535586; incidence rate ratio, 0.76; P = 0.05) polymorphisms. Although significantly correlated (P DNMT3b, and EHMT2 polymorphisms are associated with colorectal cancer, and rare variants of MTR and MTRR may reduce promoter hypermethylation. The incomplete overlap between CIMP, MLH1 hypermethylation, and microsatellite instability indicates that these related "methylation phenotypes" may not be similar and should be investigated separately.

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

    Science.gov (United States)

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

    2011-01-01

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

  12. Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Li Xu; Jing-De Zhu; Jian Yu; Hong-Yu Zhang; Meng-Hong Sun; Jun Gu; Xiang Du; Da-Ren Shi; Peng Wang; Zhen-Hua Yang

    2004-01-01

    AIM: To establish the methylation profile of the promoter CpG islands of 31 genes that might play etiological roles in colon carcinogenesis.METHODS: The methylation specific PCR in conjunction of sequencing verification was used to establish the methylationprofile of the promoter CpG islands of 31 genes in colorectal cancer (n = 65), the neighboring non-cancerous tissues (n = 5), colorectal adenoma (n = 8), and normal mucosa (n = 1). Immunohistochemically, expression of 10 genes was assessed on the home-made tissue microarrays of tissues from 58 patients. The correlation of tumor specific changes with each of clinical-pathologic features was scrutinized with relevant statistic tools.RESULTS: In comparison with the normal mucosa of the non-cancer patients, the following 14 genes displayed no tumor associated changes: breast cancer 1, early onset (BRCA1), cadherin 1, type 1, E-cadherin (epithelial) (CDH1),death-associated protein kinase 1 (DAPK1), DNA (cytosine5-)-methyltransferase 1 (DNMT1), melanoma antigen, family A, 1 (directs expression of antigen MZ2-E) (MAGEA1), tumor suppressor candidate 3 (N33), cyclin-dependent kinase inhibitor 1A (p21, Cip1) (p21WAF1), cyclin-dependent kinase inhibitor 1B (p27, Kip1) (p27KIP1) , phosphatase and tensin homlog (mutated in multiple advanced cancers 1) (PTEN), retinoic acid receptor, beta (RAR-, Ras association (RalGDS/AF-6)domain family 1 C (RASSF1C), secreted frizzled-related protein 1 (SFRP1), tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinfiammatory) (TIMP3),and von Hippel-Lindau syndrome (VHL). The rest 17 targets exhibited to various extents the tumor associated changes.As changes in methylation of the following genes occurred marginally, their impact on the formation of colorectal cancer were trivial: adenomatous polyposis coli (APC) (8%, 5/65),Ras association (RalGDS/AF-6) domain family 1A (RASSF1A) (3%, 2/65) and cyclin-dependent kinase inhibitor 2A,alternated reading frame (p14ARF) (6%, 4

  13. Methylation profile of the promoter CpG islands of 14"drug-resistance" genes in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Sheng Ding; Bang-Dong Gong; Jian Yu; Jun Gu; Hong-Yu Zhang; Zu-Bin Shang; Qi Fei; Peng Wang; Jing-De Zhu

    2004-01-01

    AIM: To establish the DNA methylation patterns of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma (HCC).METHODS: The methylation specific polymerase chain reaction in conjunction with sequencing verification was used to establish the methylation patterns of the 14 genes in the liver tissues of four healthy liver donors, as well as tumor and the paired non-cancerous tissues of 30 HCC patients.RESULTS: While 11 genes (ATP-binding cassette, sub-family G (WHITE), member 2(ABCG2), activating transcription factor (ATF2), beta-2-microglobulin (B2M), deoxycytidine kinase (DCK), occludin (OCLN), v-raf-1 murine leukemia viral oncogene homolog (RAF1), ralA binding protein 1 (RALBP1),splicing factor (45 kD) (SPF45), S-phase kinase-associated protein 2 (p45) (SKP2), tumor protein p53 (Li-Fraumeni syndrome) (TP53) and topoisomerase (DNA) Ⅱ beta (TOP2B))maintained the unmethylated patterns, three genes displayed to various extents the hypermethylation state in tumor tissues in comparison with the normal counterparts. The catalase (CAT) was hypermethylated in tumor and the neighboring non-cancerous tissue of one case (3.3%). Both glutathione S-transferase pi (GSTpi) (80%, 24/30 in tumor and 56.7%,17/30 in the paired non-cancerous tissues) and cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) (CFTR) (77%, 23/30 in tumor and 50%, 15/30 in the paired non-cancerous tissues) genes were prevalently hypermethylated in HCC as well as their neighboring non-cancerous tissues. No significant difference in the hypermethylation occurrence was observed between the HCC and its neighboring non-cancerous tissues.CONCLUSION: Hypermethylation of promoter CpG islands of both CFTR and GSTpi genes occurs prevalently in HCC,which may correlate with the low expression of these two genes at the mRNA level and has the profound etiological and clinical implications. It is likely to be specific to the early phase of HCC

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

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    Wang, Tingting [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Chen, Man; Liu, Lian [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Cheng, Huaiyan [Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Yan, You-E [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Feng, Ying-Hong, E-mail: yhfeng@usuhs.edu [Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Wang, Hui, E-mail: wanghui19@whu.edu.cn [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2011-12-15

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

  15. Methylation of specific CpG sites in the P2 promoter of parathyroid hormone-related protein determines the invasive potential of breast cancer cell lines.

    Science.gov (United States)

    Tost, Jörg; Hamzaoui, Hinda; Busato, Florence; Neyret, Aymeric; Mourah, Samia; Dupont, Jean-Michel; Bouizar, Zhor

    2011-08-01

    Parathyroid hormone-related protein (PTHrP) is upregulated in primary breast cancers and a major candidate for osteoclastic bone resorption present at sites of breast cancer to bone metastases. Using a human model of mammary epithelial cell lines differing in tumorigenicity and PTHrP expression, we investigated the role of epigenetic modifications for PTHrP expression. Quantitative analysis of the DNA methylation patterns at a total of 104 CpGs in the promoter region of PTHrP by pyrosequencing showed the absence of methylation in all analyzed cell lines in the large CpG island upstream of exon 1C. In the second intron of promoter 2 (P2) a region was identified containing 4 CpG nucleotides for which differential methylation correlated with the PTHrP expression level. The functional importance of this control mechanism was confirmed by the ability of the demethylating agent 5'-azacytidine to induce PTHrP mRNA and iPTHrP protein expression in previously non-expressing cell lines and increase their production by metastatic NS2T2A1 cells. In particular, transcription from P2 was activated non-tumoral S1T3 cells upon treatment with 5'-azacytidine. Our findings support the hypothesis that the methylation status of specific CpG dinucleotides is the dominant mechanism involved in silencing of PTHrP expression rather than the overall methylation of the CpG island. Methylation of the PTHrP P2 is a potential marker of breast cancer progression and might be used to evaluate the metastatic potential of breast tumors.

  16. JC Virus T-Antigen in Colorectal Cancer Is Associated with p53 Expression and Chromosomal Instability, Independent of CpG Island Methylator Phenotype

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    Katsuhiko Nosho

    2009-01-01

    Full Text Available JC virus has a transforming gene encoding JC virus T-antigen (JCVT. JCVT may inactivate wild-type p53, cause chromosomal instability (CIN, and stabilize β-catenin. A link between JCVT and CpG island methylator phenotype (CIMP has been suggested. However, no large-scale study has examined the relations of JCVT with molecular alterations, clinical outcome, or prognosis in colon cancer. We detected JCVT expression (by immunohistochemistry in 271 (35% of 766 colorectal cancers. We quantified DNA methylation in eight CIMP-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1 and eight other loci (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, WRN by MethyLight. We examined loss of heterozygosity in 2p, 5q, 17q, and 18q. JCVT was significantly associated with p53 expression (P < .0001, p21 loss (P < .0001, CIN (≥2 chromosomal segments with LOH; P < .0001, nuclear β-catenin (P = .006, LINE-1 hypomethylation (P = .002, and inversely with CIMP-high (P = .0005 and microsatellite instability (MSI (P < .0001, but not with PIK3CA mutation. In multivariate logistic regression analysis, the associations of JCVT with p53 [adjusted odds ratio (OR, 8.45; P < .0001], CIN (adjusted OR, 2.53; P = .003, cyclin D1 (adjusted OR, 1.57; P = .02, LINE-1 hypomethylation (adjusted OR, 1.97 for a 30% decline as a unit; P = .03, BRAF mutation (adjusted OR, 2.20; P = .04, and family history of colorectal cancer (adjusted OR, 0.64; P = .04 remained statistically significant. However, JCVT was no longer significantly associated with CIMP, MSI, β-catenin, or cyclooxygenase-2 expression in multivariate analysis. JCVT was unrelated with patient survival. In conclusion, JCVT expression in colorectal cancer is independently associated with p53 expression and CIN, which may lead to uncontrolled cell proliferation.

  17. CpG methylation differences between neurons and glia are highly conserved from mouse to human

    Science.gov (United States)

    Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study used genome-wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That stud...

  18. Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists

    Directory of Open Access Journals (Sweden)

    Kim Kyu-Tae

    2010-07-01

    Full Text Available Abstract Background DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC and Trichostatin A (TSA respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. Method Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC. Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. Results Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS. In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. Conclusion These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically

  19. Epstein-barr virus latency in B cells leads to epigenetic repression and CpG methylation of the tumour suppressor gene Bim.

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    Kostas Paschos

    2009-06-01

    Full Text Available In human B cells infected with Epstein-Barr virus (EBV, latency-associated virus gene products inhibit expression of the pro-apoptotic Bcl-2-family member Bim and enhance cell survival. This involves the activities of the EBV nuclear proteins EBNA3A and EBNA3C and appears to be predominantly directed at regulating Bim mRNA synthesis, although post-transcriptional regulation of Bim has been reported. Here we show that protein and RNA stability make little or no contribution to the EBV-associated repression of Bim in latently infected B cells. However, treatment of cells with inhibitors of histone deacetylase (HDAC and DNA methyltransferase (DNMT enzymes indicated that epigenetic mechanisms are involved in the down-regulation of Bim. This was initially confirmed by chromatin immunoprecipitation analysis of histone acetylation levels on the Bim promoter. Consistent with this, methylation-specific PCR (MSP and bisulphite sequencing of regions within the large CpG island located at the 5' end of Bim revealed significant methylation of CpG dinucleotides in all EBV-positive, but not EBV-negative B cells examined. Genomic DNA samples exhibiting methylation of the Bim promoter included extracts from a series of explanted EBV-positive Burkitt's lymphoma (BL biopsies. Subsequent analyses of the histone modification H3K27-Me3 (trimethylation of histone H3 lysine 27 and CpG methylation at loci throughout the Bim promoter suggest that in EBV-positive B cells repression of Bim is initially associated with this repressive epigenetic histone mark gradually followed by DNA methylation at CpG dinucleotides. We conclude that latent EBV initiates a chain of events that leads to epigenetic repression of the tumour suppressor gene Bim in infected B cells and their progeny. This reprogramming of B cells could have important implications for our understanding of EBV persistence and the pathogenesis of EBV-associated disease, in particular BL.

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

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    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.

  1. Body size, physical activity and risk of colorectal cancer with or without the CpG island methylator phenotype (CIMP.

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    Laura A E Hughes

    Full Text Available BACKGROUND: We investigated how body size and physical activity influence the risk of the CpG island methylator phenotype (CIMP in colorectal cancer (CRC. METHODS: In the Netherlands Cohort Study (n = 120,852, risk factors were self-reported at baseline in 1986. After 7.3 years of follow-up, 603 cases and 4,631 sub-cohort members were available. CIMP status according to the Weisenberger markers was determined using methylation specific PCR on DNA from paraffin embedded tumor tissue. Hazard rate ratios (HR and 95% confidence intervals for CIMP (27.7% and non-CIMP (72.3% tumors were calculated according to BMI, BMI at age 20, BMI change, trouser/skirt size, height, and physical activity. RESULTS: BMI modeled per 5 kg/m(2 increase was associated with both CIMP and non-CIMP tumors, however, HRs were attenuated when additionally adjusted for trouser/skirt size. Trouser/skirt size, per 2 size increase, was associated with both tumor subtypes, even after adjustment for BMI (CIMP HR: 1.20, 95%CI: 1.01-1.43; non-CIMP HR: 1.14, 95%CI: 1.04-1.28. Height per 5 cm was associated with both tumor sub-types, but HRs were attenuated when adjusted for body weight. BMI at age 20 was positively associated with increased risk of CIMP tumors and the association was significantly less pronounced for non-CIMP tumors (P-heterogeneity = 0.01. Physical activity was inversely associated with both subtypes, but a dose-response association was observed only for non-CIMP tumors (P-trend = 0.02. CONCLUSIONS: Body size, especially central adiposity, may increase the risk of both CIMP and non-CIMP tumors. Body fat at young age may differentially influence risk. Physical activity appears to decrease the risk of CRC regardless of these molecular subtypes.

  2. Detection of CpG methylations in human mismatch repair gene hMLH1 promoter by denaturing high-performance liquid chromatography (DHPLC)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objectives: To develop a novel method to detect CpG methylation by DHPLC. Methods: After DNA was treated with sodium bisulfite, mismatch repair gene hMLH1 promoter was amplified by polymerase chain reaction (PCR). DHPLC was used to separate the PCR products at their partially denaturing temperatures. BstUI digestion assay was also used for comparison study. Results: A 294bp band was obtained by PCR from each DNA samples of colon cancer cell line RKO and gastric cancer cell line PACM82. These two bands could be separated completely by DHPLC at 53° C (retention time 6.7 min for RKO vs. 6.2 min for PACM82). We concluded that the hMLH1 promoter in RKO cells is methylated, while PACM82 is not methylated, since methylation can protect the conversion of C to T and keep higher C/G content after bisulfite treatment, leading to the delayed time. These results consistent with those from BstUI digestion assay. Conclusion: Methylation in CpG islands of hMLH1 could be detected conveniently by DHPLC after bisulfite modification.

  3. AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins.

    Science.gov (United States)

    Parida, Adwaita Prasad; Sharma, Amrapali; Sharma, Arun Kumar

    2017-03-01

    DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

  4. A dual epigenomic approach for the search of obesity biomarkers: DNA methylation in relation to diet-induced weight loss.

    Science.gov (United States)

    Milagro, Fermin I; Campión, Javier; Cordero, Paúl; Goyenechea, Estíbaliz; Gómez-Uriz, Ana M; Abete, Itziar; Zulet, Maria A; Martínez, J Alfredo

    2011-04-01

    Epigenetics could help to explain individual differences in weight loss after an energy-restriction intervention. Here, we identify novel potential epigenetic biomarkers of weight loss, comparing DNA methylation patterns of high and low responders to a hypocaloric diet. Twenty-five overweight or obese men participated in an 8-wk caloric restriction intervention. DNA was isolated from peripheral blood mononuclear cells and treated with bisulfite. The basal and endpoint epigenetic differences between high and low responders were analyzed by methylation microarray, which was also useful in comparing epigenetic changes due to the nutrition intervention. Subsequently, MALDI-TOF mass spectrometry was used to validate several relevant CpGs and the surrounding regions. DNA methylation levels in several CpGs located in the ATP10A and CD44 genes showed statistical baseline differences depending on the weight-loss outcome. At the treatment endpoint, DNA methylation levels of several CpGs on the WT1 promoter were statistically more methylated in the high than in the low responders. Finally, different CpG sites from WT1 and ATP10A were significantly modified as a result of the intervention. In summary, hypocaloric-diet-induced weight loss in humans could alter DNA methylation status of specific genes. Moreover, baseline DNA methylation patterns may be used as epigenetic markers that could help to predict weight loss.

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

    Directory of Open Access Journals (Sweden)

    Dong Chan Moon

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

  6. CpG demethylation in the neurotoxicity of 1-methyl-4-phenylpyridinium might mediate transcriptional up-regulation of α-synuclein in SH-SY5Y cells.

    Science.gov (United States)

    Yang, Jian; Yang, Zhaofei; Wang, Xuan; Sun, Min; Wang, Yong; Wang, Xiaomin

    2017-08-12

    The accumulation of α-synuclein is the primary pathological hallmark of Parkinson's disease (PD). In PD patients, CpG demethylation of intron-1 has been reported to be associated with α-synuclein up-regulation. Environmental factor, for example neurotoxin, is a major etiological risk factor in PD pathogenesis. However, the role of CpG methylation in neurotoxin-induced PD has not been addressed completely yet. To explore CpG methylation associating with α-synuclein transcription and its underlying mechanisms in the neurotoxin-induced PD pathology, human neuroblastoma SH-SY5Y cells were treated with neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP(+)). Results showed that MPP(+) induced demethylation of the whole length of the CpG island around SNCA promoter, and both 6-OHDA and MPP(+) resulted in up-regulation of SNCA transcription. The CpG demethylation around promoter resulted in up-regulation of SNCA transcriptional activity. In addition, 6-OHDA and MPP(+) induced the reduced levels of DNA methyltransferase (DNMT) 3a and DNMT3b but not DNMT1. These data suggested that CpG demethylation was induced by MPP(+) and might mediate up-regulation of SNCA transcription in neurotoxin-induced PD. And down-regulation of both DNMT3a and DNMT3b, but not DNMT1, might contribute to CpG demethylation of the SNCA promoter. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins

    Indian Academy of Sciences (India)

    ADWAITA PRASAD PARIDA; AMRAPALI SHARMA; ARUN KUMAR SHARMA

    2017-03-01

    DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome fromtransposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG bindingdomain proteins are members of a class of proteins that bind tomethylated DNA. The Arabidopsis thaliana genome encodes13 methyl CpG binding domain (MBD) proteins, but themolecular/biological functions of most of these proteins are still notclear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNAbinding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40Sribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physicallyinteracts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component ofthe RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci,pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved inRNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 alsointeracts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

  8. Methylation on the Circadian Gene BMAL1 Is Associated with the Effects of a Weight Loss Intervention on Serum Lipid Levels.

    Science.gov (United States)

    Samblas, Mirian; Milagro, Fermin I; Gómez-Abellán, Purificación; Martínez, J Alfredo; Garaulet, Marta

    2016-06-01

    The circadian clock system has been linked to the onset and development of obesity and some accompanying comorbidities. Epigenetic mechanisms, such as DNA methylation, are putatively involved in the regulation of the circadian clock system. The aim of this study was to investigate the influence of a weight loss intervention based on an energy-controlled Mediterranean dietary pattern in the methylation levels of 3 clock genes, BMAL1, CLOCK, and NR1D1, and the association between the methylation levels and changes induced in the serum lipid profile with the weight loss treatment. The study sample enrolled 61 women (body mass index = 28.6 ± 3.4 kg/m(2); age: 42.2 ± 11.4 years), who followed a nutritional program based on a Mediterranean dietary pattern. DNA was isolated from whole blood obtained at the beginning and end point. Methylation levels at different CpG sites of BMAL1, CLOCK, and NR1D1 were analyzed by Sequenom's MassArray. The energy-restricted intervention modified the methylation levels of different CpG sites in BMAL1 (CpGs 5, 6, 7, 9, 11, and 18) and NR1D1 (CpGs 1, 10, 17, 18, 19, and 22). Changes in cytosine methylation in the CpG 5 to 9 region of BMAL1 with the intervention positively correlated with the eveningness profile (p = 0.019). The baseline methylation of the CpG 5 to 9 region in BMAL1 positively correlated with energy (p = 0.047) and carbohydrate (p = 0.017) intake and negatively correlated with the effect of the weight loss intervention on total cholesterol (p = 0.032) and low-density lipoprotein cholesterol (p = 0.005). Similar significant and positive correlations were found between changes in methylation levels in the CpG 5 to 9 region of BMAL1 due to the intervention and changes in serum lipids (p < 0.05). This research describes apparently for the first time an association between changes in the methylation of the BMAL1 gene with the intervention and the effects of a weight loss intervention on blood lipids levels.

  9. Diagnosis and Prognostication of Ductal Adenocarcinomas of the Pancreas Based on Genome-Wide DNA Methylation Profiling by Bacterial Artificial Chromosome Array-Based Methylated CpG Island Amplification

    Directory of Open Access Journals (Sweden)

    Masahiro Gotoh

    2011-01-01

    Full Text Available To establish diagnostic criteria for ductal adenocarcinomas of the pancreas (PCs, bacterial artificial chromosome (BAC array-based methylated CpG island amplification was performed using 139 tissue samples. Twelve BAC clones, for which DNA methylation status was able to discriminate cancerous tissue (T from noncancerous pancreatic tissue in the learning cohort with a specificity of 100%, were identified. Using criteria that combined the 12 BAC clones, T-samples were diagnosed as cancers with 100% sensitivity and specificity in both the learning and validation cohorts. DNA methylation status on 11 of the BAC clones, which was able to discriminate patients showing early relapse from those with no relapse in the learning cohort with 100% specificity, was correlated with the recurrence-free and overall survival rates in the validation cohort and was an independent prognostic factor by multivariate analysis. Genome-wide DNA methylation profiling may provide optimal diagnostic markers and prognostic indicators for patients with PCs.

  10. Comparison of CpG island methylator phenotype (CIMP frequency in colon cancer using different probe- and gene-specific scoring alternatives on recommended multi-gene panels.

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    Marianne Berg

    Full Text Available BACKGROUND: In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP. However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer. METHODS: Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared. RESULTS: For 47 samples (71%, the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20% consistently scored as CIMP positive. Only four of 31 probes (13% investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using 'the most stringent' as compared to 'the least stringent' criteria (20% vs 46%, respectively; p<0.005 was demonstrated. CONCLUSIONS: A statistical significant variation in the frequency of CIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition

  11. Environmental stress affects DNA methylation of a CpG rich promoter region of serotonin transporter gene in a nurse cohort.

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    Jukka S Alasaari

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

  12. Prognostic significance of promoter CpG island methylation of obesity-related genes in patients with nonmetastatic renal cell carcinoma.

    Science.gov (United States)

    Mendoza-Pérez, Julia; Gu, Jian; Herrera, Luis A; Tannir, Nizar M; Zhang, Shanyu; Matin, Surena; Karam, Jose A; Wood, Christopher G; Wu, Xifeng

    2017-09-15

    Greater than 40% of renal cell carcinoma (RCC) cases in the United States are attributed to excessive body weight. Moreover, obesity also may be linked to RCC prognosis. However, the molecular mechanisms underlying these associations are unclear. In the current study, the authors evaluated the role of promoter methylation in obesity-related genes in RCC tumorigenesis and disease recurrence. Paired tumors (TU) and normal adjacent (N-Adj) tissues from 240 newly diagnosed and previously untreated white patients with RCC were examined. For the discovery phase, 63 RCC pairs were analyzed. An additional 177 RCC pairs were evaluated for validation. Pyrosequencing was used to determine CpG methylation in 20 candidate obesity-related genes. An independent data set from The Cancer Genome Atlas also was analyzed for functional validation. The association between methylation and disease recurrence was analyzed using multivariate Cox proportional hazards models and Kaplan-Meier survival analysis. Methylation in neuropeptide Y (NPY), leptin (LEP), and leptin receptor (LEPR) was significantly higher in TU compared with N-Adj tissues (Pgenes are involved in RCC tumorigenesis. Furthermore, LEPR methylation appears to be associated with RCC recurrence. Future research to elucidate the biology underlying this association is warranted. Cancer 2017;123:3617-27. © 2017 American Cancer Society. © 2017 American Cancer Society.

  13. Systematic CpG islands methylation profiling of genes in the wnt pathway in epithelial ovarian cancer identifies biomarkers of progression-free survival.

    Science.gov (United States)

    Dai, Wei; Teodoridis, Jens M; Zeller, Constanze; Graham, Janet; Hersey, Jenny; Flanagan, James M; Stronach, Euan; Millan, David W; Siddiqui, Nadeem; Paul, Jim; Brown, Robert

    2011-06-15

    Wnt pathways control key biological processes that potentially impact on tumor progression and patient survival. We aimed to evaluate DNA methylation at promoter CpG islands (CGI) of Wnt pathway genes in ovarian tumors at presentation and identify biomarkers of patient progression-free survival (PFS). Epithelial ovarian tumors (screening study n = 120, validation study n = 61), prospectively collected through a cohort study, were analyzed by differential methylation hybridization at 302 loci spanning 189 promoter CGIs at 137 genes in Wnt pathways. The association of methylation and PFS was examined by Cox proportional hazards model. DNA methylation is associated with PFS at 20 of 302 loci (P < 0.05, n = 111), with 5 loci significant at false discovery rate (FDR) less than 10%. A total of 11 of 20 loci retain significance in an independent validation cohort (n = 48, P ≤ 0.05, FDR ≤ 10%), and 7 of these loci, at FZD4, DVL1, NFATC3, ROCK1, LRP5, AXIN1, and NKD1 genes, are independent from clinical parameters (adjusted P < 0.05). Increased methylation at these loci associates with increased hazard of disease progression. A multivariate Cox model incorporates only NKD1 and DVL1, identifying two groups differing in PFS [HR = 2.09; 95% CI (1.39-3.15); permutation test P < 0.005]. Methylation at DVL1 and NFATC3 show significant association with response. Consistent with their epigenetic regulation, reduced expression of FZD4, DVL1, and ROCK1 is an indicator of early-disease relapse in an independent ovarian tumor cohort (n = 311, adjusted P < 0.05). The data highlight the importance of epigenetic regulation of multiple promoter CGIs of Wnt pathway genes in ovarian cancer and identify methylation at NKD1 and DVL1 as independent predictors of PFS. ©2011 AACR.

  14. Methylation of CpG island of p14(ARK), p15(INK4b) and p16(INK4a) genes in coke oven workers.

    Science.gov (United States)

    Zhang, H; Li, X; Ge, L; Yang, J; Sun, J; Niu, Q

    2015-02-01

    To detect the blood genomic DNA methylation in coke oven workers and find a possible early screening index for occupational lung cancer, 74 coke oven workers as the exposed group and 47 water pump workers as the controls were surveyed, and urine samples and peripheral blood mononuclear cells (PBMCs) were collected. Airborne benzo[a]pyrene (B[a]P) levels in workplace and urinary 1-hydroxypyrene (1-OH-Py) levels were determined by high-performance liquid chromatography. DNA damage of PBMCs and the p14(ARK), p15(INK4b) and p16(INK4a) gene CpG island methylation in the promoter region were detected by comet assay and methylation-specific polymerase chain reaction techniques, respectively. Results show that compared with the controls, concentration of airborne B[a]Ps was elevated in the coke plant, and urinary 1-OH-Py's level and DNA olive tail moment in comet assay were significantly increased in the coke oven workers, and p14(ARK), p15(INK4b) and p16(INK4a) gene methylation rates were also significantly increased. With the working years and urinary 1-OH-Py's level, the rates of p14(ARK) and p16(INK4a) gene methylation were significantly increased while that of p15(INK4b) gene methylation displayed no statistical change. We conclude that PBMCs' p14(ARK) and p16(INK4a) gene methylation may be used for screening and warning lung cancer in coke oven workers.

  15. 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

  16. CpG Island Methylator Phenotype-High Colorectal Cancers and Their Prognostic Implications and Relationships with the Serrated Neoplasia Pathway

    Science.gov (United States)

    Rhee, Ye-Young; Kim, Kyung-Ju; Kang, Gyeong Hoon

    2017-01-01

    The concept of a CpG island methylator phenotype (CIMP) was first introduced by Toyota and Issa to describe a subset of colorectal cancers (CRCs) with concurrent hypermethylation of multiple CpG island loci. The concept of CIMP as a molecular carcinogenesis mechanism was consolidated by the identification of the serrated neoplasia pathway, in which CIMP participates in the initiation and progression of serrated adenomas. Distinct clinicopathological and molecular features of CIMP-high (CIMP-H) CRCs have been characterized, including proximal colon location, older age of onset, female preponderance, and frequent associations of high-level microsatellite instability and BRAF mutations. CIMP-H CRCs arise in sessile or traditional serrated adenomas and thus tend to display the morphological characteristics of serrated adenomas, including epithelial serration, vesicular nuclei, and abundant cytoplasm. Both the frequent association of CIMP and poor prognosis and different responses of CRCs to adjuvant therapy depending on CIMP status indicate clinical implications. In this review, we present an overview of the literature documenting the relevant findings of CIMP-H CRCs and their relationships with the serrated neoplasia pathway. PMID:27885175

  17. Screening for CPG Methylation and Allele Loss on Chromosome 14q

    Science.gov (United States)

    A study to follow-up on chromosomal regions identified from a genome-wide linkage analysis of nasopharyngeal carcinoma (NPC) families in Taiwan and an independent gene expression analysis of sporadic NPC tumors collected from a case-control study

  18. CpG methylation plays a vital role in determining tissue- and cell-specific expression of the human cell-death-inducing DFF45-like effector A gene through the regulation of Sp1/Sp3 binding.

    Science.gov (United States)

    Li, Dong; Da, Liang; Tang, Hong; Li, Tsaiping; Zhao, Mujun

    2008-01-01

    Cell-death-inducing DFF45-like effector A (CIDE-A) belongs to a family of proapoptotic proteins, the expression of which is highly restricted in human tissues and cells. Here, the core region of the human CIDE-A promoter was characterized. Surprisingly, two Sp1/Sp3-binding sites, rather than tissue-specific transcription factors, were found to be required for the promoter activity. Although the ubiquitously expressed Sp1 and Sp3 were crucial, they alone could not adequately regulate the specific expression of CIDE-A. We found that the expression of CIDE-A was further regulated by CpG methylation of the promoter region. By performing bisulfite sequencing, we observed dense CpG methylation of the promoter region in tissues and cells with low or no expression of CIDE-A but not in tissues with high level of CIDE-A expression. In vitro methylation of this region showed significantly reduced transcriptional activity. Treatment of CIDE-A-negative cells with 5-aza-2'-deoxycytidine demethylated the CpG sites; this opened the closed chromatin conformation and markedly enhanced the binding affinity of Sp1/Sp3 to the promoter in vivo, thereby restoring CIDE-A expression. These data indicated that CpG methylation plays a crucial role in establishing and maintaining tissue- and cell-specific transcription of the CIDE-A gene through the regulation of Sp1/Sp3 binding.

  19. An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss.

    Science.gov (United States)

    Benton, Miles C; Johnstone, Alice; Eccles, David; Harmon, Brennan; Hayes, Mark T; Lea, Rod A; Griffiths, Lyn; Hoffman, Eric P; Stubbs, Richard S; Macartney-Coxson, Donia

    2015-01-22

    Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3' untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

  20. Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype.

    Science.gov (United States)

    Whitehall, V L J; Dumenil, T D; McKeone, D M; Bond, C E; Bettington, M L; Buttenshaw, R L; Bowdler, L; Montgomery, G W; Wockner, L F; Leggett, B A

    2014-11-01

    The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.

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

    Science.gov (United States)

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

    2012-03-01

    Coenzyme Q(10) is an essential cofactor in the respiratory chain and serves as a potent antioxidant in biological membranes. Recent studies in vitro and in vivo provide evidence that Coenzyme Q(10) is involved in inflammatory processes and lipid metabolism via gene expression. To study these effects at the epigenomic level, C57BL6J mice were supplemented for one week with reduced Coenzyme Q(10) (ubiquinol). Afterwards, gene expression signatures and DNA promoter methylation patterns of selected genes were analysed. Genome-wide transcript profiling in the liver identified 1112 up-regulated and 571 down-regulated transcripts as differentially regulated between ubiquinol-treated and control animals. Text mining and GeneOntology analysis revealed that the "top 20" ubiquinol-regulated genes play a role in lipid metabolism and are functionally connected by the PPARα signalling pathway. With regard to the ubiquinol-induced changes in gene expression of about +3.14-fold (p≤0.05), +2.18-fold (p≤0.01), and -2.13-fold (p≤0.05) for ABCA1, ACYP1, and ACSL1 genes, respectively, hepatic DNA methylation analysis of 282 (sense orientation) and 271 (antisense) CpG units in the respective promoter islands revealed no significant effect of ubiquinol. In conclusion, ubiquinol affects the expression of genes involved in PPARα signalling and lipid metabolism without changing the promoter DNA methylation status in the liver of mice.

  2. A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome.

    Science.gov (United States)

    Kelly, A D; Kroeger, H; Yamazaki, J; Taby, R; Neumann, F; Yu, S; Lee, J T; Patel, B; Li, Y; He, R; Liang, S; Lu, Y; Cesaroni, M; Pierce, S A; Kornblau, S M; Bueso-Ramos, C E; Ravandi, F; Kantarjian, H M; Jelinek, J; Issa, J-Pj

    2017-01-31

    Genetic changes are infrequent in acute myeloid leukemia (AML) compared with other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CGI methylator phenotype (CIMP) (A-CIMP(+)). A-CIMP was associated with longer overall survival (OS) in this data set (median OS, years: A-CIMP(+)=not reached, CIMP(-)=1.17; P=0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP(+)=2.34, A-CIMP(-)=1.00; P=0.01). Hypermethylation in A-CIMP(+) favored CGIs (OR: CGI/non-CGI=5.21), and while A-CIMP(+) was enriched in CEBPA (P=0.002) and WT1 mutations (P=0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP(+) function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple data sets. We conclude that CIMP in AML cannot be explained solely by gene mutations (for example, IDH1/2, TET2), and that curability in A-CIMP(+) AML should be validated prospectively.Leukemia advance online publication, 31 January 2017; doi:10.1038/leu.2017.12.

  3. Dietary methyl donors, methyl metabolizing enzymes, and epigenetic regulators: Diet-gene interactions and 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; Engeland, M. van; Weijenberg, M.P.

    2011-01-01

    Dietary methyl donors might influence DNA methylation during carcinogenesis of colorectal cancer (CRC). Among 609 CRC cases and 1,663 subcohort members of the Netherlands Cohort Study on diet and cancer (n = 120,852), we estimated CRC risk according to methyl donor intake across genotypes of folate

  4. G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

    Directory of Open Access Journals (Sweden)

    Aaron J Stevens

    Full Text Available We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

  5. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.

    Science.gov (United States)

    Doi, Akiko; Park, In-Hyun; Wen, Bo; Murakami, Peter; Aryee, Martin J; Irizarry, Rafael; Herb, Brian; Ladd-Acosta, Christine; Rho, Junsung; Loewer, Sabine; Miller, Justine; Schlaeger, Thorsten; Daley, George Q; Feinberg, Andrew P

    2009-12-01

    Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P cancer-specific DMRs (C-DMRs; 3.6-fold, P cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer.

  6. G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter

    Science.gov (United States)

    Cree, Simone L.; Gibb, Andrew; Miller, Allison L.; Doudney, Kit; Aitchison, Alan; Eccles, Michael R.; Joyce, Peter R.; Filichev, Vyacheslav V.; Kennedy, Martin A.

    2014-01-01

    We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings. PMID:25437198

  7. Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State.

    Directory of Open Access Journals (Sweden)

    Steve Pells

    Full Text Available Human embryonic stem cells (hESCs undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency "signature" would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5 induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells.

  8. Differential DNA methylation patterns between high and low responders to a weight loss intervention in overweight or obese adolescents: the EVASYON study.

    Science.gov (United States)

    Moleres, Adriana; Campión, Javier; Milagro, Fermín I; Marcos, Ascensión; Campoy, Cristina; Garagorri, Jesús M; Gómez-Martínez, Sonia; Martínez, J Alfredo; Azcona-Sanjulián, M Cristina; Martí, Amelia

    2013-06-01

    In recent years, epigenetic markers emerged as a new tool to understand the influence of lifestyle factors on obesity phenotypes. Adolescence is considered an important epigenetic window over a human's lifetime. The objective of this work was to explore baseline changes in DNA methylation that could be associated with a better weight loss response after a multidisciplinary intervention program in Spanish obese or overweight adolescents. Overweight or obese adolescents (n=107) undergoing 10 wk of a multidisciplinary intervention for weight loss were assigned as high or low responders to the treatment. A methylation microarray was performed to search for baseline epigenetic differences between the 2 groups (12 subjects/group), and MALDI-TOF mass spectrometry was used to validate (n=107) relevant CpG sites and surrounding regions. After validation, 5 regions located in or near AQP9, DUSP22, HIPK3, TNNT1, and TNNI3 genes showed differential methylation levels between high and low responders to the multidisciplinary weight loss intervention. Moreover, a calculated methylation score was significantly associated with changes in weight, BMI-SDS, and body fat mass loss after the treatment. In summary, we have identified 5 DNA regions that are differentially methylated depending on weight loss response. These methylation changes may help to better understand the weight loss response in obese adolescents.

  9. DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

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    Shimrat Mamrut

    Full Text Available Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

  10. HOXB13, a target of DNMT3B, is methylated at an upstream CpG island, and functions as a tumor suppressor in primary colorectal tumors.

    Directory of Open Access Journals (Sweden)

    Kalpana Ghoshal

    Full Text Available BACKGROUND: A hallmark of cancer cells is hypermethylation of CpG islands (CGIs, which probably arises from upregulation of one or more DNA methyltransferases. The purpose of this study was to identify the targets of DNMT3B, an essential DNA methyltransferase in mammals, in colon cancer. METHODOLOGY/PRINCIPAL FINDINGS: Chromatin immunoprecipitation with DNMT3B specific antibody followed by CGI microarray identified genes with or without CGIs, repeat elements and genomic contigs in RKO cells. ChIP-Chop analysis showed that the majority of the target genes including P16, DCC, DISC1, SLIT1, CAVEOLIN1, GNA11, TBX5, TBX18, HOXB13 and some histone variants, that harbor CGI in their promoters, were methylated in multiple colon cancer cell lines but not in normal colon epithelial cells. Further, these genes were reactivated in RKO cells after treatment with 5-aza-2'-deoxycytidine, a DNA hypomethylating agent. COBRA showed that the CGIs encompassing the promoter and/or coding region of DCC, TBX5, TBX18, SLIT1 were methylated in primary colorectal tumors but not in matching normal colon tissues whereas GNA11 was methylated in both. MassARRAY analysis demonstrated that the CGI located approximately 4.5 kb upstream of HOXB13 +1 site was tumor-specifically hypermethylated in primary colorectal cancers and cancer cell lines. HOXB13 upstream CGI was partially hypomethylated in DNMT1(-/- HCT cells but was almost methylation free in cells lacking both DNMT1 and DNMT3B. Analysis of tumor suppressor properties of two aberrantly methylated transcription factors, HOXB13 and TBX18, revealed that both inhibited growth and clonogenic survival of colon cancer cells in vitro, but only HOXB13 abolished tumor growth in nude mice. CONCLUSIONS/SIGNIFICANCE: This is the first report that identifies several important tumor suppressors and transcription factors as direct DNMT3B targets in colon cancer and as potential biomarkers for this cancer. Further, this study shows that

  11. Correlation of β-Catenin Localization with Cyclooxygenase-2 Expression and CpG Island Methylator Phenotype (CIMP in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Takako Kawasaki

    2007-07-01

    Full Text Available The WNT/β-catenin (CTNNB1 pathway is commonly activated in the carcinogenic process. Cross-talks between the WNT and cyclooxygenase-2 (COX-2 or PTGS2/prostaglandin pathways have been suggested. The relationship between (3-catenin activation and microsatellite instability (MSI in colorectal cancer has been controversial. The CpG island methylator phenotype (CIMP or CIMP-high with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, which is associated with MSI-high. However, no study has examined the relationship between (β-catenin activation and CIMP status. Using 832 population-based colorectal cancer specimens, we assessed (3-catenin localization by immunohistochemistry. We quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A(p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight. MSI-high, CIMP-high, and BRAF mutation were associated inversely with cytoplasmic and nuclear (β-catenin expressions (i.e., β-catenin activation and associated positively with membrane expression. The inverse relation between (β-catenin activation and CIMP was independent of MSI. COX-2 overexpression correlated with cytoplasmic (β-catenin expression (even after tumors were stratified by CIMP status, but did not correlate significantly with nuclear or membrane expression. In conclusion, β-catenin activation is inversely associated with CIMP-high independent of MSI status. Cytoplasmic β-catenin is associated with COX-2 overexpression, supporting the role of cytoplasmic β-catenin in stabilizing PTGS2(COX-2 mRNA.

  12. B vitamins, methionine and alcohol intake and risk of colon cancer in relation to BRAF mutation and CpG island methylator phenotype (CIMP.

    Directory of Open Access Journals (Sweden)

    Eva S Schernhammer

    Full Text Available BACKGROUND: One-carbon metabolism appears to play an important role in DNA methylation reaction. Evidence suggests that a low intake of B vitamins or high alcohol consumption increases colorectal cancer risk. How one-carbon nutrients affect the CpG island methylator phenotype (CIMP or BRAF mutation status in colon cancer remains uncertain. METHODS: Utilizing incident colon cancers in a large prospective cohort of women (the Nurses' Health Study, we determined BRAF status (N = 386 and CIMP status (N = 375 by 8 CIMP-specific markers [CACNA1G, CDKN2A (p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and 8 other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT-1, MINT-31, p14, and WRN. We examined the relationship between intake of one-carbon nutrients and alcohol and colon cancer risk, by BRAF mutation or CIMP status. RESULTS: Higher folate intake was associated with a trend towards low risk of CIMP-low/0 tumors [total folate intake ≥400 µg/day vs. <200 µg/day; the multivariate relative risk = 0.73; 95% CI = 0.53-1.02], whereas total folate intake had no influence on CIMP-high tumor risks (P(heterogeneity = 0.73. Neither vitamin B(6, methionine or alcohol intake appeared to differentially influence risks for CIMP-high and CIMP-low/0 tumors. Using the 16-marker CIMP panel did not substantially alter our results. B vitamins, methionine or alcohol intake did not affect colon cancer risk differentially by BRAF status. CONCLUSIONS: This molecular pathological epidemiology study suggests that low level intake of folate may be associated with an increased risk of CIMP-low/0 colon tumors, but not that of CIMP-high tumors. However, the difference between CIMP-high and CIMP-low/0 cancer risks was not statistically significant, and additional studies are necessary to confirm these observations.

  13. Methylation at the CpG island shore region upregulates Nr3c1 promoter activity after early-life stress.

    Science.gov (United States)

    Bockmühl, Yvonne; Patchev, Alexandre V; Madejska, Arleta; Hoffmann, Anke; Sousa, Joao C; Sousa, Nuno; Holsboer, Florian; Almeida, Osborne F X; Spengler, Dietmar

    2015-01-01

    Early-life stress (ELS) induces long-lasting changes in gene expression conferring an increased risk for the development of stress-related mental disorders. Glucocorticoid receptors (GR) mediate the negative feedback actions of glucocorticoids (GC) in the paraventricular nucleus (PVN) of the hypothalamus and anterior pituitary and therefore play a key role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the endocrine response to stress. We here show that ELS programs the expression of the GR gene (Nr3c1) by site-specific hypermethylation at the CpG island (CGI) shore in hypothalamic neurons that produce corticotropin-releasing hormone (Crh), thus preventing Crh upregulation under conditions of chronic stress. CpGs mapping to the Nr3c1 CGI shore region are dynamically regulated by ELS and underpin methylation-sensitive control of this region's insulation-like function via Ying Yang 1 (YY1) binding. Our results provide new insight into how a genomic element integrates experience-dependent epigenetic programming of the composite proximal Nr3c1 promoter, and assigns an insulating role to the CGI shore.

  14. A downstream CpG island controls transcript initiation and elongation and the methylation state of the imprinted Airn macro ncRNA promoter.

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    Martha V Koerner

    Full Text Available A CpG island (CGI lies at the 5' end of the Airn macro non-protein-coding (nc RNA that represses the flanking Igf2r promoter in cis on paternally inherited chromosomes. In addition to being modified on maternally inherited chromosomes by a DNA methylation imprint, the Airn CGI shows two unusual organization features: its position immediately downstream of the Airn promoter and transcription start site and a series of tandem direct repeats (TDRs occupying its second half. The physical separation of the Airn promoter from the CGI provides a model to investigate if the CGI plays distinct transcriptional and epigenetic roles. We used homologous recombination to generate embryonic stem cells carrying deletions at the endogenous locus of the entire CGI or just the TDRs. The deleted Airn alleles were analyzed by using an ES cell imprinting model that recapitulates the onset of Igf2r imprinted expression in embryonic development or by using knock-out mice. The results show that the CGI is required for efficient Airn initiation and to maintain the unmethylated state of the Airn promoter, which are both necessary for Igf2r repression on the paternal chromosome. The TDRs occupying the second half of the CGI play a minor role in Airn transcriptional elongation or processivity, but are essential for methylation on the maternal Airn promoter that is necessary for Igf2r to be expressed from this chromosome. Together the data indicate the existence of a class of regulatory CGIs in the mammalian genome that act downstream of the promoter and transcription start.

  15. INACTIVATION OF THE CDKN2/pl6 GENE INDUCED BY METHYLATION AT 5'-CpG ISLAND AND ITS RELATION TO LUNG CANCER

    Institute of Scientific and Technical Information of China (English)

    SU; Chang-qing

    2001-01-01

    [1]Kempster S, Phillips WA, Baindur-Hudson S, et al. Methylation of exon2 of pl6 is associated with late stage esophageal cancer [J]. Cancer Lett 2000; 150:57.[2]Liggett WH Jr, Sidransky D. Role of the p16 tumor suppressor gene in cancer [J]. J Clin Oncol 1998; 16:1197.[3]Gonzalez-Zulueta M, Bender CM, Yang AS, et al. Methylation of the 5'CpG island of the pl6/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing [J]. Cancer Res 1995; 55:4531.[4]Itoh N, Kakehe Y, Akao T, et al. Concomitant presence of P16/Cyclin-dependent kinase 4 and cyclin D/cyclin-dependent kinase 4 complexes in LNCaP prostatic cancer cell line [J]. Jpn J Cancer Res 1997; 88:229.[5]Stone S, Jiang P, Dayananth P, et al. Complex structure and regulation of the P16 (MTS1) locus [J]. Cancer Res 1995; 55:2988.[6]Migeon BR. Insights into X chromosome inactivation from studies of species variation DNA methylation and replication and vice versa [J]. Genet Res 1990; 56:91.[7]Coleman A, Fountain JW, Nobori T, et al. Distinct deletion of chromosome 9p associated with melanoma versus glioma, lung cancer, and leukemia [J]. Cancer Res 1994; 54:344.[8]Quelle DE, Cheng M, Ashman R, et al. Cancer-associated mutations at the INK4a locus causes cell cycle arrest by p16INK4a but not by the alternative reading frame protein p19ARF [J]. Proc Natl Acad Sci USA 1997; 94:669.[9]Herman JG, Merlo A, Mao L, et al. Inactivation of the CDKN2/pl6/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers [J]. Cancer Res 1995; 55:4525.[10]Mateos MV, Gonzalez M, Balanzategui A, et al. Status of methylation of pl6 gene in multiple myeloma: a comparative study of three methods for its detection [J]. Clin Biochem 2000; 33: 415.[11]Lukas J, Aagaard L, Strauss M, et al. Oncogenic aberrations of P16INK4/CDKN2 and cyclinD1 cooperate to deregulate G1 control [J]. Cancer Res 1995; 55:4818.[12]Grana X, Reddy EP

  16. CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker.

    Science.gov (United States)

    Molano, Monica; Tabrizi, Sepehr N; Garland, Suzanne M; Roberts, Jennifer M; Machalek, Dorothy A; Phillips, Samuel; Chandler, David; Hillman, Richard J; Grulich, Andrew E; Jin, Fengyi; Poynten, I Mary; Templeton, David J; Cornall, Alyssa M

    2016-01-01

    Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.

  17. Decrease of 5hmC in gastric cancers is associated with TET1 silencing due to with DNA methylation and bivalent histone marks at TET1 CpG island 3'-shore.

    Science.gov (United States)

    Park, Jong-Lyul; Kim, Hee-Jin; Seo, Eun-Hye; Kwon, Oh-Hyung; Lim, Byungho; Kim, Mirang; Kim, Seon-Young; Song, Kyu-Sang; Kang, Gyeong Hoon; Kim, Hyun Ja; Choi, Bo Youl; Kim, Yong Sung

    2015-11-10

    Recent evidence has shown that the level of 5-hydroxymethylcytosine (5 hmC) in chromosomal DNA is aberrantly decreased in a variety of cancers, but whether this decrease is a cause or a consequence of tumorigenesis is unclear. Here we show that, in gastric cancers, the 5 hmC decrease correlates with a decrease in ten-eleven translocation 1 (TET1) expression, which is strongly associated with metastasis and poor survival in patients with gastric cancer. In gastric cancer cells, TET1-targeted siRNA induced a decrease in 5 hmC, whereas TET1 overexpression induced an increase in 5 hmC and reduced cell proliferation, thus correlating decreased 5 hmC with gastric carcinogenesis. We also report the epigenetic signatures responsible for regulating TET1 transcription. Methyl-CpG Binding Domain Sequencing and Reduced Representation Bisulfite Sequencing identified unique CpG methylation signatures at the CpG island 3'-shore region located 1.3 kb from the transcription start site of TET1 in gastric tumor cells but not in normal mucosa. The luciferase activity of constructs with a methylated 3'-shore sequence was greatly decreased compared with that of an unmethylated sequence in transformed gastric cancer cells. In gastric cancer cells, dense CpG methylation in the 3'-shore was strongly associated with TET1 silencing and bivalent histone marks. Thus, a decrease in 5 hmC may be a cause of gastric tumorigenesis owing to a decrease in TET1 expression through DNA methylation coupled with bivalent marks in the 3'-shore of TET1.

  18. Using peripheral blood circulating DNAs to detect CpG global methylation status and genetic mutations in patients with myelodysplastic syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Iriyama, Chisako [Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Tomita, Akihiro, E-mail: atomita@med.nagoya-u.ac.jp [Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Hoshino, Hideaki; Adachi-Shirahata, Mizuho [Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Furukawa-Hibi, Yoko; Yamada, Kiyofumi [Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University School of Medicine, Nagoya (Japan); Kiyoi, Hitoshi; Naoe, Tomoki [Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya (Japan)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Circulating DNAs (CDs) can be used to detect genetic/epigenetic abnormalities in MDS. Black-Right-Pointing-Pointer Epigenetic changes can be detected more sensitively when using plasma DNA than PBMNC. Black-Right-Pointing-Pointer Mutation ratio in CDs may reflect the ratio in stem cell population in bone marrow. Black-Right-Pointing-Pointer Using CDs can be a safer alternate strategy compared to bone marrow aspiration. -- Abstract: Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder. Several genetic/epigenetic abnormalities are deeply associated with the pathogenesis of MDS. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic abnormalities, BM aspiration is difficult to perform repeatedly to obtain serial samples because of pain and safety concerns. Here, we report that circulating cell-free DNAs from plasma and serum of patients with MDS can be used to detect genetic/epigenetic abnormalities. The plasma DNA concentration was found to be relatively high in patients with higher blast cell counts in BM, and accumulation of DNA fragments from mono-/di-nucleosomes was confirmed. Using serial peripheral blood (PB) samples from patients treated with hypomethylating agents, global methylation analysis using bisulfite pyrosequencing was performed at the specific CpG sites of the LINE-1 promoter. The results confirmed a decrease of the methylation percentage after treatment with azacitidine (days 3-9) using DNAs from plasma, serum, and PB mono-nuclear cells (PBMNC). Plasma DNA tends to show more rapid change at days 3 and 6 compared with serum DNA and PBMNC. Furthermore, the TET2 gene mutation in DNAs from plasma, serum, and BM cells was quantitated by pyrosequencing analysis. The existence ratio of mutated genes in plasma and serum DNA showed almost equivalent level with that in the CD34+/38- stem cell population in BM. These data suggest that genetic

  19. CpG traffic lights are markers of regulatory regions in humans

    KAUST Repository

    Khamis, Abdullah M.

    2016-12-29

    DNA methylation is involved in regulation of gene expression. Although modern methods profile DNA methylation at single CpG sites, methylation levels are usually averaged over genomic regions in the downstream analyses. In this study we demonstrate that single CpG methylation can serve as a more accurate predictor of gene expression compared to average promoter / gene body methylation. CpG positions with significant correlation between methylation and expression of a gene nearby (named CpG traffic lights) are evolutionary conserved and enriched for exact TSS positions and active enhancers. Among all promoter types, CpG traffic lights are especially enriched in poised promoters. Genes that harbor CpG traffic lights are associated with development and signal transduction. Methylation levels of individual CpG traffic lights vary between cell types dramatically with the increased frequency of intermediate methylation levels, indicating cell population heterogeneity in CpG methylation levels. Being in line with the concept of the inherited stochastic epigenetic variation, methylation of such CpG positions might contribute to transcriptional regulation. Alternatively, one can hypothesize that traffic lights are markers of absent gene expression resulting from inactivation of their regulatory elements. The CpG traffic lights provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to expression.

  20. Toxicity of methyl parathion to bats: Mortality and coordination loss

    Science.gov (United States)

    Clark, D.R.

    1986-01-01

    The 24-h oral LD50 of methyl parathion (phosphorothioic acid O,O-dimethyl O-(4-nitrophenyl) ester) to little brown bats (Myotis lucifugus) (372 mg/kg) was 8.5 times the LD50 for mice (Mus musculus) (44 mg/kg). However, orally dosed mice either died or appeared behaviorally normal after 2 to 3 h, whereas many dosed bats, although alive at 24 h, could not right themselves when placed on their backs. The oral dose estimated to cause this loss of coordination in 50% of a sample of big brown bats (Eptesicus fuscus) was one-third or less the LD50 of this species. Cholinesterase activity depression in brains of little brown bats was similar whether dosage was oral or dermal. With death as the criterion, bats proved relatively insensitive to methyl parathion in 24-h tests, but considerations of the chemical's potential to cause coordination loss, leading to capture and death by predators, coupled with bats' naturally low reproductive rates, suggest possible injury to exposed bat populations.

  1. The polymorphism (-600 C>A) of CpG methylation site at the promoter region of CYP17A1 and its association of male infertility and testosterone levels.

    Science.gov (United States)

    Park, Jung-Hoon; Lee, Jinu; Kim, Cheol-Hee; Lee, Suman

    2014-01-15

    Cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1) is a key regulatory enzyme in the steroidogenic pathway. The functional and clinical relevance of novel CYP17A1 promoter single nucleotide polymorphism (-600 C>A, rs17115149) was investigated with male infertility. Case-control association study of CYP17A1 from 456 infertile men performed with 465 normal fertile men. The rs17115149 at the promoter region of CYP17A1 was significantly associated with Oligoasthenoteratozoospermia (OAT, P=0.0015, n=265). 5-aza-dC treatment to B lymphocyte cells increased the CYP17A1 expression. Direct bisulfite sequencing of five human tissues showed that the rs17115149 is located at -600bp (-600C>A) before transcription start site within the CpG islands of CYP17A1 promoter. This -600 Cytosine of CpG site was highly methylated in colon and stomach tissues, but low methylated in adrenal gland, kidney and testis with higher CYP17A1 RNA expression. Especially, this polymorphism is statistically significant associated with testosterone levels from infertile males (n=197, PA) is a functional regulatory SNP which associated with its expression possibly by epigenetic pathway, which may signify a genetic risk factor for male infertility. © 2013 Elsevier B.V. All rights reserved.

  2. CpG + CpNpG Analysis of Protein-Coding Sequences from Tomato

    DEFF Research Database (Denmark)

    Hobolth, Asger; Nielsen, Rasmus; Wang, Ying

    2006-01-01

    We develop codon-based models for simultaneously inferring the mutational effects of CpG and CpNpG methylation in coding regions. In a data set of 369 tomato genes, we show that there is very little effect of CpNpG methylation but a strong effect of CpG methylation affecting almost all genes. We...... further show that the CpNpG and CpG effects are largely uncorrelated. Our results suggest different roles of CpG and CpNpG methylation, with CpNpG methylation possibly playing a specialized role in defense against transposons and RNA viruses....

  3. HOXB13, a Target of DNMT3B, Is Methylated at an Upstream CpG Island, and Functions as a Tumor Suppressor in Primary Colorectal Tumors

    OpenAIRE

    Kalpana Ghoshal; Tasneem Motiwala; Rainer Claus; Pearlly Yan; Huban Kutay; Jharna Datta; Sarmila Majumder; Shoumei Bai; Arnab Majumder; Tim Huang; Christoph Plass; Jacob, Samson T.

    2010-01-01

    BACKGROUND: A hallmark of cancer cells is hypermethylation of CpG islands (CGIs), which probably arises from upregulation of one or more DNA methyltransferases. The purpose of this study was to identify the targets of DNMT3B, an essential DNA methyltransferase in mammals, in colon cancer. METHODOLOGY/PRINCIPAL FINDINGS: Chromatin immunoprecipitation with DNMT3B specific antibody followed by CGI microarray identified genes with or without CGIs, repeat elements and genomic contigs in RKO cells....

  4. DNA methylation changes in ovarian cancer are cumulative with disease progression and identify tumor stage

    Directory of Open Access Journals (Sweden)

    DeGeest Koen

    2008-09-01

    Full Text Available Abstract Background Hypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers. Methods The methylation of 6,502 CpG-rich sequences spanning the genome was analyzed in 137 ovarian samples (ten normal, 23 low malignant potential, 18 stage I, 16 stage II, 54 stage III, and 16 stage IV ranging from normal tissue through to stage IV cancer using a sequence-validated human CpG island microarray. The microarray contained 5' promoter-associated CpG islands as well as CpG-rich satellite and Alu repetitive elements. Results Results showed a progressive de-evolution of normal CpG methylation patterns with disease progression; 659 CpG islands showed significant loss or gain of methylation. Satellite and Alu sequences were primarily associated with loss of methylation, while promoter CpG islands composed the majority of sequences with gains in methylation. Since the majority of ovarian tumors are late stage when diagnosed, we tested whether DNA methylation profiles could differentiate between normal and low malignant potential (LMP compared to stage III ovarian samples. We developed a class predictor consisting of three CpG-rich sequences that was 100% sensitive and 89% specific when used to predict an independent set of normal and LMP samples versus stage III samples. Bisulfite sequencing confirmed the NKX-2-3 promoter CpG island was hypermethylated with disease progression. In addition, 5-aza-2'-deoxycytidine treatment of the ES2 and OVCAR ovarian cancer cell lines re-expressed NKX-2-3. Finally, we merged our CpG methylation results with previously published ovarian expression microarray data and identified correlated expression changes. Conclusion Our results show that changes in CpG methylation are cumulative with ovarian cancer progression in a sequence-type dependent manner, and that CpG

  5. Methylation of CpG islands of p16(INK4a) and cyclinD1 overexpression associated with progression of intraductal proliferative lesions of the breast.

    Science.gov (United States)

    Liu, Tieju; Niu, Yun; Feng, Yumei; Niu, Ruifang; Yu, Yong; Lv, Ajuan; Yang, Yi

    2008-11-01

    P16(INK4a) is a tumor suppressor gene frequently inactivated by aberrant promoter hypermethylation. In this study, p16(INK4a) methylation was evaluated in intraductal proliferative lesions of the breast, using real-time quantitative polymerase chain reaction (MethyLight) and methylation-sensitive restriction endonuclease polymerase chain reaction. Immunohistochemistry was performed to compare and validate the methylation analysis. P16(INK4a) methylation associated with oncogene cyclinD1 expression, detected through the use of in situ hybridization and immunohistochemistry, was likewise characterized. P16(INK4a) methylation displayed varying significance among different types of intraductal proliferative lesions. Both the positive rate and the median quantitative methylation value increased with the evolution of intraductal proliferative lesions through the use of quantitative and qualitative assays. P16(INK4a) methylation was positively correlated to cyclinD1 overexpression. This study demonstrated that p16(INK4a) methylation served as the silencing mechanism of p16(INK4a) protein expression and played a crucial role in the intraductal proliferative lesions' progression. In the differential diagnosis of intraductal proliferative lesions, quantitative DNA methylation analysis of p16(INK4a) by MethyLight may be used as a surrogate, especially to distinguish atypical ductal hyperplasia from usual ductal hyperplasia and low-grade ductal carcinoma in situ. Furthermore, this study discovered that flat epithelial atypia do not share similar molecular profiles of p16(INK4a) epigenetic modification with atypical ductal hyperplasia and low-grade ductal carcinoma in situ.

  6. Molecular insights into the association of obesity with breast cancer risk: relevance to xenobiotic metabolism and CpG island methylation of tumor suppressor genes.

    Science.gov (United States)

    Naushad, Shaik Mohammad; Hussain, Tajamul; Al-Attas, Omar S; Prayaga, Aruna; Digumarti, Raghunadha Rao; Gottumukkala, Suryanarayana Raju; Kutala, Vijay Kumar

    2014-07-01

    Obesity, genetic polymorphisms of xenobiotic metabolic pathway, hypermethylation of tumor suppressor genes, and hypomethylation of proapoptotic genes are known to be independent risk factors for breast cancer. The objective of this study is to evaluate the combined effect of these environmental, genetic, and epigenetic risk factors on the susceptibility to breast cancer. PCR-RFLP and multiplex PCR were used for the genetic analysis of six variants of xenobiotic metabolic pathway. Methylation-specific PCR was used for the epigenetic analysis of four genetic loci. Multifactor dimensionality reduction analysis revealed a significant interaction between the body mass index (BMI) and catechol-O-methyl transferase H108L variant alone or in combination with cytochrome P450 (CYP) 1A1m1 variant. Women with "Luminal A" breast cancer phenotype had higher BMI compared to other phenotypes and healthy controls. There was no association between the BMI and tumor grade. The post-menopausal obese women exhibited lower glutathione levels. BMI showed a positive association with the methylation of extracellular superoxide dismutase (r = 0.21, p obesity increases the breast cancer susceptibility by two possible mechanisms: (i) by interacting with xenobiotic genetic polymorphisms in inducing increased oxidative DNA damage and (ii) by altering the methylome of several tumor suppressor genes.

  7. 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.

  8. Synthetic CpG islands reveal DNA sequence determinants of chromatin structure

    Science.gov (United States)

    Wachter, Elisabeth; Quante, Timo; Merusi, Cara; Arczewska, Aleksandra; Stewart, Francis; Webb, Shaun; Bird, Adrian

    2014-01-01

    The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG. CGIs often include transcription initiation sites and display ‘active’ histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a ‘bivalent’ chromatin state bearing simultaneous ‘active’ and ‘inactive’ chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G + C-rich DNA. A high CpG density alone is not sufficient for this effect, as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs. DOI: http://dx.doi.org/10.7554/eLife.03397.001 PMID:25259796

  9. 5-羟色胺转运体基因启动子区CpG岛甲基化状态与精神分裂症Ⅰ型和Ⅱ型的关联%Relationship Between Methylation Status of CpG Islands within the Promoter Region of Serotonin Transporter Gene and Type Ⅰ or Type Ⅱ Schizophrenia

    Institute of Scientific and Technical Information of China (English)

    张海生; 王伟; 余道军; 张顺泉; 谢健

    2011-01-01

    目的:探讨5-羟色胺转运体(5-HTT)基因启动子区CpG岛甲基化状态与精神分裂症Ⅰ型和Ⅱ型的关联.方法:运用特异性甲基检测PCR和直接测序法对62例精神分裂症Ⅰ型患者、38例Ⅱ型患者和50例健康被试5-HTT 基因启动子区CpG岛甲基化状态进行检测.结果:三组5-HTT基因启动子区CpC岛甲基化阳性率无显著性差异;精神分裂症Ⅰ型患者5-HTT基因启动子区CpC岛内位点甲基化率显著高于精神分裂症Ⅱ型患者和健康被试组.结论:5-HTT基因启动子区CpG岛内位点高甲基化可能是精神分裂症Ⅰ型的发病机制之一.%Objective: To explore the relationship between methylation status of CpG islands in the promoter region of serotonin transporter gene (5-HTT) and type Ⅰ or Ⅱ schizophrenia. Methods: Methylation Specific PCR and DNA sequencing were used to detect the methylation status of CpG islands in 62 type Ⅰ schizophrenia patients, 38 type Ⅱ schizophrenia patients and 50 health subjects. Results: The methylation rates of CpG islands within the promoter region of 5-HTT of the three groups are 52% (32/62), 47% (18/38) and 50% (25/50), respectively. The methylation rates of loci in CpG islands within the promoter region of 5-HTT in type Ⅰ schizophrenia group is significantly higher than those in type Ⅱ schizophrenia group and health subjects. Conclusion: The high methylation rate of loci in CpG islands within the promoter region of 5-HTT should be a possible factor to influence the nosogenesis of type Ⅰ schizophrenia.

  10. MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status

    NARCIS (Netherlands)

    P. Bady (Pierre); D. Sciuscio (Davide); A.C. Diserens; J. Bloch (Jocelyne); M.J. van den Bent (Martin); C. Marosi (Christine); P-Y. Dietrich (Pierre Yves); M. Weller (Michael); L. Mariani (Luigi); F.L. Heppner (Frank ); D.R. Mcdonald (David ); D. Lacombe (Denis); R. Stupp (Roger); M. Delorenzi (Mauro); M.E. Hegi (Monika)

    2012-01-01

    textabstractThe methylation status of the O6-methylguanine- DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogen

  11. Methylation of the claudin 1 promoter is associated with loss of expression in estrogen receptor positive breast cancer.

    Directory of Open Access Journals (Sweden)

    Francescopaolo Di Cello

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

  12. Effects of methylation status of CpG islands on results of luciferase reporter assay%细胞荧光素酶报告基因试验受CpG岛甲基化状态影响

    Institute of Scientific and Technical Information of China (English)

    张宝珍; 邓大君

    2009-01-01

    Objective To investigate the effects of methylation status of CpG islands of endogenous E-cadherin (CDH1) gene on the promoter activity of corresponding genes in reporter assays. Methods The methylation statuses of CpG island of CDHI in 8 different cell lines were detected by methylation-specific PCR. CDH1 protein was analyzed by Western blotting. Two sets of pGL3 reporter vectors with different genotypes/haplotypes of the CDH1 promoter were constructed [pGI3-A(-73)/-C(-73)pGL3-H1/-H4]and used to transfect these cell lines. The differences between these promoter reporter vectors were analyzed by t-test. Results (1) CDH1 CpG island was unmethylated in AGS, MCF7, MKN74, and PC-3 cell lines,expressed in MCF7, MKN74, and PC-3 ,but not in AGS. Expression of CDH1 was silenced by methylation in HeLa, BGC823, A549, and RKO cell lines. (2) In the four CDH1 -unmethylated MCFT, M KN74, PC-3, and AGS cell lines ,the promoter activities of pGI3-C(-73)(as 0. 78±0. 10,0. 17±0.01,0. 11±0. 01,1.19±0. 18)were significantly higher than those of pGL3-A(-73)(as 0. 30±0. 08,0. 07±0. 01,0. 07±0. 01,0. 39±0. 04) (t values are -6. 298, -12. 349, -8. 128, -7.388, and P<0. 01). However, in the four C DH1 -methylated HeLa, BGC823, A549, and RKO cell lines, the promoter activity of pGL3-C(-73)(as 0. 09±0. 02,0. 13±0. 02,0. 05±0. 01,0. 01±0. 00) was significantly lower than that of pGL3-A(-73)(as 0. 16±0. 01,0.25±0.01,0. 11±0.03,0.03±0.00) (t valued at 5.958,11. 189,3. 661,13. 866,and P<0.05). (3) In the unmethylated MKN74 and methylated RKO cell lines, the promoter activities of pGI3-H1/-H4 were obviously and contrarily different(as 1.57±0. 23/0. 94±0. 06 and 0. 38±0. 02/0. 50±0. 04 ,t values were 4. 577 and -4. 915 ,P values were 0. 010 and 0. 003). Conclusion The methylation status of CpG island of the target gene in the tested cell lines affects the promoter activity in Reporter Assay significantly. The most active one may be the most suppressive one.%目的 研究细

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

    Science.gov (United States)

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

    2009-06-01

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

  14. Nucleosome dynamics and maintenance of epigenetic states of CpG islands

    Science.gov (United States)

    Sneppen, Kim; Dodd, Ian B.

    2016-06-01

    Methylation of mammalian DNA occurs primarily at CG dinucleotides. These CpG sites are located nonrandomly in the genome, tending to occur within high density clusters of CpGs (islands) or within large regions of low CpG density. Cluster methylation tends to be bimodal, being dominantly unmethylated or mostly methylated. For CpG clusters near promoters, low methylation is associated with transcriptional activity, while high methylation is associated with gene silencing. Alternative CpG methylation states are thought to be stable and heritable, conferring localized epigenetic memory that allows transient signals to create long-lived gene expression states. Positive feedback where methylated CpG sites recruit enzymes that methylate nearby CpGs, can produce heritable bistability but does not easily explain that as clusters increase in size or density they change from being primarily methylated to primarily unmethylated. Here, we show that an interaction between the methylation state of a cluster and its occupancy by nucleosomes provides a mechanism to generate these features and explain genome wide systematics of CpG islands.

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

    Directory of Open Access Journals (Sweden)

    Andrew D Beggs

    2013-05-01

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

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

    Science.gov (United States)

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

    2013-05-01

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

  17. A DNA methylation fingerprint of 1628 human samples

    Science.gov (United States)

    Fernandez, Agustin F.; Assenov, Yassen; Martin-Subero, Jose Ignacio; Balint, Balazs; Siebert, Reiner; Taniguchi, Hiroaki; Yamamoto, Hiroyuki; Hidalgo, Manuel; Tan, Aik-Choon; Galm, Oliver; Ferrer, Isidre; Sanchez-Cespedes, Montse; Villanueva, Alberto; Carmona, Javier; Sanchez-Mut, Jose V.; Berdasco, Maria; Moreno, Victor; Capella, Gabriel; Monk, David; Ballestar, Esteban; Ropero, Santiago; Martinez, Ramon; Sanchez-Carbayo, Marta; Prosper, Felipe; Agirre, Xabier; Fraga, Mario F.; Graña, Osvaldo; Perez-Jurado, Luis; Mora, Jaume; Puig, Susana; Prat, Jaime; Badimon, Lina; Puca, Annibale A.; Meltzer, Stephen J.; Lengauer, Thomas; Bridgewater, John; Bock, Christoph; Esteller, Manel

    2012-01-01

    Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases. PMID:21613409

  18. CpG islands undermethylation in human genomic regions under selective pressure.

    Directory of Open Access Journals (Sweden)

    Sergio Cocozza

    Full Text Available DNA methylation at CpG islands (CGIs is one of the most intensively studied epigenetic mechanisms. It is fundamental for cellular differentiation and control of transcriptional potential. DNA methylation is involved also in several processes that are central to evolutionary biology, including phenotypic plasticity and evolvability. In this study, we explored the relationship between CpG islands methylation and signatures of selective pressure in Homo Sapiens, using a computational biology approach. By analyzing methylation data of 25 cell lines from the Encyclopedia of DNA Elements (ENCODE Consortium, we compared the DNA methylation of CpG islands in genomic regions under selective pressure with the methylation of CpG islands in the remaining part of the genome. To define genomic regions under selective pressure, we used three different methods, each oriented to provide distinct information about selective events. Independently of the method and of the cell type used, we found evidences of undermethylation of CGIs in human genomic regions under selective pressure. Additionally, by analyzing SNP frequency in CpG islands, we demonstrated that CpG islands in regions under selective pressure show lower genetic variation. Our findings suggest that the CpG islands in regions under selective pressure seem to be somehow more "protected" from methylation when compared with other regions of the genome.

  19. Downstream targets of methyl CpG binding protein 2 and their abnormal expression in the frontal cortex of the human Rett syndrome brain

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    Minchenko Dimitri

    2010-04-01

    Full Text Available Abstract Background The Rett Syndrome (RTT brain displays regional histopathology and volumetric reduction, with frontal cortex showing such abnormalities, whereas the occipital cortex is relatively less affected. Results Using microarrays and quantitative PCR, the mRNA expression profiles of these two neuroanatomical regions were compared in postmortem brain tissue from RTT patients and normal controls. A subset of genes was differentially expressed in the frontal cortex of RTT brains, some of which are known to be associated with neurological disorders (clusterin and cytochrome c oxidase subunit 1 or are involved in synaptic vesicle cycling (dynamin 1. RNAi-mediated knockdown of MeCP2 in vitro, followed by further expression analysis demonstrated that the same direction of abnormal expression was recapitulated with MeCP2 knockdown, which for cytochrome c oxidase subunit 1 was associated with a functional respiratory chain defect. Chromatin immunoprecipitation (ChIP analysis showed that MeCP2 associated with the promoter regions of some of these genes suggesting that loss of MeCP2 function may be responsible for their overexpression. Conclusions This study has shed more light on the subset of aberrantly expressed genes that result from MECP2 mutations. The mitochondrion has long been implicated in the pathogenesis of RTT, however it has not been at the forefront of RTT research interest since the discovery of MECP2 mutations. The functional consequence of the underexpression of cytochrome c oxidase subunit 1 indicates that this is an area that should be revisited.

  20. The Study of CpG Island Methylation of BRCA1 Gene Promoter in a Taxol Induced Drug-resistant Human Lung Aadenocarcinoma Cell Line A549%耐紫杉醇人肺腺癌A549细胞株中BRCA1基因启动子CpG岛甲基化的研究

    Institute of Scientific and Technical Information of China (English)

    尹红英; 王红兵

    2012-01-01

    目的 检测耐紫杉醇人肺腺癌A549细胞株(A549/Taxol)中BRCA1基因启动子CpG岛甲基化状态,探讨A549/Taxol细胞对紫杉醇的耐药机制.方法 应用甲基化特异性聚合酶链反应(MSP)技术,检测耐紫杉醇人肺腺癌A549细胞株BRCA1基因启动子CpG岛甲基化状态.结果 A549/Taxol细胞存在BRCA1基因异常甲基化,呈部分甲基化.结论 A549/Taxol细胞存在BRCA1基因异常甲基化,可能是A549/Taxol细胞对紫杉醇耐药的机制之一.%Objective To detect the CpG island methylation status of BRCA1 gene promoter in the Taxol induced drug-resistant human lung adenocarcinoma cell line A549 ( A549/Taxol ), and to explore the resistance mechanisms of A549/Taxol. Methods A549/Taxol were examined CpG island methylation of BRCA1 gene promoter by methylation specific PCR ( MSP ). Results IBRCA1 gene aberrant methylation of A549/Taxol cells is part of methylation. Conclusion BRCA1 gene aberrant methylation of A549/Taxol may be one of the resistance mechanisms of taxol in A549/Taxol.

  1. Epigenetic analysis of HIV-1 proviral genomes from infected individuals: predominance of unmethylated CpG's.

    Science.gov (United States)

    Weber, Stefanie; Weiser, Barbara; Kemal, Kimdar S; Burger, Harold; Ramirez, Christina M; Korn, Klaus; Anastos, Kathryn; Kaul, Rupert; Kovacs, Colin; Doerfler, Walter

    2014-01-20

    Efforts to cure HIV-1 infections aim at eliminating proviral DNA. Integrated DNA from various viruses often becomes methylated de novo and transcriptionally inactivated. We therefore investigated CpG methylation profiles of 55 of 94 CpG's (58.5%) in HIV-1 proviral genomes including ten CpG's in each LTR and additional CpG's in portions of gag, env, nef, rev, and tat genes. We analyzed 33 DNA samples from PBMC's of 23 subjects representing a broad spectrum of HIV-1 disease. In 22 of 23 HIV-1-infected individuals, there were only unmethylated CpG's regardless of infection status. In one long term nonprogressor, however, methylation of proviral DNA varied between 0 and 75% over an 11-year period although the CD4+ counts remained stable. Hence levels of proviral DNA methylation can fluctuate. The preponderance of unmethylated CpG's suggests that proviral methylation is not a major factor in regulating HIV-1 proviral activity in PBMC's. Unmethylated CpG's may play a role in HIV-1 immunopathogenesis. © 2013 Elsevier Inc. All rights reserved.

  2. CpG demethylation enhances alpha-synuclein expression and affects the pathogenesis of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Lumine Matsumoto

    Full Text Available BACKGROUND: Alpha-synuclein (SNCA gene expression is an important factor in the pathogenesis of Parkinson's disease (PD. Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD. CpG methylation in the promoter region may also influence SNCA expression. METHODOLOGY/PRINCIPAL FINDINGS: By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression. Postmortem brain analysis revealed regional non-specific methylation differences in this CpG region in the anterior cingulate and putamen among controls and PD; however, in the substantia nigra of PD, methylation was significantly decreased. CONCLUSIONS/SIGNIFICANCE: This CpG region may function as an intronic regulatory element for SNCA gene. Our findings suggest that a novel epigenetic regulatory mechanism controlling SNCA expression influences PD pathogenesis.

  3. Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors

    OpenAIRE

    CAI, JINQUAN; Chen, Jing; Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang., Tao

    2015-01-01

    Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher...

  4. 上皮性卵巢癌组织中WWOX基因启动子区域CpG岛的甲基化状态及临床意义%Methylation State of WWOX Gene Promoter CpG Islands in Epithelial Ovarian Cancer and Its Clinical Significance

    Institute of Scientific and Technical Information of China (English)

    闫洪超; 孙洁芸; 陆晓媛; 韩秋峪; 魏敏

    2012-01-01

    目的 研究上皮性卵巢癌组织中WWOX基因启动子区CpG岛的甲基化状态,并分析WWOX基因的甲基化与上皮性卵巢癌的临床病理指标之间的关系.方法 采用甲基化特异性PCR(methylation specific polymerase chain reaction,MSP)方法检测48例上皮性卵巢癌、18例卵巢交界性上皮性肿瘤、26例卵巢良性上皮性肿瘤及33例正常卵巢组织中WWOX基因CpG岛甲基化状态.结果 上皮性卵巢癌、卵巢交界性上皮性肿瘤、卵巢良性上皮性肿瘤组织中WWOX基因启动子区CpG岛甲基化率分别为43.75%、26.32%、3.84%,正常卵巢组织中未检测到WWOX基因CpG岛甲基化.上皮性卵巢癌组织中WWOX基因CpG岛的甲基化率明显高于其他卵巢组织,差异有统计学意义(P<0.01).晚期(Ⅲ期、Ⅳ期)上皮性卵巢癌组织中WWOX基因CpG岛的甲基化率高于早期(Ⅰ期、Ⅱ期)上皮性卵巢癌组织,差异有统计学意义(P<0.05).结论 上皮性卵巢癌组织中广泛存在着WWOX基因启动子区CpG岛甲基化,可能是导致WWOX基因失活的重要机制.WWOX基因的异常甲基化可能与上皮性卵巢癌的发生发展密切相关,其可能成为上皮性卵巢癌的早期诊断和评估预后的重要指标.%Objective To evaluate the methylation status of CpG islands in WWOX gene promoter region of epithelial ovarian cancer, and to explore the relationship between methylation state of WWOX gene CpG island and clinicopathological indexes in epithelial ovarian cancer. Methods The methylation state of WWOX gene CpG island was evaluated by methylation specific polymerase chain reaction(MSP) in 48 patients with epithelial ovarian cancer, 18 patients with borderline epithelial ovarian tumors,26 patients with epithelial benign tumors,and 33 patients with normal ovarian tissues. Results The rates of CpG island methylation in WWOX gene promoter region in epithelial ovarian cancer tissues,borderline ovarian tumor tissues and benign ovarian tumor

  5. Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors.

    Science.gov (United States)

    Cai, Jinquan; Chen, Jing; Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

    2015-07-20

    Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma.

  6. CPG OLIGONUCLEOTIDES REGULATE OSTEOCLAST DIFFERENTIATION

    Institute of Scientific and Technical Information of China (English)

    Zhao Weigong; Han Xuezhe; Li Xinyou; Guo Xong; Liu Miao

    2005-01-01

    Objective Bacterial DNA is a pathogen-derived molecule which can regulate the innate immune system by stimulating NF-κB activation. The activity of bacterial DNA relies on its content of unmethylated CpG dinucleotides in particular base contexts("CpG motif"). In light of the pivotal role played by NF-κB in osteoclast differentiation, the ability of CpG oligodeoxynucleotides (CpG ODN) coming from bacterial DNA to modulate osteoclastogenesis was studied. Methods Bone marrow mononuclear cells (BMM) were purified from Balb/c mice, cultured in α-MEM media containing 10% FCS in the presence of mouse M-CSF, with either RANKL or ODNs for 5 days. Osteoclast formation was evaluated on day 5 according to TRAP and May-Grunwald-Giemsa staining. Results CpG ODN alone could induce osteoclast formation in the low degree in BMM culture. The relationship between CpG ODN and RANKL was that CpG ODN could inhibit RANKL-induced osteoclastogenesis when present from the beginning of BMM culture, but strongly increased RANKL-induced osteoclastogenesis in RANKL-pretreated BMMs. Conclusion The mechanism of CpG ODN regulating osteoclast differentiation was bidirectional, which might be a potential therapy for treating metabolic bone disease.

  7. Surface water seal application to minimize volatilization loss of methyl isothiocyanate from soil columns.

    Science.gov (United States)

    Simpson, Catherine R; Nelson, Shad D; Stratmann, Jerry E; Ajwa, Husein A

    2010-06-01

    Metam-sodium (MS, sodium methyldithiocarbamate) has been identified as a promising alternative chemical to replace methyl bromide (MeBr) in soil preplant fumigation. One degradation product of MS in soil is the volatile gas methyl isothiocyanate (MITC) which controls soilborne pests. Inconsistent results associated with MS usage indicate that there is a need to determine cultural practices that increase pest control efficacy. Sealing the soil surface with water after MS application may be a sound method to reduce volatilization loss of MITC from soils and increase the contact time necessary for MITC to control pests. The objective of this research was to develop a preliminary soil surface water application amount that would potentially inhibit the off-gassing rate of MITC. Off-gassing rate was consistently reduced with increasing water seal application. The application of a 2.5-3.8 cm water seal provided significantly lower (71-74% reduction in MITC volatilization) total fumigant loss compared with no water seal. The most favorable reduction in MITC off-gassing was observed in the 2.5 cm water seal. This suggests that volatilization of MITC-generating compounds can be highly suppressed using adequate surface irrigation following chemical application in this soil type (sandy clay loam), based on preliminary bench-scale soil column studies. .

  8. LINE-1 methylation levels, a biomarker of weight loss in obese subjects, are influenced by dietary antioxidant capacity.

    Science.gov (United States)

    Garcia-Lacarte, Marcos; Milagro, Fermin I; Zulet, Maria A; Martinez, J Alfredo; Mansego, Maria L

    2016-03-01

    Epigenetic markers, and in particular DNA methylation, have come to the fore as new tools in the personalization of the treatment of obesity and its comorbidities. The objectives of the current investigation were to identify epigenetic biomarkers that might be predictive of response to a weight-loss intervention, and to better understand the influence of certain nutrients (particularly antioxidants) on the epigenome. Global DNA (LINE-1) methylation levels were assessed in peripheral blood mononuclear cells (PBMCs) from 96 obese volunteers of the Metabolic Syndrome Reduction in Navarra study, using a methylation-sensitive high resolution melting approach after bisulfite modification. Baseline LINE-1 DNA methylation levels were significantly higher (5.41%) in high responders (>8% of weight loss) as compared to low responders (response to the hypocaloric diet. Statistically significant correlations were found between LINE-1 baseline DNA methylation levels and the response to the treatment involving total fat mass and body weight. Furthermore, LINE-1 baseline methylation levels positively correlated with baseline dietary total antioxidant capacity (TAC). LINE-1 methylation levels in PBMCs might be used to predict response to a dietary weight-loss intervention, and seem to be related to the dietary TAC. www.clinicaltrials.gov : NCT01087086.

  9. The dynamic DNA methylation cycle from egg to sperm in the honey bee Apis mellifera.

    Science.gov (United States)

    Drewell, Robert A; Bush, Eliot C; Remnant, Emily J; Wong, Garrett T; Beeler, Suzannah M; Stringham, Jessica L; Lim, Julianne; Oldroyd, Benjamin P

    2014-07-01

    In honey bees (Apis mellifera), the epigenetic mark of DNA methylation is central to the developmental regulation of caste differentiation, but may also be involved in additional biological functions. In this study, we examine the whole genome methylation profiles of three stages of the haploid honey bee genome: unfertilised eggs, the adult drones that develop from these eggs and the sperm produced by these drones. These methylomes reveal distinct patterns of methylation. Eggs and sperm show 381 genes with significantly different CpG methylation patterns, with the vast majority being more methylated in eggs. Adult drones show greatly reduced levels of methylation across the genome when compared with both gamete samples. This suggests a dynamic cycle of methylation loss and gain through the development of the drone and during spermatogenesis. Although fluxes in methylation during embryogenesis may account for some of the differentially methylated sites, the distinct methylation patterns at some genes suggest parent-specific epigenetic marking in the gametes. Extensive germ line methylation of some genes possibly explains the lower-than-expected frequency of CpG sites in these genes. We discuss the potential developmental and evolutionary implications of methylation in eggs and sperm in this eusocial insect species.

  10. Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures.

    Directory of Open Access Journals (Sweden)

    Mohita Upadhyay

    Full Text Available Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses.We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC, differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses.All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses.The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the evolutionary lineage of the

  11. Promoter CpG island hypermethylation in the development of cutaneous melanoma

    NARCIS (Netherlands)

    Gao, Linda

    2015-01-01

    Epigenetic mechanisms such as promoter methylation affect gene expression by inducing changes in organizational structure of chromatin rather than in the DNA sequence. Increased methylation of cytosine within cystosine-guanine (CpG) dinucleotides that are located at higher density in gene promoter r

  12. Promoter CpG island hypermethylation in the development of cutaneous melanoma

    NARCIS (Netherlands)

    Gao, Linda

    2015-01-01

    Epigenetic mechanisms such as promoter methylation affect gene expression by inducing changes in organizational structure of chromatin rather than in the DNA sequence. Increased methylation of cytosine within cystosine-guanine (CpG) dinucleotides that are located at higher density in gene promoter

  13. Clinical analysis of Rett syndrome and a research of methylation CpG binding protein-2 gene%Rett综合征临床分析及甲基化CpG结合蛋白-2基因调查

    Institute of Scientific and Technical Information of China (English)

    程芹; 吕康模; 康涵; 邓佳; 刘平; 蒋利萍

    2015-01-01

    目的:探讨甲基化CpG结合蛋白2(MECP2)基因突变在4例疑似Rett综合征(RTT)患者中的致病作用及基因测序对协助诊断RTT的意义。方法选择2014年4~8月重庆医科大学附属成都市妇女儿童中心医院收治的4例疑似RTT患者,即病例1、2、3、4,病例1为家族先证者,病例2为病例1的母亲,病例3、4为散发患者,家系依次为A、B、C。对4例患者进行临床资料分析、家系调查、核型分析等,并对患者及其父母MECP2基因进行聚合酶链反应扩增测序。结果病例1、4通过2010年最新RTT的修订诊断标准及基因诊断确诊,病例2、3的临床诊断不成立但发现致病基因突变,病例1、2为国内首例母女同患RTT,其MECP2基因测序为同一个国内未报道的突变位点,病例3、4家系中发现已有文献报道的致病位点突变。结论 RTT临床诊断可能与基因诊断结果不一致,基因诊断对确诊RTT,尤其是临床症状不典型的病例具有重要价值;疑诊患者早期完成基因诊断可降低医疗成本、早期开始康复训练并辅助优生优育。%Objective To explore methylation CpG binding protein-2 gene mutation′s pathopoiesia effect on 4 suspected Rett syndrome (RTT) patients and gene sequencing′s significance in assist diagnosing RTT. Methods Selected 4 suspected RTT patients admitted in Chengdu Women′s&Children′s Central Hospital from April to August,2014,i.e. case 1,2,3,4. Case 1 was fam-ily propositus,case 2 was case 1′s mother,case 3 and 4 were sporadic patients,pedigree was successively A,B,C. Did clinical data analysis,pedigree investigation,karyotype analysis on 4 patients. Did polymerase chain reaction amplification sequencing on patients and their parents' MECP2 gene. Results Case 1 and 4 were confirmed by the latest 2010 Revised RTT diagnostic criteria and genet-ic diagnosis. Case 2 and 3′s clinical diagnosis was disconfirmed but virulence gene mutation

  14. N-methyl-D-aspartate receptor encephalitis mediates loss of intrinsic activity measured by functional MRI.

    Science.gov (United States)

    Brier, Matthew R; Day, Gregory S; Snyder, Abraham Z; Tanenbaum, Aaron B; Ances, Beau M

    2016-06-01

    Spontaneous brain activity is required for the development and maintenance of normal brain function. Many disease processes disrupt the organization of intrinsic brain activity, but few pervasively reduce the amplitude of resting state blood oxygen level dependent (BOLD) fMRI fluctuations. We report the case of a female with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, longitudinally studied during the course of her illness to determine the contribution of NMDAR signaling to spontaneous brain activity. Resting state BOLD fMRI was measured at the height of her illness and 18 weeks following discharge from hospital. Conventional resting state networks were defined using established methods. Correlation and covariance matrices were calculated by extracting the BOLD time series from regions of interest and calculating either the correlation or covariance quantity. The intrinsic activity was compared between visits, and to expected activity from 45 similarly aged healthy individuals. Near the height of the illness, the patient exhibited profound loss of consciousness, high-amplitude slowing of the electroencephalogram, and a severe reduction in the amplitude of spontaneous BOLD fMRI fluctuations. The patient's neurological status and measures of intrinsic activity improved following treatment. We conclude that NMDAR-mediated signaling plays a critical role in the mechanisms that give rise to organized spontaneous brain activity. Loss of intrinsic activity is associated with profound disruptions of consciousness and cognition.

  15. Small deletions but not methylation underlie CDKN2A/p16 loss of expression in conventional osteosarcoma.

    Science.gov (United States)

    Mohseny, Alexander B; Tieken, Chris; van der Velden, Pieter A; Szuhai, Karoly; de Andrea, Carlos; Hogendoorn, Pancras C W; Cleton-Jansen, Anne-Marie

    2010-12-01

    Conventional osteosarcoma is characterized by rapid growth, high local aggressiveness, and metastasizing potential. Patients developing lung metastases experience poor prognosis despite extensive chemotherapy regimens and surgical interventions. Previously we identified a subgroup of osteosarcoma patients with loss of CDKN2A/p16 protein expression in the primary tumor biopsies which was significantly predictive of a very poor prognosis. Here we aimed to identify the underlying mechanism(s) of this protein loss in relation to osteosarcoma behavior. The CDKN2A locus was analyzed in osteosarcoma cases with total loss of CDKN2A/p16 expression and in cases with high protein expression using melting curve analysis-methylation assay (MCA-Meth), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and mutation analysis. All cases with complete CDKN2A/p16 protein loss showed homozygous deletions at the CDKN2A locus. In none of the cases hyper methylation of the promoter region was seen which was confirmed by sequencing this region. Taken together we show that large or smaller deletions of the CDKN2A locus are evident in patient samples and underlie the CDKN2A/p16 protein expression loss while promoter methylation does not appear to be a mechanism of this expression loss. Genomic loss of CDKN2A instead of promoter methylation might be a plausible explanation for the rapid proliferation and high aggressiveness of osteosarcoma by simultaneous impairment CDKN2A/p14(ARF) function. © 2010 Wiley-Liss, Inc.

  16. CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas.

    Science.gov (United States)

    Paz, Maria F; Yaya-Tur, Ricard; Rojas-Marcos, Iñigo; Reynes, Gaspar; Pollan, Marina; Aguirre-Cruz, Lucinda; García-Lopez, Jose Luis; Piquer, Jose; Safont, María-Jose; Balaña, Carmen; Sanchez-Cespedes, Montserrat; García-Villanueva, Mercedes; Arribas, Leoncio; Esteller, Manel

    2004-08-01

    The DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents, and its loss in cancer cells is associated with hypermethylation of the MGMT CpG island. Thus, methylation of MGMT has been correlated with the clinical response to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in primary gliomas. Here, we investigate whether the presence of MGMT methylation in gliomas is also a good predictor of response to another emergent alkylating agent, temozolomide. Using a methylation-specific PCR approach, we assessed the methylation status of the CpG island of MGMT in 92 glioma patients who received temozolomide as first-line chemotherapy or as treatment for relapses. Methylation of the MGMT promoter positively correlated with the clinical response in the glioma patients receiving temozolomide as first-line chemotherapy (n = 40). Eight of 12 patients with MGMT-methylated tumors (66.7%) had a partial or complete response, compared with 7 of 28 patients with unmethylated tumors (25.0%; P = 0.030). We also found a positive association between MGMT methylation and clinical response in those patients receiving BCNU (n = 35, P = 0.041) or procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (n = 17, P = 0.043) as first-line chemotherapy. Overall, if we analyze the clinical response of all of the first-line chemotherapy treatments with temozolomide, BCNU, and procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea as a group in relation to the MGMT methylation status, MGMT hypermethylation was strongly associated with the presence of partial or complete clinical response (P < 0.001). Finally, the MGMT methylation status determined in the initial glioma tumor did not correlate with the clinical response to temozolomide when this drug was administered as treatment for relapses (P = 0.729). MGMT methylation predicts the clinical response of primary gliomas to first-line chemotherapy with the alkylating agent

  17. Technology evaluation: CpG-7909, Coley.

    Science.gov (United States)

    Paul, Stéphane

    2003-10-01

    Coley Pharmaceutical (formerly CpG ImmunoPharmaceuticals) is developing CpG-7909 (ProMune) for use in the potential treatment of cancer and as a vaccine adjuvant. By April 2000, CpG-7909 had entered phase I/II trials for cancer and in March 2002, Coley initiated a phase I trial in non-Hodgkin's lymphoma in combination with rituximab (Rituxan). By October 2002, CpG-7909 was in phase II trials as a vaccine adjuvant. Cpg-7909 is currently also undergoing phase II trials for melanoma.

  18. DNA Methylation Profiles of Protease Nexin 1 (SERPINE2) Gene in Human Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Shan Gao; Peter A. Andreasen

    2011-01-01

    Objective: To investigated whether epigenetic mechanisms contribute to the variable expression of variable protease nexin1(PN-1) encoded by the SERPINE2 gene in different cell types. Methods: Working with 5 human cell lines, we determined the CpG methylation status within two CpG islands in the SERPINE2 gene by bisulphate sequencing and the PN-1 mRNA level by Q-RT PCR. Results: A CpG island spanning the transcription initiation site showed little methylation in 3 of the cell lines and substantial methylation in 2 of the cell lines. A CpG island covering the translation starting site showed full methylation in all investigated cell lines. Methylation within the CpG island was not randomly distributed, but showed accumulation at specific sites. However, we were not able to distinguish any patterns which related the methylation frequency to the gene expression level. Inhibition of CpG methylation with 5-aza-2′-deoxycytidine led to a several fold increase in PN-1 mRNA levels, but based on the results on CpG methylation in the CpG island spanning the transcript, the effect is most likely indirect. Conclusion: We have carefully mapped the CpG methylation pattern in two CpG islands in the 5′ part of the SERPINE2 gene without finding any obvious inverse correlation between methylation frequency and expression level.

  19. Interleukin-4 Induces CpG Site-Specific Demethylation of the Pendrin Promoter in Primary Human Bronchial Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Giada Scantamburlo

    2017-03-01

    Full Text Available Pendrin is upregulated in bronchial epithelial cells following IL-4 stimulation via binding of STAT6 to an N4 GAS motif. Basal CpG methylation of the pendrin promoter is cell-specific. We studied if a correlation exists between IL-4 sensitivity and the CpG methylation status of the pendrin promoter in human bronchial epithelial cell models. Methods: Real-time PCR and pyrosequencing were used to respectively quantify pendrin mRNA levels and methylation of pendrin promoter, with and without IL-4 stimulation, in healthy and diseased primary HBE cells, as well as NCI-H292 cells. Results: Increases in pendrin mRNA after IL-4 stimulation was more robust in NCI-H292 cells than in primary cells. The amount of gDNA methylated varied greatly between the cell types. In particular, CpG site 90 located near the N4 GAS motif was highly methylated in the primary cells. An additional CpG site (90bis, created by a SNP, was found only in the primary cells. IL-4 stimulation resulted in dramatic demethylation of CpG sites 90 and 90bis in the primary cells. Conclusions: IL-4 induces demethylation of specific CpG sites within the pendrin promoter. These epigenetic alterations are cell type specific, and may in part dictate pendrin mRNA transcription.

  20. Characterization of tumor cells and stem cells by differential nuclear methylation imaging

    Science.gov (United States)

    Tajbakhsh, Jian; Wawrowsky, Kolja A.; Gertych, Arkadiusz; Bar-Nur, Ori; Vishnevsky, Eugene; Lindsley, Erik H.; Farkas, Daniel L.

    2008-02-01

    DNA methylation plays a key role in cellular differentiation. Aberrant global methylation patterns are associated with several cancer types, as a result of changes in long-term activation status of up to 50% of genes, including oncogenes and tumor-suppressor genes, which are regulated by methylation and demethylation of promoter region CpG dinucleotides (CpG islands). Furthermore, DNA methylation also occurs in nonisland CpG sites (> 95% of the genome), present once per 80 dinucleotides on average. Nuclear DNA methylation increases during the course of cellular differentiation while cancer cells usually show a net loss in methylation. Given the large dynamic range in DNA methylation load, the methylation pattern of a cell can provide a valuable distinction as to its status during differentiation versus the disease state. By applying immunofluorescence, confocal microscopy and 3D image analysis we assessed the potential of differential nuclear distribution of methylated DNA to be utilized as a biomarker to characterize cells during development and when diseased. There are two major fields that may immediately benefit from this development: (1) the search for factors that contribute to pluripotency and cell fate in human embryonic stem cell expansion and differentiation, and (2) the characterization of tumor cells with regard to their heterogeneity in molecular composition and behavior. We performed topological analysis of the distribution of methylated CpG-sites (MeC) versus heterochromatin. This innovative approach revealed significant differences in colocalization patterns of MeC and heterochromatin-derived signals between undifferentiated and differentiated human embryonic stem cells, as well as untreated AtT20 mouse pituitary tumor cells compared to a subpopulation of these cells treated with 5-azacytidine for 48 hours.

  1. Quantitative, high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans.

    Science.gov (United States)

    Fryer, Anthony A; Emes, Richard D; Ismail, Khaled M K; Haworth, Kim E; Mein, Charles; Carroll, William D; Farrell, William E

    2011-01-01

    Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypo-methylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period.

  2. Comprehensive analysis of CpG islands in human chromosomes 21 and 22

    Science.gov (United States)

    Takai, Daiya; Jones, Peter A.

    2002-03-01

    CpG islands are useful markers for genes in organisms containing 5-methylcytosine in their genomes. In addition, CpG islands located in the promoter regions of genes can play important roles in gene silencing during processes such as X-chromosome inactivation, imprinting, and silencing of intragenomic parasites. The generally accepted definition of what constitutes a CpG island was proposed in 1987 by Gardiner-Garden and Frommer [Gardiner-Garden, M. & Frommer, M. (1987) J. Mol. Biol. 196, 261-282] as being a 200-bp stretch of DNA with a C+G content of 50% and an observed CpG/expected CpG in excess of 0.6. Any definition of a CpG island is somewhat arbitrary, and this one, which was derived before the sequencing of mammalian genomes, will include many sequences that are not necessarily associated with controlling regions of genes but rather are associated with intragenomic parasites. We have therefore used the complete genomic sequences of human chromosomes 21 and 22 to examine the properties of CpG islands in different sequence classes by using a search algorithm that we have developed. Regions of DNA of greater than 500 bp with a G+C equal to or greater than 55% and observed CpG/expected CpG of 0.65 were more likely to be associated with the 5' regions of genes and this definition excluded most Alu-repetitive elements. We also used genome sequences to show strong CpG suppression in the human genome and slight suppression in Drosophila melanogaster and Saccharomyces cerevisiae. This finding is compatible with the recent detection of 5-methylcytosine in Drosophila, and might suggest that S. cerevisiae has, or once had, CpG methylation.

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

    OpenAIRE

    Nilsen, Frances M.; Benjamin B Parrott; 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

    2015-01-01

    Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry...

  4. Epigenetic repression of RARRES1 is mediated by methylation of a proximal promoter and a loss of CTCF binding.

    Directory of Open Access Journals (Sweden)

    Zhengang Peng

    Full Text Available BACKGROUND: The cis-acting promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1 by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We first compared methylation occurring at the sequences (-664~+420 flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region (-664~-86, while the downstream element (-85~+420 proximal to the transcriptional start site (+1 remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter bound with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effect. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores. CONCLUSION/SIGNIFICANCE: This report delineated silencing of RARRES1 by hypermethylation is occurring at a proximal promoter element and is associated with a loss of binding to CTCF, an activator for

  5. Circulating Differentially Methylated Amylin DNA as a Biomarker of β-Cell Loss in Type 1 Diabetes.

    Directory of Open Access Journals (Sweden)

    John A Olsen

    Full Text Available In type 1 diabetes (T1D, β-cell loss is silent during disease progression. Methylation-sensitive quantitative real-time PCR (qPCR of β-cell-derived DNA in the blood can serve as a biomarker of β-cell death in T1D. Amylin is highly expressed by β-cells in the islet. Here we examined whether demethylated circulating free amylin DNA (cfDNA may serve as a biomarker of β-cell death in T1D. β cells showed unique methylation patterns within the amylin coding region that were not observed with other tissues. The design and use of methylation-specific primers yielded a strong signal for demethylated amylin in purified DNA from murine islets when compared with other tissues. Similarly, methylation-specific primers detected high levels of demethylated amylin DNA in human islets and enriched human β-cells. In vivo testing of the primers revealed an increase in demethylated amylin cfDNA in sera of non-obese diabetic (NOD mice during T1D progression and following the development of hyperglycemia. This increase in amylin cfDNA did not mirror the increase in insulin cfDNA, suggesting that amylin cfDNA may detect β-cell loss in serum samples where insulin cfDNA is undetected. Finally, purified cfDNA from recent onset T1D patients yielded a high signal for demethylated amylin cfDNA when compared with matched healthy controls. These findings support the use of demethylated amylin cfDNA for detection of β-cell-derived DNA. When utilized in conjunction with insulin, this latest assay provides a comprehensive multi-gene approach for the detection of β-cell loss.

  6. Methylation-Specific PCR Unraveled

    Directory of Open Access Journals (Sweden)

    Sarah Derks

    2004-01-01

    Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

  7. DNA methylation pattern in overweight women under an energy-restricted diet supplemented with fish oil.

    Science.gov (United States)

    do Amaral, Cátia Lira; Milagro, Fermín I; Curi, Rui; Martínez, J Alfredo

    2014-01-01

    Dietary factors modulate gene expression and are able to alter epigenetic signatures in peripheral blood mononuclear cells (PBMC). However, there are limited studies about the effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) on the epigenetic mechanisms that regulate gene expression. This research investigates the effects of n-3-rich fish oil supplementation on DNA methylation profile of several genes whose expression has been reported to be downregulated by n-3 PUFA in PBMC: CD36, FFAR3, CD14, PDK4, and FADS1. Young overweight women were supplemented with fish oil or control in a randomized 8-week intervention trial following a balanced diet with 30% energy restriction. Fatty acid receptor CD36 decreased DNA methylation at CpG +477 due to energy restriction. Hypocaloric diet-induced weight loss also reduced the methylation percentages of CpG sites located in CD14, PDK4, and FADS1. The methylation patterns of these genes were only slightly affected by the fish oil supplementation, being the most relevant to the attenuation of the weight loss-induced decrease in CD36 methylation after adjusting by baseline body weight. These results suggest that the n-3 PUFA-induced changes in the expression of these genes in PBMC are not mediated by DNA methylation, although other epigenetic mechanisms cannot be discarded.

  8. CpG islands influence chromatin structure via the CpG-binding protein Cfp1.

    Science.gov (United States)

    Thomson, John P; Skene, Peter J; Selfridge, Jim; Clouaire, Thomas; Guy, Jacky; Webb, Shaun; Kerr, Alastair R W; Deaton, Aimée; Andrews, Rob; James, Keith D; Turner, Daniel J; Illingworth, Robert; Bird, Adrian

    2010-04-15

    CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides. Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity. In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectively binds to non-methylated CpGs in vitro. Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo. High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7, 8). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins.

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

    Directory of Open Access Journals (Sweden)

    Catia Pilon

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains

    DEFF Research Database (Denmark)

    Crowther, P J; Doherty, J P; Linsenmeyer, M E

    1991-01-01

    preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts...... from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site...... duplication in the insertion site as commonly observed with truncated L1 elements. These data would be consistent with two mechanisms of integration of transposing L1 elements with different mechanisms predominating for full length and truncated elements. Udgivelsesdato: 1991-May-11...

  12. 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.

  13. DNA methylation is widespread and associated with differential gene expression in castes of the honeybee, Apis mellifera.

    Science.gov (United States)

    Elango, Navin; Hunt, Brendan G; Goodisman, Michael A D; Yi, Soojin V

    2009-07-07

    The recent, unexpected discovery of a functional DNA methylation system in the genome of the social bee Apis mellifera underscores the potential importance of DNA methylation in invertebrates. The extent of genomic DNA methylation and its role in A. mellifera remain unknown, however. Here we show that genes in A. mellifera can be divided into 2 distinct classes, one with low-CpG dinucleotide content and the other with high-CpG dinucleotide content. This dichotomy is explained by the gradual depletion of CpG dinucleotides, a well-known consequence of DNA methylation. The loss of CpG dinucleotides associated with DNA methylation also may explain the unusual mutational patterns seen in A. mellifera that lead to AT-rich regions of the genome. A detailed investigation of this dichotomy implicates DNA methylation in A. mellifera development. High-CpG genes, which are predicted to be hypomethylated in germlines, are enriched with functions associated with developmental processes, whereas low-CpG genes, predicted to be hypermethylated in germlines, are enriched with functions associated with basic biological processes. Furthermore, genes more highly expressed in one caste than another are overrepresented among high-CpG genes. Our results highlight the potential significance of epigenetic modifications, such as DNA methylation, in developmental processes in social insects. In particular, the pervasiveness of DNA methylation in the genome of A. mellifera provides fertile ground for future studies of phenotypic plasticity and genomic imprinting.

  14. Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Asmar, Fazila; Punj, Vasu; Christensen, Jesper Aagaard;

    2013-01-01

    The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers......% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P...

  15. 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.

  16. Dual effects of superovulation: loss of maternal and paternal imprinted methylation in a dose-dependent manner.

    Science.gov (United States)

    Market-Velker, Brenna A; Zhang, Liyue; Magri, Lauren S; Bonvissuto, Anne C; Mann, Mellissa R W

    2010-01-01

    Superovulation or ovarian stimulation is currently an indispensable assisted reproductive technology (ART) for human subfertility/infertility treatment. Recently, increased frequencies of imprinting disorders have been correlated with ARTs. Significantly, for Angelman and Beckwith-Wiedemann Syndromes, patients have been identified where ovarian stimulation was the only procedure used by the couple undergoing ART. In many cases, increased risk of genomic imprinting disorders has been attributed to superovulation in combination with inherent subfertility. To distinguish between these contributing factors, carefully controlled experiments are required on spontaneously ovulated, in vivo-fertilized oocytes and their induced-ovulated counterparts, thereby minimizing effects of in vitro manipulations. To this end, effects of superovulation on genomic imprinting were evaluated in a mouse model, where subfertility is not a confounding issue. This work represents the first comprehensive examination of the overall effects of superovulation on imprinted DNA methylation for four imprinted genes in individual blastocyst stage embryos. We demonstrate that superovulation perturbed genomic imprinting of both maternally and paternally expressed genes; loss of Snrpn, Peg3 and Kcnq1ot1 and gain of H19 imprinted methylation were observed. This perturbation was dose-dependent, with aberrant imprinted methylation more frequent at the high hormone dosage. Superovulation is thought to primarily affect oocyte development; thus, effects were expected to be limited to maternal alleles. Our study revealed that maternal as well as paternal H19 methylation was perturbed by superovulation. We postulate that superovulation has dual effects during oogenesis, disrupting acquisition of imprints in growing oocytes, as well as maternal-effect gene products subsequently required for imprint maintenance during pre-implantation development.

  17. DNA Methylation as a Biomarker for Preeclampsia

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-01

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

  18. Chicken spermatogenesis is accompanied by a genomic-wide loss of DNA methylation.

    Science.gov (United States)

    Rocamora, N; Mezquita, C

    1989-04-24

    Renaturation kinetics of DNA obtained from chicken testis cell nuclei separated by sedimentation at unit gravity showed that the undermethylation, previously observed in meiotic and postmeiotic cells, is not a peculiarity of repetitive sequences, but is also a feature of unique sequences. The large proportion of slowly renaturing, intermediately renaturing and rapidly renaturing DNAs contain 27, 32 and 31% less methylcytosines in meiotic and postmeiotic cells than the corresponding fractions of premeiotic cells. DNA methyltransferase activity is lower in meiotic cells containing undermethylated DNA than in immature testis, enriched in spermatogonia, with higher levels of DNA methylation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Haruta, Mayumi [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Nishiyama, Atsuya; Johmura, Yoshikazu [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Le Tallec, Benoît; Debatisse, Michelle [Institut Curie, Centre de Recherche, 26 rue d’Ulm, CNRS UMR 3244, 75248 ParisCedex 05 (France); Nakanishi, Makoto, E-mail: mkt-naka@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

    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. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

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

    Science.gov (United States)

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

    2012-08-01

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

  1. Transcriptional Regulation of Brain-Derived Neurotrophic Factor (BDNF) by Methyl CpG Binding Protein 2 (MeCP2): a Novel Mechanism for Re-Myelination and/or Myelin Repair Involved in the Treatment of Multiple Sclerosis (MS).

    Science.gov (United States)

    KhorshidAhmad, Tina; Acosta, Crystal; Cortes, Claudia; Lakowski, Ted M; Gangadaran, Surendiran; Namaka, Michael

    2016-03-01

    Multiple sclerosis (MS) is a chronic progressive, neurological disease characterized by the targeted immune system-mediated destruction of central nervous system (CNS) myelin. Autoreactive CD4+ T helper cells have a key role in orchestrating MS-induced myelin damage. Once activated, circulating Th1-cells secrete a variety of inflammatory cytokines that foster the breakdown of blood-brain barrier (BBB) eventually infiltrating into the CNS. Inside the CNS, they become reactivated upon exposure to the myelin structural proteins and continue to produce inflammatory cytokines such as tumor necrosis factor α (TNFα) that leads to direct activation of antibodies and macrophages that are involved in the phagocytosis of myelin. Proliferating oligodendrocyte precursors (OPs) migrating to the lesion sites are capable of acute remyelination but unable to completely repair or restore the immune system-mediated myelin damage. This results in various permanent clinical neurological disabilities such as cognitive dysfunction, fatigue, bowel/bladder abnormalities, and neuropathic pain. At present, there is no cure for MS. Recent remyelination and/or myelin repair strategies have focused on the role of the neurotrophin brain-derived neurotrophic factor (BDNF) and its upstream transcriptional repressor methyl CpG binding protein (MeCP2). Research in the field of epigenetic therapeutics involving histone deacetylase (HDAC) inhibitors and lysine acetyl transferase (KAT) inhibitors is being explored to repress the detrimental effects of MeCP2. This review will address the role of MeCP2 and BDNF in remyelination and/or myelin repair and the potential of HDAC and KAT inhibitors as novel therapeutic interventions for MS.

  2. Formulation of vaccines containing CpG oligonucleotides and alum

    OpenAIRE

    Aebig, Joan A.; Mullen, Gregory E. D.; Dobrescu, Gelu; Rausch, Kelly; Lambert, Lynn; Ajose-Popoola, Olubunmi; Long, Carole A.; Saul, Allan; Miles, Aaron P.

    2007-01-01

    CpG oligodeoxynucleotides are potent immunostimulants. For parenterally delivered alum based vaccines, the immunostimulatory effect of CpG depends on the association of the CpG and antigen to the alum. We describe effects of buffer components on the binding of CPG 7909 to aluminum hydroxide (Alhydrogel), assays for measuring binding of CPG 7909 to alum and CPG 7909 induced dissociation of antigen from the alum. Free CPG 7909 is a potent inducer of IP-10 in mice. However the lack of IP-10 prod...

  3. Formulation of vaccines containing CpG oligonucleotides and alum.

    Science.gov (United States)

    Aebig, Joan A; Mullen, Gregory E D; Dobrescu, Gelu; Rausch, Kelly; Lambert, Lynn; Ajose-Popoola, Olubunmi; Long, Carole A; Saul, Allan; Miles, Aaron P

    2007-06-30

    CpG oligodeoxynucleotides are potent immunostimulants. For parenterally delivered alum-based vaccines, the immunostimulatory effect of CpG depends on the association of the CpG and antigen to the alum. We describe effects of buffer components on the binding of CPG 7909 to aluminum hydroxide (Alhydrogel), assays for measuring binding of CPG 7909 to alum and CPG 7909 induced dissociation of antigen from the alum. Free CPG 7909 is a potent inducer of IP-10 in mice. However the lack of IP-10 production from formulations containing bound CPG 7909 suggested that CPG 7909 does not rapidly dissociate from the alum after injection. It also suggests that IP-10 assays are not a good basis for potency assays for alum-based vaccines containing CPG 7909.

  4. Germline methylation patterns determine the distribution of recombination events in the dog genome.

    Science.gov (United States)

    Berglund, Jonas; Quilez, Javier; Arndt, Peter F; Webster, Matthew T

    2014-12-19

    The positive-regulatory domain containing nine gene, PRDM9, which strongly associates with the location of recombination events in several vertebrates, is inferred to be inactive in the dog genome. Here, we address several questions regarding the control of recombination and its influence on genome evolution in dogs. First, we address whether the association between CpG islands (CGIs) and recombination hotspots is generated by lack of methylation, GC-biased gene conversion (gBGC), or both. Using a genome-wide dog single nucleotide polymorphism data set and comparisons of the dog genome with related species, we show that recombination-associated CGIs have low CpG mutation rates, and that CpG mutation rate is negatively correlated with recombination rate genome wide, indicating that nonmethylation attracts the recombination machinery. We next use a neighbor-dependent model of nucleotide substitution to disentangle the effects of CpG mutability and gBGC and analyze the effects that loss of PRDM9 has on these rates. We infer that methylation patterns have been stable during canid genome evolution, but that dog CGIs have experienced a drastic increase in substitution rate due to gBGC, consistent with increased levels of recombination in these regions. We also show that gBGC is likely to have generated many new CGIs in the dog genome, but these mostly occur away from genes, whereas the number of CGIs in gene promoter regions has not increased greatly in recent evolutionary history. Recombination has a major impact on the distribution of CGIs that are detected in the dog genome due to the interaction between methylation and gBGC. The results indicate that germline methylation patterns are the main determinant of recombination rates in the absence of PRDM9. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. Hypermethylation of CpG island loci and hypomethylation of LINE-1 and Alu repeats in prostate adenocarcinoma and their relationship to clinicopathological features.

    Science.gov (United States)

    Cho, N-Y; Kim, B-H; Choi, M; Yoo, E J; Moon, K C; Cho, Y-M; Kim, D; Kang, G H

    2007-02-01

    Promoter CpG island hypermethylation is an important carcinogenic event in prostate adenocarcinoma. Regardless of tissue type, human cancers have in common both focal CpG island hypermethylation and global genomic hypomethylation. The present study evaluated CpG island loci hypermethylation and LINE-1 and Alu repeat hypomethylation in prostate adenocarcinoma, analysed the relationship between them, and correlated these findings with clinicopathological features. We examined 179 cases of prostate adenocarcinoma and 30 cases of benign prostate hypertrophy for the methylation status of 22 CpG island loci and the methylation levels of LINE-1 and Alu repeats using methylation-specific polymerase chain reaction and combined bisulphite restriction analysis, respectively. The following 16 CpG island loci were found to display cancer-related hypermethylation: RASSF1A, GSTP1, RARB, TNFRSF10C, APC, BCL2, MDR1, ASC, TIG1, RBP1, COX2, THBS1, TNFRSF10D, CD44, p16, and RUNX3. Except for the last four CpG island loci, hypermethylation of each of the remaining 12 CpG island loci displayed a close association with one or more of the prognostic parameters (ie preoperative serum prostate specific antigen level, Gleason score sum, and clinical stage). Prostate adenocarcinoma with hypermethylation of each of ASC, COX2, RARB, TNFRSF10C, MDR1, TIG1, RBP1, NEUROG1, RASSF1A, and GSTP1 showed a significantly lower methylation level of Alu or LINE-1 than prostate adenocarcinoma without hypermethylation. In addition, hypomethylation of Alu or LINE-1 was closely associated with one or more of the above prognostic parameters. These data suggest that in tumour progression a close relationship exists between CpG island hypermethylation and the hypomethylation of repetitive elements, and that CpG island hypermethylation and DNA hypomethylation contribute to cancer progression.

  6. Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis

    National Research Council Canada - National Science Library

    Newman, Michelle; Blyth, Benjamin J; Hussey, Damian J; Jardine, Daniel; Sykes, Pamela J; Ormsby, Rebecca J

    2012-01-01

    We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1...

  7. 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

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

    Directory of Open Access Journals (Sweden)

    Chiara Grasso

    Full Text Available Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis.Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results.We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1 by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36 and DNA from blood fractions of healthy people (DD study, N = 28, respectively.We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites.The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

  9. Relationship between gene body DNA methylation and intragenic H3K9me3 and H3K36me3 chromatin marks.

    Directory of Open Access Journals (Sweden)

    Maria A Hahn

    Full Text Available To elucidate the relationship between intragenic DNA methylation and chromatin marks, we performed epigenetic profiling of chromosome 19 in human bronchial epithelial cells (HBEC and in the colorectal cancer cell line HCT116 as well as its counterpart with double knockout of DNMT1 and DNMT3B (HCT116-DKO. Analysis of H3K36me3 profiles indicated that this intragenic mark of active genes is associated with two categories of genes: (i genes with low CpG density and H3K9me3 in the gene body or (ii genes with high CpG density and DNA methylation in the gene body. We observed that a combination of low CpG density in gene bodies together with H3K9me3 and H3K36me3 occupancy is a specific epigenetic feature of zinc finger (ZNF genes, which comprise 90% of all genes carrying both histone marks on chromosome 19. For genes with high intragenic CpG density, transcription and H3K36me3 occupancy were not changed in conditions of partial or intensive loss of DNA methylation in gene bodies. siRNA knockdown of SETD2, the major histone methyltransferase responsible for production of H3K36me3, did not reduce DNA methylation in gene bodies. Our study suggests that the H3K36me3 and DNA methylation marks in gene bodies are established largely independently of each other and points to similar functional roles of intragenic DNA methylation and intragenic H3K9me3 for CpG-rich and CpG-poor genes, respectively.

  10. Evaluation of methylation pattern in promoter region of E-cadherin ...

    African Journals Online (AJOL)

    user

    2011-03-07

    Mar 7, 2011 ... promoter methylation in CDH1 gene inactivation in breast cancer, the CpG methylation status of E- cadherin promoter ... The data indicate that CDH1 promoter methylation might be a potential ... Complex genetic and epigenetic alterations affect the ... DNA methylation is the covalent addition of a methyl.

  11. Primer design versus PCR bias in methylation independent PCR amplifications.

    Science.gov (United States)

    Wojdacz, Tomasz K; Borgbo, Tanni; Hansen, Lise Lotte

    2009-05-16

    Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten "CpG-free" primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the "CpG-free" primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1-0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.

  12. CpG hypomethylation in a large domain encompassing the embryonic beta-like globin genes in primitive erythrocytes.

    Science.gov (United States)

    Hsu, Mei; Mabaera, Rodwell; Lowrey, Christopher H; Martin, David I K; Fiering, Steven

    2007-07-01

    There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the beta-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine beta-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large ( approximately 20-kb) domain is hypomethylated only in primitive erythroid cells; it extends from the region just past the locus control region to before beta-major and encompasses the embryonic genes Ey, beta h1, and beta h0. Even retrotransposons in this region are hypomethylated in primitive erythroid cells. The existence of this large developmentally regulated domain of hypomethylation supports a mechanistic role for DNA methylation in developmental regulation of globin genes.

  13. CpG Hypomethylation in a Large Domain Encompassing the Embryonic β-Like Globin Genes in Primitive Erythrocytes▿ †

    Science.gov (United States)

    Hsu, Mei; Mabaera, Rodwell; Lowrey, Christopher H.; Martin, David I. K.; Fiering, Steven

    2007-01-01

    There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the β-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine β-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large (∼20-kb) domain is hypomethylated only in primitive erythroid cells; it extends from the region just past the locus control region to before β-major and encompasses the embryonic genes Ey, βh1, and βh0. Even retrotransposons in this region are hypomethylated in primitive erythroid cells. The existence of this large developmentally regulated domain of hypomethylation supports a mechanistic role for DNA methylation in developmental regulation of globin genes. PMID:17452448

  14. Methyl sulfone induces loss of metastatic properties and reemergence of normal phenotypes in a metastatic cloudman S-91 (M3 murine melanoma cell line.

    Directory of Open Access Journals (Sweden)

    Joan McIntyre Caron

    Full Text Available BACKGROUND: The most deadly form of cancer is not lung or colon, breast or prostate; it is any cancer that has become metastatic. Mortality due to metastatic melanoma, one of the most aggressive and deadly cancers, has increased steadily over the last several decades. Unfortunately, the arsenal of chemotherapeutic agents available today is most often unsuccessful at extending and improving the life expectancy of afflicted individuals. We sought to identify an effective and nontoxic agent against metastatic melanoma. METHODOLOGY/PRINCIPAL FINDINGS: We chose to study Cloudman S-91 mouse melanoma cells (sub-clone M3, CCL53.1 because these cells are highly aggressive and metastatic, representing one of the deadliest types of cancer. Melanoma cells also had an experimental advantage because their morphology, which is easily monitored, relates to the physiology of metastatic cells and normal melanocytes. We chose to test methyl sulfone as a chemotherapeutic agent for two reasons. Because of its chemical structure, we speculated a potential anti-cancer activity by targeting microtubules. Equally important, methyl sulfone has a well-established safety profile in humans. Surprisingly, we found that malignant melanoma cells exposed to methyl sulfone demonstrated the loss of phenotypes characteristic of malignant cells, and the reemergence of phenotypes characteristic of healthy melanocytes. Briefly, over time methyl sulfone induced contact inhibition, loss of ability to migrate through an extracellular matrix, loss of anchorage-independent growth, proper wound healing followed by contact inhibition, irreversible senescence followed by arborization with melanosomes in arbors as seen in normal melanocytes. CONCLUSIONS/SIGNIFICANCE: Methyl sulfone may have clinical potential as a non-toxic agent effective against metastatic melanoma. Additionally, methyl sulfone has promise as a tool to explore molecular mechanisms of metastatic transformation as well as

  15. 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....

  16. All and only CpG containing sequences are enriched in promoters abundantly bound by RNA polymerase II in multiple tissues

    Directory of Open Access Journals (Sweden)

    Myakishev Maxim V

    2008-02-01

    Full Text Available Abstract Background The promoters of housekeeping genes are well-bound by RNA polymerase II (RNAP in different tissues. Although the promoters of these genes are known to contain CpG islands, the specific DNA sequences that are associated with high RNAP binding to housekeeping promoters has not been described. Results ChIP-chip experiments from three mouse tissues, liver, heart ventricles, and primary keratinocytes, indicate that 94% of promoters have similar RNAP binding, ranging from well-bound to poorly-bound in all tissues. Using all 8-base pair long sequences as a test set, we have identified the DNA sequences that are enriched in promoters of housekeeping genes, focusing on those DNA sequences which are preferentially localized in the proximal promoter. We observe a bimodal distribution. Virtually all sequences enriched in promoters with high RNAP binding values contain a CpG dinucleotide. These results suggest that only transcription factor binding sites (TFBS that contain the CpG dinucleotide are involved in RNAP binding to housekeeping promoters while TFBS that do not contain a CpG are involved in regulated promoter activity. Abundant 8-mers that are preferentially localized in the proximal promoters and exhibit the best enrichment in RNAP bound promoters are all variants of six known CpG-containing TFBS: ETS, NRF-1, BoxA, SP1, CRE, and E-Box. The frequency of these six DNA motifs can predict housekeeping promoters as accurately as the presence of a CpG island, suggesting that they are the structural elements critical for CpG island function. Experimental EMSA results demonstrate that methylation of the CpG in the ETS, NRF-1, and SP1 motifs prevent DNA binding in nuclear extracts in both keratinocytes and liver. Conclusion In general, TFBS that do not contain a CpG are involved in regulated gene expression while TFBS that contain a CpG are involved in constitutive gene expression with some CpG containing sequences also involved in

  17. Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Miralem Mrkonjic

    Full Text Available We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734 and microsatellite unstable (MSI-H colorectal cancers (CRCs in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability.We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls and from Seattle (591 cases, 629 controls. Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohistochemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10(-4 when the SNP was examined alone.The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

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

    Science.gov (United States)

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

    2014-05-14

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

  19. 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.

  20. Association of AGTR1 Promoter Methylation Levels with Essential Hypertension Risk: A Matched Case-Control Study.

    Science.gov (United States)

    Fan, Rui; Mao, Shuqi; Zhong, Fade; Gong, Minli; Yin, Fengying; Hao, Lingmei; Zhang, Lina

    2015-01-01

    The purpose of the present study was to investigate whether methylation of the angiotensin II type 1 receptor (AGTR1) promoter contributed to the risk of essential hypertension (EH). A total of 96 EH cases and 96 gender- and age-matched healthy controls were recruited. Methylation of 8 CpG dinucleotides (CpG1-8) in the AGTR1 promoter was examined using the bisulphite pyrosequencing technology. Three CpG dinucleotides (CpG6-8) could not be well sequenced, therefore only the remaining 5 CpG sites were analysed. A significantly lower CpG1 methylation level was identified in EH cases than in controls (cases vs. 6.74 ± 4.32% vs. 9.66 ± 5.45%, p = 0.007), and no significant association was observed in the remaining analyses. In addition, significantly lower CpG1 (p = 0.028) and higher CpG2 (p = 0.032) methylation levels were observed in males than in females. In the breakdown association test by gender, a higher CpG1 methylation level was also identified in EH in both males (p = 0.034) and females (p = 0.020). Receiver operating characteristic curves showed that CpG1 methylation was a significant predictor of EH. Furthermore, CpG1 methylation was inversely correlated with uric acid levels in controls. The present study suggests that CpG1 hypomethylation in the AGTR1 promoter is likely associated with the risk of EH in the population assessed. © 2015 S. Karger AG, Basel.

  1. The Role of RASSF1A Methylation in Cancer

    Directory of Open Access Journals (Sweden)

    Luke B. Hesson

    2007-01-01

    Full Text Available Tumour suppressor gene inactivation is critical to the pathogenesis of cancers; such loss of function may be mediated by irreversible processes such as gene deletion or mutation. Alternatively tumour suppressor genes may be inactivated via epigenetic processes a reversible mechanism that promises to be more amenable to treatment by therapeutic agents. The CpG dinucleotide is under-represented in the genome, but it is found in clusters within the promoters of some genes, and methylation of these CpG islands play a critical role in the control of gene expression. Inhibitors of the DNA methyltransferases DNMT1 and DNMT3b have been used in a clinical setting, these nucleotide analogues lack specificity but the side effects of low dose treatments were minimal and in 2004 Vidaza (5-azacitidine was licensed for use in myelodysplastic syndrome. Methylation inhibitors are also entering trials in conjunction with another class of epigenetic modifiers, the histone deacetylase inhibitors and this epigenetic double bullet offers hope of improved treatment regimes. Recently there has been a plethora of reports demonstrating epigenetic inactivation of genes that play important roles in development of cancer, including Ras-association domain family of genes. Epigenetic inactivation of RASSF1A (Ras-association domain family 1, isoform A is one of the most common molecular changes in cancer. Hypermethylation of the RASSF1A promoter CpG island silences expression of the gene in many cancers including lung, breast, prostate, glioma, neuroblastoma and kidney cancer. Several recent studies have illustrated the diagnostic and prognostic potential of RASSF1A methylation. This presents RASSF1A methylation as an attractive biomarker for early cancer detection which, for most cancers, results in improved clinical outcome. DNA methylation analysis is applicable to a range of body fluids including serum, urine, bronchioalveolar lavage and sputum. The ease with which these

  2. Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40

    Directory of Open Access Journals (Sweden)

    Wajdi Ayadi

    2014-01-01

    Full Text Available To further explore the epigenetic changes in nasopharyngeal carcinoma (NPC, methylation-sensitive arbitrarily primed PCR was performed on NPC biopsies and nontumor nasopharyngeal samples. We have shown mainly two DNA fragments that appeared to be differentially methylated in NPCs versus nontumors. The first, defined as hypermethylated, corresponds to a CpG island at the 5′-end of the tetratricopeptide repeat domain 40 (TTC40 gene, whereas the second, defined as hypo-methylated, is located on repetitive sequences at chromosomes 16p11.1 and 13.1. Thereafter, the epigenetic alteration on the 5′-TTC40 gene was confirmed by methylation-specific PCR, showing a significant aberrant methylation in NPCs, compared to nontumors. In addition, the bisulfite sequencing analysis has shown a very high density of methylated cytosines in C15, C17, and X666 NPC xenografts. To assess whether TTC40 gene is silenced by aberrant methylation, we examined the gene expression by reverse transcription-PCR. Our analysis showed that the mRNA expression was significantly lower in tumors than in nontumors, which is associated with 5′-TTC40 gene hypermethylation. In conclusion, we found that the 5′-TTC40 gene is frequently methylated and is associated with the loss of mRNA expression in NPCs. Hypermethylation of 5′-TTC40 gene might play a role in NPC development; nevertheless, other studies are needed.

  3. Distinct Methylation of IFNG in the Gut

    OpenAIRE

    Gonsky, Rivkah; Deem, Richard L; Targan, Stephan R.

    2009-01-01

    Mucosal expression of proinflammatory cytokines plays a pivotal role in inflammatory bowel disease (IBD) pathogenesis. Epigenetic remodeling of chromatin via DNA methylation regulates gene expression. In this study, IFNG DNA methylation was analyzed within the mucosal compartment in both normal and IBD populations and compared to its peripheral counterparts. Overall IFNG methylation (across eight CpG sites) was significantly lower in lamina propria (LP) T cells compared to peripheral blood (P...

  4. 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

  5. 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...

  6. A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies.

    Directory of Open Access Journals (Sweden)

    David S Shames

    2006-12-01

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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Hellman Asaf

    2010-05-01

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

  9. Age-Associated Methylation Suppresses SPRY1, Leading to a Failure of Re-quiescence and Loss of the Reserve Stem Cell Pool in Elderly Muscle

    Directory of Open Access Journals (Sweden)

    Anne Bigot

    2015-11-01

    Full Text Available The molecular mechanisms by which aging affects stem cell number and function are poorly understood. Murine data have implicated cellular senescence in the loss of muscle stem cells with aging. Here, using human cells and by carrying out experiments within a strictly pre-senescent division count, we demonstrate an impaired capacity for stem cell self-renewal in elderly muscle. We link aging to an increased methylation of the SPRY1 gene, a known regulator of muscle stem cell quiescence. Replenishment of the reserve cell pool was modulated experimentally by demethylation or siRNA knockdown of SPRY1. We propose that suppression of SPRY1 by age-associated methylation in humans inhibits the replenishment of the muscle stem cell pool, contributing to a decreased regenerative response in old age. We further show that aging does not affect muscle stem cell senescence in humans.

  10. Age-Associated Methylation Suppresses SPRY1, Leading to a Failure of Re-quiescence and Loss of the Reserve Stem Cell Pool in Elderly Muscle.

    Science.gov (United States)

    Bigot, Anne; Duddy, William J; Ouandaogo, Zamalou G; Negroni, Elisa; Mariot, Virginie; Ghimbovschi, Svetlana; Harmon, Brennan; Wielgosik, Aurore; Loiseau, Camille; Devaney, Joe; Dumonceaux, Julie; Butler-Browne, Gillian; Mouly, Vincent; Duguez, Stéphanie

    2015-11-10

    The molecular mechanisms by which aging affects stem cell number and function are poorly understood. Murine data have implicated cellular senescence in the loss of muscle stem cells with aging. Here, using human cells and by carrying out experiments within a strictly pre-senescent division count, we demonstrate an impaired capacity for stem cell self-renewal in elderly muscle. We link aging to an increased methylation of the SPRY1 gene, a known regulator of muscle stem cell quiescence. Replenishment of the reserve cell pool was modulated experimentally by demethylation or siRNA knockdown of SPRY1. We propose that suppression of SPRY1 by age-associated methylation in humans inhibits the replenishment of the muscle stem cell pool, contributing to a decreased regenerative response in old age. We further show that aging does not affect muscle stem cell senescence in humans.

  11. 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...

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

    NARCIS (Netherlands)

    E.H.J. van Roon (Eddy); A. Boot (Arnoud); A.A. Dihal (Ashwin); R.F. Ernst (Robert); T. van Wezel (Tom); H. Morreau (Hans); J.M. Boer (Judith)

    2013-01-01

    textabstractBackground: Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have

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

    Science.gov (United States)

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

    2013-09-10

    Specific methylation of appetite-related genes in leukocytes could serve as a useful biomarker to predict weight regain after an energy restriction program. We aimed to evaluate whether the pre-intervention DNA methylation patterns involved in the epigenetic control of appetite-regulatory genes in leukocytes are associated with the weight regain process. Eighteen men who lost ≥5% of body weight after an 8-week nutritional intervention were categorized as "regainers" (≥10% weight regain) and "non-regainers" (weight regain) 32weeks after stopping dieting. At baseline, leukocytes were isolated and DNA was analyzed for epigenetic methylation patterns of appetite-related gene promoters by MALDI-TOF mass spectrometry. Regainers showed higher methylation levels than non-regainers in proopiomelanocortin (POMC) CpG sites +136bp and +138bp (fold change from non-regainers=26%; p=0.020) and lower methylation of the whole analyzed region of neuropeptide Y (NPY; fold change from non-regainers=-22%; p=0.033), as well as of several individual NPY-promoter CpG sites. Importantly, total baseline NPY methylation was associated with weight-loss regain (r=-0.76; pweight-loss maintenance (odds ratio=0.042 [95% CI 0.01-0.57]; p=0.018), whereas lower total methylation levels in NPY promoter were associated with higher risk of weight regain (odds ratio=14.0 [95% CI 1.13-172]; p=0.039). Therefore, the study of leukocyte methylation levels reflects a putative epigenetic regulation of NPY and POMC, which might be implicated in the weight regain process and be used as biomarkers for predicting weight regain after dieting. © 2013.

  14. CpG DNA: A pathogenic factor in systemic lupus erythematosus?

    Energy Technology Data Exchange (ETDEWEB)

    Krieg, A.M. [Univ. of Iowa College of Medicine, Iowa City, IA (United States)

    1995-11-01

    Systemic lupus erythematosus (SLE) is a multifactorial disease of unknown etiology. Characteristic features of SLE include (1) polyclonal B cell activation, (2) overexpression of the immune stimulatory cytokine interleukin-6 (IL-6), (3) defective tolerance to self antigens, and (4) production of anti-DNA antibodies (Ab). Bacterial infection has been suspected as a triggering factor for lupus. Bacterial DNA differs from vertebrate DNA in the frequency and methylation of CpG dinucleotides. These CpG motifs in bacterial DNA induce a variety of immune effects, including (1) polyclonal activation of murine and human B cells, (2) IL-6 secretion, and (3) resistance to apoptosis, thereby potentially allowing the survival of autoreactive cells. These results suggest that microbial DNA could therefore be a pathogenic factor in SLE. SLE patients have elevated levels of circulating plasma DNA which is reportedly enriched in hypomethylated CpGs. Genomic DNA is also hypomethylated in SLE. The purpose of this review is to summarize the immune effects of CpG motifs and to present the evidence for their possible involvement in the pathogenesis of SLE. 77 refs.

  15. Loss of CclA, required for histone 3 lysine 4 methylation, decreases growth but increases secondary metabolite production in Aspergillus fumigatus

    Directory of Open Access Journals (Sweden)

    Jonathan M. Palmer

    2013-02-01

    Full Text Available Secondary metabolite (SM production in filamentous fungi is mechanistically associated with chromatin remodeling of specific SM clusters. One locus recently shown to be involved in SM suppression in Aspergillus nidulans was CclA, a member of the histone 3 lysine 4 methylating COMPASS complex. Here we examine loss of CclA and a putative H3K4 demethylase, HdmA, in the human pathogen Aspergillus fumigatus. Although deletion of hdmA showed no phenotype under the conditions tested, the cclA deletant was deficient in tri- and di-methylation of H3K4 and yielded a slowly growing strain that was rich in the production of several SMs, including gliotoxin. Similar to deletion of other chromatin modifying enzymes, ΔcclA was sensitive to 6-azauracil indicating a defect in transcriptional elongation. Despite the poor growth, the ΔcclA mutant had wild-type pathogenicity in a murine model and the Toll-deficient Drosophila model of invasive aspergillosis. These data indicate that tri- and di-methylation of H3K4 is involved in the regulation of several secondary metabolites in A. fumigatus, however does not contribute to pathogenicity under the conditions tested.

  16. The rates of G:C[yields]T:A and G:C[yields]C:G transversions at CpG dinucleotides in the human factor IX gene

    Energy Technology Data Exchange (ETDEWEB)

    Ketterling, R.P.; Vielhaber, E.; Sommer, S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))

    1994-05-01

    The authors have identified eight independent transversions at CpG in 290 consecutive families with hemophilia B. These eight transversions account for 16.3% of all independent transversions in the sample, yet the expected frequency of CpG transversions at random in the factor IX gene is only 2.6% (P<0.1). The aggregate data suggest that the two types of CpG transversions (G:C[yields]T:A and G:C[yields]C:G) possess similar mutation rates (24.8 [times] 10[sup [minus]10] and 20.6 [times] 10[sup [minus]10], respectively), which are about fivefold greater than the comparable rates for transversions at non-CpG dinucleotides. The enhancement of transversions at CpG suggest that the model by which mutations occur at CpG may need to be reevaluated. The relationship, if any, between deamination of 5-methyl cytosine and enhancement of transversions at CpG remains to be defined. 28 refs., 2 tabs.

  17. The protection of CpG ODNs and Yarrowia lipolytica harboring VP28 for shrimp Litopenaeus vannamei against White spot syndrome virus infection

    Directory of Open Access Journals (Sweden)

    Q Yi

    2014-04-01

    Full Text Available The white spot syndrome is one of the most serious disease which has caused high mortalities and huge economic losses to shrimp culture. In the present study, the oral administrations with CpG ODNs and Yarrowia lipolytica harboring VP28 (rVP28-yl as dietary supplement for shrimp Litopenaeus vannamei were conducted to evaluate their protective effects against WSSV. After feeding for 15 days, the cumulative mortality and the copy number of WSSV in CpG and rVP28-yl feeding shrimps were significantly lower when they were challenged by WSSV, compared with those in control shrimps (p < 0.05. The caspase-3 activity was suppressed in rVP28-yl feeding shrimps but ascended in CpG feeding shrimps after WSSV challenge. Besides, the PO activity in CpG feeding shrimps was significantly increased after feeding trial, and kept increasing post WSSV challenge (p < 0.05. While the increased NO production was observed both in CpG and rVP28-yl feeding shrimps after feeding trial and WSSV challenge. In addition, increased mRNA expression levels of STAT and Dicer were observed in CpG group post WSSV challenge. These results together indicated that oral feeding of CpG ODNs and rVP28-yl could enhance the innate non-specific immune responses especially antiviral immunity of shrimps in varying degrees, and increase their resistance against WSSV infection

  18. Lsh Is Essential for Maintaining Global DNA Methylation Levels in Amphibia and Fish and Interacts Directly with Dnmt1

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    Donncha S. Dunican

    2015-01-01

    Full Text Available Eukaryotic genomes are methylated at cytosine bases in the context of CpG dinucleotides, a pattern which is maintained through cell division by the DNA methyltransferase Dnmt1. Dramatic methylation losses are observed in plant and mouse cells lacking Lsh (lymphoid specific helicase, predominantly at repetitive sequences and gene promoters. However, the mechanism by which Lsh contributes to the maintenance of DNA methylation is unknown. Here we show that DNA methylation is lost in Lsh depleted frog and fish embryos, both of which exhibit developmental delay. Additionally, we show that both Lsh and Dnmt1 are associated with chromatin and that Lsh knockdown leads to a decreased Dnmt1-chromatin association. Coimmunoprecipitation experiments reveal that Lsh and Dnmt1 are found in the same protein complex, and pulldowns show this interaction is direct. Our data indicate that Lsh is usually diffuse in the nucleus but can be recruited to heterochromatin in a HP1α-dependent manner. These data together (a show that the role of Lsh in DNA methylation is conserved in plants, amphibian, fish, and mice and (b support a model in which Lsh contributes to Dnmt1 binding to chromatin, explaining how its loss can potentially lead to perturbations in DNA methylation maintenance.

  19. Methylation-mediated transcriptional repression of microRNAs during cervical carcinogenesis

    Science.gov (United States)

    Wilting, Saskia M.; Verlaat, Wina; Jaspers, Annelieke; Makazaji, Nour A.; Agami, Reuven; Meijer, Chris J.L.M.; Snijders, Peter J.F.

    2013-01-01

    Deregulated expression of microRNAs (miRNAs) is common and biologically relevant in cervical carcinogenesis and appears only partly related to chromosomal changes. We recently identified 32 miRNAs showing decreased expression in high-grade cervical intraepithelial neoplasia (CIN) and carcinomas not associated with a chromosomal loss, 6 of which were located within a CpG island. This study aimed to investigate to what extent these miRNAs are subject to DNA methylation-mediated transcriptional repression in cervical carcinogenesis.   Methylation-specific PCR (MSP) analysis on a cell line panel representing different stages of human papillomavirus (HPV) induced transformation revealed an increase in methylation of hsa-miR-149, -203 and -375 with progression to malignancy, whereas expression of these miRNAs was restored upon treatment with a demethylating agent. All three miRNAs showed significantly increased levels of methylation in cervical carcinomas, whereas methylation levels of hsa-miR-203 and -375 were also significantly increased in high-grade CIN. A pilot analysis showed that increased hsa-miR-203 methylation was also detectable in HPV-positive cervical scrapes of women with high-grade CIN compared with controls. Similar to recent findings on hsa-miR-375, ectopic expression of hsa-miR-203 in cervical cancer cells decreased both the proliferation rate and anchorage independent growth. We found evidence for methylation-mediated transcriptional repression of hsa-miR-149, -203 and -375 in cervical cancer. Methylation of the latter two was already apparent in precancerous lesions and represent functionally relevant events in HPV-mediated transformation. Increased hsa-miR-203 methylation was detectable in scrapes of women with high-grade CIN, indicating that methylated miRNAs may provide putative markers to assess the presence of (pre)cancerous lesions. PMID:23324622

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

    NARCIS (Netherlands)

    Ligthart, Symen; Steenaard, Rebecca V.; Peters, Marjolein J.; van Meurs, Joyce B. J.; Sijbrands, Eric J. G.; Uitterlinden, Andre G.; Bonder, Marc J.; Hofman, Albert; Franco, Oscar H.; Dehghan, Abbas

    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. We annotated CpG sites available on the Illumina Human Methylation 450K array to diabetes genes

  1. Role for DNA methylation in the regulation of miR-200c and miR-141 expression in normal and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Vrba, Lukas; Jensen, Taylor J.; Garbe, James C.; Heimark, Ronald L.; Cress, Anne E.; Dickinson, Sally; Stampfer, Martha R.; Futscher, Bernard W.

    2009-12-23

    BACKGROUND: The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood. METHODOLOGY/ PRINCIPAL FINDINGS: Epigenetic mechanisms participate in the control of miR-200c and miR-141 expression in both normal and cancer cells. A CpG island near the predicted mir-200c/mir-141 transcription start site shows a striking correlation between miR-200c and miR-141 expression and DNA methylation in both normal and cancer cells, as determined by MassARRAY technology. The CpG island is unmethylated in human miR-200/miR-141 expressing epithelial cells and in miR-200c/miR-141 positive tumor cells. The CpG island is heavily methylated in human miR-200c/miR-141 negative fibroblasts and miR-200c/miR-141 negative tumor cells. Mouse cells show a similar inverse correlation between DNA methylation and miR-200c expression. Enrichment of permissive histone modifications, H3 acetylation and H3K4 trimethylation, is seen in normal miR-200c/miR-141-positive epithelial cells, as determined by chromatin immunoprecipitation coupled to real-time PCR. In contrast, repressive H3K9 dimethylation marks are present in normal miR-200c/miR-141-negative fibroblasts and miR-200c/miR-141 negative cancer cells and the permissive histone modifications are absent. The epigenetic modifier drug, 5-aza-2'-deoxycytidine, reactivates miR-200c/miR-141 expression showing that epigenetic mechanisms play a functional role in their transcriptional control. CONCLUSIONS/ SIGNIFICANCE: We report that DNA methylation plays a role in the normal cell type-specific expression of miR-200c and miR-141 and this role appears evolutionarily conserved, since similar results were obtained in mouse. Aberrant DNA methylation

  2. Absence of MGMT promoter methylation in endometrial cancer.

    Science.gov (United States)

    Rimel, B J; Huettner, Phyllis; Powell, Matthew A; Mutch, David G; Goodfellow, Paul J

    2009-01-01

    O(6)-methylguanine-DNA methyltransferase (MGMT) acts to repair DNA damaged by alkylation of guanine residues. MGMT promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes [Ogino S, Meyerhardt JA, Kawasaki T, et al. CpG island methylation, response to combination chemotherapy, and patient survival in advanced microsatellite stable colorectal carcinoma. Virchows Arch 2007;450:529-37; Rodriguez MJ, Acha A, Ruesga MT, Rodriguez C, Rivera JM, Aguirre JM. Loss of expression of DNA repair enzyme MGMT in oral leukoplakia and early oral squamous cell carcinoma. A prognostic tool? Cancer Lett 2007;245:263-8; Ishii T, Murakami J, Notohara K, et al. Oesophageal squamous cell carcinoma may develop within a background of accumulating DNA methylation in normal and dysplastic mucosa. Gut 2007;56:13-9]. The loss of MGMT activity and promoter methylation is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas [Weaver KD, Grossman SA, Herman JG. Methylated tumor-specific DNA as a plasma biomarker in patients with glioma. Cancer Invest 2006;24:35-40; Esteller M, Garcia-Foncillas J, Andion E, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000;343:1350-4; Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005;352:997-1003]. We sought to determine if MGMT promoter methylation plays a role in endometrial cancer. One hundred and twenty primary endometrial cancers were analyzed for MGMT promoter methylation by combined bisulfite restriction analysis (COBRA). The cohort included 77 endometrioid endometrial cancers, 43 endometrial tumors of adverse histologic type, and 6 endometrial cancer cell lines. Twenty-one endometrioid and mixed endometrioid ovarian cancers were also analyzed. A subset of the primary tumors was analyzed for MGMT expression by

  3. A differentially methylated region of the DAZ1 gene in spermatic and somatic cells

    Institute of Scientific and Technical Information of China (English)

    Zuo-Xiang Li; Xu Ma; Zhao-Hui Wang

    2006-01-01

    Aim: To investigate the methylation status of the deleted in azoospermia 1 (DAZ1) gene promoter region in different cell types. Methods: Using CpG island Searcher software, a CpG island was found in the promoter region of the DAZ1 gene. The methylation status of this region was analyzed in sperm and leukocytes by bisulfited sequencing.Results: The methylation status of the CpG island in the DAZ1 gene promoter region differed in leukocytes and sperm: it was methylated in leukocytes, but unmethylated in sperm. Conclusion: A differentially methylated region of the DAZ1 gene exists in spermatic and somatic cells, suggesting that methylation of this region may regulate DAZ1 gene expression in different tissues.

  4. Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis

    Institute of Scientific and Technical Information of China (English)

    Li-hong FU; Chun-ling MA; Bin CONG; Shu-jin LI; Hai-ying CHEN; Jing-ge ZHANG

    2011-01-01

    Aim:The promoter of human interleukin-10 (IL10),a cytokine crucial for suppressing inflammation and regulating immune responses,contains an interspecies-conserved sequence with CpG motifs.The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA).Methods:Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human,macaque and mouse IL10 genes.Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected.The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 μmol/L).The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA,respectively.The methylation of CpGs in the IL10 promoter was determined by pyrosequencing.Chromatin immunoprecipitation (CHIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions.Results:One interspecies-conserved sequence was found within the IL10 promoter.The upstream CpGs at -408,-387,-385,and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls.In contrast,the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016),which was correlated with higher IL10 mRNA and serum levels.In the 5-azacytidine-treated PBMCs,the CpG motifs were demethylated,and the expression levels of IL10 mRNA and protein was significantly increased.CHIP assays revealed increased phospho-CREB binding to the IL10 promoter.Conclusion:The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.

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

    Directory of Open Access Journals (Sweden)

    V Shilpa

    2014-01-01

    Full Text Available Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6 -methyguanine-DNA methyltransferase (MGMT is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC tissue samples, 14 low malignant potential (LMP tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

  6. Identifying differentially methylated genes using mixed effect and generalized least square models

    OpenAIRE

    2009-01-01

    Abstract Background DNA methylation plays an important role in the process of tumorigenesis. Identifying differentially methylated genes or CpG islands (CGIs) associated with genes between two tumor subtypes is thus an important biological question. The methylation status of all CGIs in the whole genome can be assayed with differential methylation hybridization (DMH) microarrays. However, patient samples or cell lines are heterogeneous, so their methylation pattern may be very different. In a...

  7. Focussing reduced representation CpG sequencing through judicious restriction enzyme choice.

    Science.gov (United States)

    Kirschner, Sophie A; Hunewald, Oliver; Mériaux, Sophie B; Brunnhoefer, Regina; Muller, Claude P; Turner, Jonathan D

    2016-04-01

    Current restriction enzyme based reduced representation methylation analyses aim for limited, but unbiased, methylome coverage. As the current best estimate suggests that only ~20% of CpGs are dynamically regulated, we characterised the CpG and genomic context surrounding all suitable restriction enzyme sites to identify those that were located in regions rich in dynamically methylated CpGs. The restriction-site distributions for MspI, BstUI, and HhaI were non-random. CpGs in CGI and shelf+shore could be enriched, particularly in gene bodies for all genomic regions, promoters (TSS1500, TSS200), intra- (1st exon, gene body, 3'UTR, 5'UTR) and inter-genic regions. HpyCH4IV enriched CpG elements in the open sea for all genomic elements. Judicious restriction enzyme choice improves the focus of reduced representation approaches by avoiding the monopolization of read coverage by genomic regions that are irrelevant, unwanted or difficult to map, and only sequencing the most informative fraction of CpGs.

  8. Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-rasG to A mutation in colorectal tumor

    Institute of Scientific and Technical Information of China (English)

    Jian Qi; You-Qing Zhu; Mei-Fang Huang; Dong Yang

    2005-01-01

    AIM: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression.METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas,62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutantallele-specific amplification was used to detect K-rasG to A point mutation in codon 12.RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation,respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-rasG to A point mutation in codon 12 had methylated MGMT(P = 0.011). The frequencies of K-rasG to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively.CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.

  9. Differential methylation of the oxytocin receptor gene in patients with anorexia nervosa: a pilot study.

    Directory of Open Access Journals (Sweden)

    Youl-Ri Kim

    Full Text Available BACKGROUND AND AIM: Recent studies in patients with anorexia nervosa suggest that oxytocin may be involved in the pathophysiology of anorexia nervosa. We examined whether there was evidence of variation in methylation status of the oxytocin receptor (OXTR gene in patients with anorexia nervosa that might account for these findings. METHODS: We analyzed the methylation status of the CpG sites in a region from the exon 1 to the MT2 regions of the OXTR gene in buccal cells from 15 patients and 36 healthy women using bisulfite sequencing. We further examined whether methylation status was associated with markers of illness severity or form. RESULTS: We identified six CpG sites with significant differences in average methylation levels between the patient and control groups. Among the six differentially methylated CpG sites, five showed higher than average methylation levels in patients than those in the control group (64.9-88.8% vs. 6.6-45.0%. The methylation levels of these five CpG sites were negatively associated with body mass index (BMI. BMI, eating disorders psychopathology, and anxiety were identified in a regression analysis as factors affecting the methylation levels of these CpG sites with more variation accounted for by BMI. CONCLUSIONS: Epigenetic misregulation of the OXTR gene may be implicated in anorexia nervosa, which may either be a mechanism linking environmental adversity to risk or may be a secondary consequence of the illness.

  10. Methylation of the SPARC gene promoter and its clinical implication in pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Lv Shunli

    2010-03-01

    Full Text Available Abstract Background The secreted protein acidic and rich in cysteine (SPARC plays a pivotal role in regulating cell-matrix interactions and tumor angiogenesis, proliferation, and migration. Detection of SPARC gene methylation may be useful as a tumorigenesis marker for early detection of pancreatic cancer. Methods Methylation of the SPARC gene transcriptional regulation region (TRR was detected using bisulfite-specific (BSP PCR-based sequencing analysis in 40 cases of pancreatic cancer and the adjacent normal tissues, 6 chronic pancreatitis tissues, and 6 normal pancreatic tissues. BSP cloning-based sequencing analysis was also performed in selected cases. Clinicopathological data from the cancer patients were collected and analyzed. Results Analysis of SPARC gene TRR methylation showed two hypermethylation wave peak regions: CpG Region 1 (CpG site 1-7 and CpG Region 2 (CpG site 8-12. Pancreatic tissues have shown methylation in both regions with gradual increases from normal, chronic pancreatitis, and adjacent normal tissues to cancerous tissues. However, Methylation of CpG Region 2 was more sensitive than CpG Region 1 in pancreatic tumorigenesis. Furthermore, the methylation level of CpG Region 2 was associated with increased tumor size and exposure to the risk factors (tobacco smoke and alcohol consumption for developing pancreatic cancer. Conclusion Methylation of the SPARC gene, specifically CpG Region 2, may be an early event during pancreatic tumorigenesis and should be further evaluated as a tumorigenesis marker for early detection of pancreatic cancer.

  11. Identification and validation of highly frequent CpG island hypermethylation in colorectal adenomas and carcinomas.

    Science.gov (United States)

    Oster, Bodil; Thorsen, Kasper; Lamy, Philippe; Wojdacz, Tomasz K; Hansen, Lise Lotte; Birkenkamp-Demtröder, Karin; Sørensen, Karina D; Laurberg, Søren; Orntoft, Torben F; Andersen, Claus L

    2011-12-15

    In our study, whole-genome methylation arrays were applied to identify novel genes with tumor specific DNA methylation of promoter CpG islands in pre-malignant and malignant colorectal lesions. Using a combination of Illumina HumanMethylation27 beadchips, Methylation-Sensitive High Resolution Melting (MS-HRM) analysis, and Exon arrays (Affymetrix) the DNA methylation pattern of ∼14,000 genes and their transcript levels were investigated in six normal mucosas, six adenomas and 30 MSI and MSS carcinomas. Sixty eight genes with tumor-specific hypermethylation were identified (p mucosas, 12 adenomas, 40 MSS and nine MSI cancer samples. The methylation patterns of 15 selected genes, hypermethylated in adenomas and carcinomas (FLI1, ST6GALNAC5, TWIST1, ADHFE1, JAM2, IRF4, CNRIP1, NRG1 and EYA4), in carcinomas only (ABHD9, AOX1 and RERG), or in MSI but not MSS carcinomas (RAMP2, DSC3 and MLH1) were validated using MS-HRM. Four of these genes (MLH1, AOX1, EYA4 and TWIST1) had previously been reported to be hypermethylated in CRC. Eleven genes, not previously known to be affected by CRC specific hypermethylation, were identified and validated. Inverse correlation to gene expression was observed for six of the 15 genes with Spearman correlation coefficients ranging from -0.39 to -0.60. For six of these genes the altered methylation patterns had a profound transcriptional association, indicating that methylation of these genes may play a direct regulatory role. The hypermethylation changes often occurred already in adenomas, indicating that they may be used as biomarkers for early detection of CRC.

  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 extracted from saliva for methylation studies of psychiatric traits: evidence tissue specificity and relatedness to brain.

    Science.gov (United States)

    Smith, Alicia K; Kilaru, Varun; Klengel, Torsten; Mercer, Kristina B; Bradley, Bekh; Conneely, Karen N; Ressler, Kerry J; Binder, Elisabeth B

    2015-01-01

    DNA methylation has become increasingly recognized in the etiology of psychiatric disorders. Because brain tissue is not accessible in living humans, epigenetic studies are most often conducted in blood. Saliva is often collected for genotyping studies but is rarely used to examine DNA methylation because the proportion of epithelial cells and leukocytes varies extensively between individuals. The goal of this study was to evaluate whether saliva DNA is informative for studies of psychiatric disorders. DNA methylation (HumanMethylation450 BeadChip) was assessed in saliva and blood samples from 64 adult African Americans. Analyses were conducted using linear regression adjusted for appropriate covariates, including estimated cellular proportions. DNA methylation from brain tissues (cerebellum, frontal cortex, entorhinal cortex, and superior temporal gyrus) was obtained from a publically available dataset. Saliva and blood methylation was clearly distinguishable though there was positive correlation overall. There was little correlation in CpG sites within relevant candidate genes. Correlated CpG sites were more likely to occur in areas of low CpG density (i.e., CpG shores and open seas). There was more variability in CpG sites from saliva than blood, which may reflect its heterogeneity. Finally, DNA methylation in saliva appeared more similar to patterns from each of the brain regions examined overall than methylation in blood. Thus, this study provides a framework for using DNA methylation from saliva and suggests that DNA methylation of saliva may offer distinct opportunities for epidemiological and longitudinal studies of psychiatric traits.

  14. Chilling-induced tomato flavor loss is associated with altered volatile synthesis and transient changes in DNA methylation

    Science.gov (United States)

    Commercial tomatoes are widely perceived by consumers as lacking flavor. A major part of that problem is a postharvest handling system that chills fruit. Low-temperature storage is widely used to slow ripening and reduce decay. However, chilling results in loss of flavor. Flavor-associated volatiles...

  15. 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...

  16. ANRIL Promoter DNA Methylation: A Perinatal Marker for Later Adiposity

    Directory of Open Access Journals (Sweden)

    Karen Lillycrop

    2017-05-01

    Full Text Available Experimental studies show a substantial contribution of early life environment to obesity risk through epigenetic processes. We examined inter-individual DNA methylation differences in human birth tissues associated with child's adiposity. We identified a novel association between the level of CpG methylation at birth within the promoter of the long non-coding RNA ANRIL (encoded at CDKN2A and childhood adiposity at age 6-years. An association between ANRIL methylation and adiposity was also observed in three additional populations; in birth tissues from ethnically diverse neonates, in peripheral blood from adolescents, and in adipose tissue from adults. Additionally, CpG methylation was associated with ANRIL expression in vivo, and CpG mutagenesis in vitro inhibited ANRIL promoter activity. Furthermore, CpG methylation enhanced binding to an Estrogen Response Element within the ANRIL promoter. Our findings demonstrate that perinatal methylation at loci relevant to gene function may be a robust marker of later adiposity, providing substantial support for epigenetic processes in mediating long-term consequences of early life environment on human health.

  17. Epigenetic Changes in Response to Tai Chi Practice: A Pilot Investigation of DNA Methylation Marks

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    Hua Ren

    2012-01-01

    Full Text Available Tai chi exercise has been shown to improve physiological and psychosocial functions, well-being, quality of life, and disease conditions. The biological mechanisms by which tai chi exerts its holistic effects remain unknown. We investigated whether tai chi practice results in positive epigenetic changes at the molecular level. Design. The DNA methylation profiles of sixty CpG-dinucleotide marks in female tai chi practitioners (N=237; 45–88 years old who have been practising tai chi for three or more years were compared with those of age-matched control females (N=263 who have never practised tai chi. Results. Six CpG marks originating from three different chromosomes reveal a significant difference (P<0.05 between the two cohorts. Four marks show losses while two marks show gains in DNA methylation with age in the controls. In the tai chi cohort all six marks demonstrate significant slowing (by 5–70% of the age-related methylation losses or gains observed in the controls, suggesting that tai chi practice may be associated with measurable beneficial epigenetic changes. Conclusions. The results implicate the potential use of DNA methylation as an epigenetic biomarker to better understand the biological mechanisms and the health and therapeutic efficacies of tai chi.

  18. Genome‐Wide DNA Methylation Study Identifies Significant Epigenomic Changes in Osteoarthritic Subchondral Bone and Similarity to Overlying Cartilage

    National Research Council Canada - National Science Library

    Jeffries, Matlock A; Donica, Madison; Baker, Lyle W; Stevenson, Michael E; Annan, Anand C; Beth Humphrey, Mary; James, Judith A; Sawalha, Amr H

    2016-01-01

    .... Genes with differentially methylated CpG sites were analyzed to identify shared pathways, upstream regulators, and overrepresented gene ontologies, and these patterns were compared with those of the overlying cartilage...

  19. Liposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce the same protection against the murine model of leishmaniasis.

    Science.gov (United States)

    Shargh, Vahid Heravi; Jaafari, Mahmoud Reza; Khamesipour, Ali; Jaafari, Iman; Jalali, Seyed Amir; Abbasi, Azam; Badiee, Ali

    2012-06-06

    First generation Leishmania vaccines consisting of whole killed parasites with or without adjuvants have reached phase 3 trial and failed to show enough efficacy mainly due to the lack of an appropriate adjuvant. In this study, the nuclease-resistant phosphorothioate CpG oligodeoxynucleotides (PS CpG) or nuclease-sensitive phosphodiester CpG ODNs (PO CpG) were used as adjuvants to enhance immunogenicity and rate of protection against leishmaniasis. Due to the susceptibility of PO CpG to nuclease degradation, an efficient liposomal delivery system was developed to protect them from degradation. 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid was used because of its unique adjuvanticity and electrostatic interaction with negatively charged CpG ODNs. To evaluate the role of liposomal formulation in protection rate and enhanced immune response, BALB/c mice were immunized subcutaneously with liposomal soluble Leishmania antigens (SLA) co-incorporated with PO CpG (Lip-SLA-PO CpG), Lip-SLA-PS CpG, SLA+PO CpG, SLA+PS CpG, SLA or buffer. As criteria for protection, footpad swelling at the site of challenge, parasite loads, the levels of IFN-γ and IL-4, and the IgG subtypes were evaluated. The groups of mice receiving Lip-SLA-PO CpG or Lip-SLA-PS CpG showed a high protection rate compared with the control groups. In addition, there was no significant difference in immune response generation between mice immunized with PS CpG and the group receiving PO CpG when incorporated into the liposomes. The results suggested that liposomal form of PO CpG might be used instead of PS CpG in future vaccine formulations as an efficient adjuvant. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. FCGR2A Promoter Methylation and Risks for Intravenous Immunoglobulin Treatment Responses in Kawasaki Disease

    Directory of Open Access Journals (Sweden)

    Ho-Chang Kuo

    2015-01-01

    Full Text Available Kawasaki disease (KD is characterized by pediatric systemic vasculitis of an unknown cause. The low affinity immunoglobulin gamma Fc region receptor II-a (FCGR2A gene was reported to be involved in the susceptibility of KD. DNA methylation is one of the epigenetic mechanisms that control gene expression; thus, we hypothesized that methylation status of CpG islands in FCGR2A promoter associates with the susceptibility and therapeutic outcomes of Kawasaki disease. In this study, 36 KD patients and 24 healthy subjects from out-patient clinic were recruited. Eleven potential methylation sites within the targeted promoter region of FCGR2A were selected for investigation. We marked the eleven methylation sites from A to K. Our results indicated that methylation at the CpG sites G, H, and J associated with the risk of KD. CpG sites B, C, E, F, H, J, and K were found to associate with the outcomes of IVIG treatment. In addition, CpG sites G, J, and K were predicted as transcription factors binding sites for NF-kB, Myc-Max, and SP2, respectively. Our study reported a significant association among the promoter methylation of FCGR2A, susceptibility of KD, and the therapeutic outcomes of IVIG treatment. The methylation levels of CpG sites of FCGR2A gene promoter should be an important marker for optimizing IVIG therapy.

  1. Combined crossed molecular beam and ab initio investigation of the multichannel reaction of boron monoxide (BO; X2Σ+) with Propylene (CH3CHCH2; X1A'): competing atomic hydrogen and methyl loss pathways.

    Science.gov (United States)

    Maity, Surajit; Dangi, Beni B; Parker, Dorian S N; Kaiser, Ralf I; An, Yi; Sun, Bing-Jian; Chang, A H H

    2014-10-16

    The reaction dynamics of boron monoxide ((11)BO; X(2)Σ(+)) with propylene (CH(3)CHCH(2); X(1)A') were investigated under single collision conditions at a collision energy of 22.5 ± 1.3 kJ mol(-1). The crossed molecular beam investigation combined with ab initio electronic structure and statistical (RRKM) calculations reveals that the reaction follows indirect scattering dynamics and proceeds via the barrierless addition of boron monoxide radical with its radical center located at the boron atom. This addition takes place to either the terminal carbon atom (C1) and/or the central carbon atom (C2) of propylene reactant forming (11)BOC(3)H(6) intermediate(s). The long-lived (11)BOC(3)H(6) doublet intermediate(s) underwent unimolecular decomposition involving at least three competing reaction mechanisms via an atomic hydrogen loss from the vinyl group, an atomic hydrogen loss from the methyl group, and a methyl group elimination to form cis-/trans-1-propenyl-oxo-borane (CH(3)CHCH(11)BO), 3-propenyl-oxo-borane (CH(2)CHCH(2)(11)BO), and ethenyl-oxo-borane (CH(2)CH(11)BO), respectively. Utilizing partially deuterated propylene (CD(3)CHCH(2) and CH(3)CDCD(2)), we reveal that the loss of a vinyl hydrogen atom is the dominant hydrogen elimination pathway (85 ± 10%) forming cis-/trans-1-propenyl-oxo-borane, compared to the loss of a methyl hydrogen atom (15 ± 10%) leading to 3-propenyl-oxo-borane. The branching ratios for an atomic hydrogen loss from the vinyl group, an atomic hydrogen loss from the methyl group, and a methyl group loss are experimentally derived to be 26 ± 8%:5 ± 3%:69 ± 15%, respectively; these data correlate nicely with the branching ratios calculated via RRKM theory of 19%:5%:75%, respectively.

  2. Loss of Dnmt1 catalytic activity reveals multiple roles for DNA methylation during pancreas development and regeneration.

    Science.gov (United States)

    Anderson, Ryan M; Bosch, Justin A; Goll, Mary G; Hesselson, Daniel; Dong, P Duc Si; Shin, Donghun; Chi, Neil C; Shin, Chong Hyun; Schlegel, Amnon; Halpern, Marnie; Stainier, Didier Y R

    2009-10-01

    Developmental mechanisms regulating gene expression and the stable acquisition of cell fate direct cytodifferentiation during organogenesis. Moreover, it is likely that such mechanisms could be exploited to repair or regenerate damaged organs. DNA methyltransferases (Dnmts) are enzymes critical for epigenetic regulation, and are used in concert with histone methylation and acetylation to regulate gene expression and maintain genomic integrity and chromosome structure. We carried out two forward genetic screens for regulators of endodermal organ development. In the first, we screened for altered morphology of developing digestive organs, while in the second we screed for the lack of terminally differentiated cell types in the pancreas and liver. From these screens, we identified two mutant alleles of zebrafish dnmt1. Both lesions are predicted to eliminate dnmt1 function; one is a missense mutation in the catalytic domain and the other is a nonsense mutation that eliminates the catalytic domain. In zebrafish dnmt1 mutants, the pancreas and liver form normally, but begin to degenerate after 84 h post fertilization (hpf). Acinar cells are nearly abolished through apoptosis by 100 hpf, though neither DNA replication, nor entry into mitosis is halted in the absence of detectable Dnmt1. However, endocrine cells and ducts are largely spared. Surprisingly, dnmt1 mutants and dnmt1 morpholino-injected larvae show increased capacity for pancreatic beta cell regeneration in an inducible model of pancreatic beta cell ablation. Thus, our data suggest that Dnmt1 is dispensable for pancreatic duct or endocrine cell formation, but not for acinar cell survival. In addition, Dnmt1 may influence the differentiation of pancreatic beta cell progenitors or the reprogramming of cells toward the pancreatic beta cell fate.

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

    Science.gov (United States)

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

    2001-01-01

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

  4. Methylation of C9orf72 expansion reduces RNA foci formation and dipeptide-repeat proteins expression in cells.

    Science.gov (United States)

    Bauer, Peter O

    2016-01-26

    A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), together referred to as c9FTD/ALS. It has been suggested that a loss of C9orf72 protein expression, the formation of toxic RNA foci and dipeptide-repeat proteins contribute to C9orf72-related diseases. Interestingly, it has been shown that trimethylation of histones and methylation of CpG islands near the repeat expansion may play a role in the pathogenesis c9FTD/ALS. Recently, methylation of expanded repeat itself has been reported. To further elucidate the mechanisms underlying these diseases, the influence of epigenetic modification in the repeat expansion on its pathogenic effect was assessed. Here, a reduced formation of toxic RNA foci and dipeptide-repeat proteins upon methylation of the GGGGCC repeat in a cellular model of c9FTD/ALS is shown. Additionally, a novel methylcytosine-capture DNA hybridization immunoassay for semi-quantitative detection of the repeat methylation levels is presented, potentially usable for methylation analysis in patients carrying C9orf72 repeat expansion carriers as a diagnostic tool. Presented results suggest that increased level of pathogenic GGGGCC expansion methylation may be sufficient to alleviate the molecular pathology of the C9orf72-related diseases.

  5. Spinal Tolerance and Dependence: Some Observations on the Role of Spinal N-Methyl-D-Aspartate Receptors and Phosphorylation in the Loss of Opioid Analgesic Responses

    Directory of Open Access Journals (Sweden)

    Tony L Yaksh

    2000-01-01

    Full Text Available The continuous delivery of opiates can lead to a reduction in analgesic effects. In humans, as in other animals, some component of this change in sensitivity seems likely to have a strong pharmacodynamic component. Such loss of effect, deemed to be tolerance in the present article, can be readily demonstrated in animals with repeated bolus and continuous intrathecal infusion of mu and delta opioids and alpha-2 adrenergic agonists. Research has shown that this loss of effect can be diminished by concurrent treatment with N-methyl-D-aspartate (NMDA receptor antagonists and by the suppression of the activity of spinal protein kinase C (PKC. This suggests in part the probable role of PKC-mediated phosphorylation in the right shift in the dose-effect curves observed with continuous opiate or adrenergic exposure. Importantly, this right shift is seen to occur in parallel with an increase in the phosphorylating activity in the dorsal horn and in the expression of several PKC isozymes. The target of this phosphorylation is not certain. Phosphorylation of the NMDA receptor enhances its functionality, while phosphorylation of the opioid receptor or associated channels seems to diminish their activity or to enhance internalization. While the focus is on several specific components, the accumulating data emphasize the biological complexity of these changes in spinal drug reactivity.

  6. Study on the polymorphisms and promoter methylation and expression of the glutathione Stransferases P1 gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    张友才

    2006-01-01

    Objective To study the relationship between hepatocellular carcinoma (HCC) and the polymorphisms, promoter methylation, and expression of glutathione S-transferases P1 gene (GST)P1 gene. Methods Using methylation -special PCR (MSP), the methylated status of CpG islands of GSTP1 gene in tumor tissues of 53 HCC and its adjacent nontumor tissues were studied. The en-

  7. 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.

  8. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing

    Directory of Open Access Journals (Sweden)

    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

  9. Distribution of CpG Motifs in Upstream Gene Domains in a Reef Coral and Sea Anemone: Implications for Epigenetics in Cnidarians.

    Directory of Open Access Journals (Sweden)

    Adam G Marsh

    Full Text Available Coral reefs are under assault from stressors including global warming, ocean acidification, and urbanization. Knowing how these factors impact the future fate of reefs requires delineating stress responses across ecological, organismal and cellular scales. Recent advances in coral reef biology have integrated molecular processes with ecological fitness and have identified putative suites of temperature acclimation genes in a Scleractinian coral Acropora hyacinthus. We wondered what unique characteristics of these genes determined their coordinate expression in response to temperature acclimation, and whether or not other corals and cnidarians would likewise possess these features. Here, we focus on cytosine methylation as an epigenetic DNA modification that is responsive to environmental stressors. We identify common conserved patterns of cytosine-guanosine dinucleotide (CpG motif frequencies in upstream promoter domains of different functional gene groups in two cnidarian genomes: a coral (Acropora digitifera and an anemone (Nematostella vectensis. Our analyses show that CpG motif frequencies are prominent in the promoter domains of functional genes associated with environmental adaptation, particularly those identified in A. hyacinthus. Densities of CpG sites in upstream promoter domains near the transcriptional start site (TSS are 1.38x higher than genomic background levels upstream of -2000 bp from the TSS. The increase in CpG usage suggests selection to allow for DNA methylation events to occur more frequently within 1 kb of the TSS. In addition, observed shifts in CpG densities among functional groups of genes suggests a potential role for epigenetic DNA methylation within promoter domains to impact functional gene expression responses in A. digitifera and N. vectensis. Identifying promoter epigenetic sequence motifs among genes within specific functional groups establishes an approach to describe integrated cellular responses to

  10. 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. Copyright © 2010 Elsevier Inc. All rights reserved.

  11. DNA Methylation Profiling of Uniparental Disomy Subjects Provides a Map of Parental Epigenetic Bias in the Human Genome.

    Science.gov (United States)

    Joshi, Ricky S; Garg, Paras; Zaitlen, Noah; Lappalainen, Tuuli; Watson, Corey T; Azam, Nidha; Ho, Daniel; Li, Xin; Antonarakis, Stylianos E; Brunner, Han G; Buiting, Karin; Cheung, Sau Wai; Coffee, Bradford; Eggermann, Thomas; Francis, David; Geraedts, Joep P; Gimelli, Giorgio; Jacobson, Samuel G; Le Caignec, Cedric; de Leeuw, Nicole; Liehr, Thomas; Mackay, Deborah J; Montgomery, Stephen B; Pagnamenta, Alistair T; Papenhausen, Peter; Robinson, David O; Ruivenkamp, Claudia; Schwartz, Charles; Steiner, Bernhard; Stevenson, David A; Surti, Urvashi; Wassink, Thomas; Sharp, Andrew J

    2016-09-01

    Genomic imprinting is a mechanism in which gene expression varies depending on parental origin. Imprinting occurs through differential epigenetic marks on the two parental alleles, with most imprinted loci marked by the presence of differentially methylated regions (DMRs). To identify sites of parental epigenetic bias, here we have profiled DNA methylation patterns in a cohort of 57 individuals with uniparental disomy (UPD) for 19 different chromosomes, defining imprinted DMRs as sites where the maternal and paternal methylation levels diverge significantly from the biparental mean. Using this approach we identified 77 DMRs, including nearly all those described in previous studies, in addition to 34 DMRs not previously reported. These include a DMR at TUBGCP5 within the recurrent 15q11.2 microdeletion region, suggesting potential parent-of-origin effects associated with this genomic disorder. We also observed a modest parental bias in DNA methylation levels at every CpG analyzed across ∼1.9 Mb of the 15q11-q13 Prader-Willi/Angelman syndrome region, demonstrating that the influence of imprinting is not limited to individual regulatory elements such as CpG islands, but can extend across entire chromosomal domains. Using RNA-seq data, we detected signatures consistent with imprinted expression associated with nine novel DMRs. Finally, using a population sample of 4,004 blood methylomes, we define patterns of epigenetic variation at DMRs, identifying rare individuals with global gain or loss of methylation across multiple imprinted loci. Our data provide a detailed map of parental epigenetic bias in the human genome, providing insights into potential parent-of-origin effects.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  14. Identification of new differentially methylated genes that have potential functional consequences in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Jin W Kim

    Full Text Available Many differentially methylated genes have been identified in prostate cancer (PCa, primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19 and adjacent normal tissues (n = 4 and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05, defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa. Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  17. 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.

  18. Modeling the mammalian locomotor CPG: insights from mistakes and perturbations.

    Science.gov (United States)

    McCrea, David A; Rybak, Ilya A

    2007-01-01

    A computational model of the mammalian spinal cord circuitry incorporating a two-level central pattern generator (CPG) with separate half-center rhythm generator (RG) and pattern formation (PF) networks is reviewed. The model consists of interacting populations of interneurons and motoneurons described in the Hodgkin-Huxley style. Locomotor rhythm generation is based on a combination of intrinsic (persistent sodium current dependent) properties of excitatory RG neurons and reciprocal inhibition between the two half-centers comprising the RG. The two-level architecture of the CPG was suggested from an analysis of deletions (spontaneous omissions of activity) and the effects of afferent stimulation on the locomotor pattern and rhythm observed during fictive locomotion in the cat. The RG controls the activity of the PF network that in turn defines the rhythmic pattern of motoneuron activity. The model produces realistic firing patterns of two antagonist motoneuron populations and generates locomotor oscillations encompassing the range of cycle periods and phase durations observed during cat locomotion. A number of features of the real CPG operation can be reproduced with separate RG and PF networks, which would be difficult if not impossible to demonstrate with a classical single-level CPG. The two-level architecture allows the CPG to maintain the phase of locomotor oscillations and cycle timing during deletions and during sensory stimulation. The model provides a basis for functional identification of spinal interneurons involved in generation and control of the locomotor pattern.

  19. Enhanced antibody production in mice to the malaria antigen AMA1 by CPG 7909 requires physical association of CpG and antigen

    OpenAIRE

    Mullen, Gregory E. D.; Aebig, Joan A.; Dobrescu, Gelu; Rausch, Kelly; Lambert, Lynn; Long, Carole A.; Miles, Aaron P.; Saul, Allan

    2007-01-01

    CpG oligodeoxynucleotides are potent immunostimulants. In this study, CPG 7909 was formulated with the recombinant Plasmodium falciparum protein AMA1-C1 adsorbed to Alhydrogel (aluminum hydroxide) and used to immunize mice. Mice receiving free CPG 7909 in a separate same site injection to the AMA1-C1/Alhydrogel had the same antibody responses as mice receiving AMA1-C1/Alhydrogel alone. For mice immunized with CPG 7909 bound to the AMA1-C1/Alhydrogel formulation, there was a bell shaped CPG 79...

  20. 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.

  1. CpG 7909: PF 3512676, PF-3512676.

    Science.gov (United States)

    2006-01-01

    CpG 7909 [PF-3512676] is an immunomodulating synthetic oligonucleotide designed to specifically agonise the Toll-like receptor 9 (TLR9). It is being developed for the treatment of cancer [ProMune] as a monotherapy and in combination with chemotherapeutic agents, and it is also under development as an adjuvant [VaxImmune] for vaccines against cancer and infectious diseases. CpG 7909, acting through the TLR9 receptor present in B cells and plasmacytoid dendritic cells, stimulates human B-cell proliferation, enhances antigen-specific antibody production and induces interferon-alpha production, interleukin-10 secretion and natural killer cell activity. Coley Pharmaceutical Group originally developed CpG 7909 using its CpG DNA technology. In March 2005, Coley granted Pfizer an exclusive global license to develop and commercialise CPG 7909 [ProMune] for the treatment, control and prevention of multiple cancer indications. Coley licensed CpG 7909 [VaxImmune] to Chiron Corporation for adjuvant use with Chiron's prophylactic vaccine candidates against infectious diseases in December 2003. Chiron was acquired by and merged into Novartis in April 2006. In 2002, GlaxoSmithKline (GSK) was granted a worldwide, non-exclusive licence to Coley's CpG immunostimulatory oligonucleotides, including CpG 7909 [VaxImmune], for their use as adjuvants for cancer vaccines. In 2000, Coley entered into a co-exclusive licensing agreement with GSK for the development of therapeutic and prophylactic vaccines against infectious diseases. This licensing agreement included CpG 7909 [VaxImmune] and other CpG-based immunostimulatory oligonucleotides. In September 2004, Coley Pharmaceuticals was awarded a 16.9 million US dollars, 5-year contract from the National Institute of Allergy and Infectious Diseases (NIAID), one of the National Institutes of Health (NIH), to support the development of novel immune-activating drugs for defense against bioterror agents. This contract will be used to expand Coley

  2. Enhanced antibody production in mice to the malaria antigen AMA1 by CPG 7909 requires physical association of CpG and antigen.

    Science.gov (United States)

    Mullen, Gregory E D; Aebig, Joan A; Dobrescu, Gelu; Rausch, Kelly; Lambert, Lynn; Long, Carole A; Miles, Aaron P; Saul, Allan

    2007-07-20

    CpG oligodeoxynucleotides are potent immunostimulants. In this study, CPG 7909 was formulated with the recombinant Plasmodium falciparum protein AMA1-C1 adsorbed to Alhydrogel (aluminum hydroxide) and used to immunize mice. Mice receiving free CPG 7909 in a separate same site injection to the AMA1-C1/Alhydrogel had the same antibody responses as mice receiving AMA1-C1/Alhydrogel alone. For mice immunized with CPG 7909 bound to the AMA1-C1/Alhydrogel formulation, there was a bell shaped CPG 7909 dose-response curve with the highest antibody response co-incident with the concentration of CPG 7909 that saturated binding to the Alhydrogel. At a higher CPG 7909 dose where 74% was unbound, there was no enhancement of response over AMA1-C1/Alhydrogel alone. Our results suggest that the adjuvant effects of CpGs are optimal when adsorbed to Alhydrogel and highlight the need for careful characterization of the vaccine formulation.

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

    NARCIS (Netherlands)

    S. Ligthart (Symen); R.V. Steenaard (Rebecca); M.J. Peters (Marjolein); J.B.J. van Meurs (Joyce); E.J.G. Sijbrands (Eric); A.G. Uitterlinden (André); M.J. Bonder (Marc); 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 (André); 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

  4. 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

  5. Correlating CpG islands, motifs, and sequence variants in human chromosome 21

    Directory of Open Access Journals (Sweden)

    Cercone Nick

    2011-07-01

    Full Text Available Abstract Background CpG islands are important regions in DNA. They usually appear at the 5’ end of genes containing GC-rich dinucleotides. When DNA methylation occurs, gene regulation is affected and it sometimes leads to carcinogenesis. We propose a new detection program using a hidden-markov model alongside the Viterbi algorithm. Methods Our solution provides a graphical user interface not seen in many of the other CGI detection programs and we unify the detection and analysis under one program to allow researchers to scan a genetic sequence, detect the significant CGIs, and analyze the sequence once the scan is complete for any noteworthy findings. Results Using human chromosome 21, we show that our algorithm finds a significant number of CGIs. Running an analysis on a dataset of promoters discovered that the characteristics of methylated and unmethylated CGIs are significantly different. Finally, we detected significantly different motifs between methylated and unmethylated CGI promoters using MEME and MAST. Conclusions Developing this new tool for the community using powerful algorithms has shown that combining analysis with CGI detection will improve the continued research within the field of epigenetics.

  6. The methyl-CpG-binding protein CIBZ suppresses myogenic differentiation by directly inhibiting myogenin expression

    Institute of Scientific and Technical Information of China (English)

    Yu Oikawa; Reiko Omori; Tomonori Nishii; Yasumasa Ishida; Masashi Kawaichi; Eishou Matsuda

    2011-01-01

    Postnatal growth and regeneration of skeletal muscle are carried out mainly by satellite cells,which,upon stimulation,begin to express myogenin (Myog),the critical determinant of myogenic differentiation.DNA methylation status has been associated with the expression of Myog,but the causative mechanism remains almost unknown.Here,we report that the level of CIBZ,a methyI-CpG-binding protein,decreases upon myogenic differentiation of satellitederived C2C12 cells,and during skeletal muscle regeneration in mice.We present data showing that the loss of CIBZ promotes myogenic differentiation,whereas exogenous expression of CIBZ impairs it,in cultured cells.CIBZ binds to a Myog promoter-proximal region and inhibits Myog transcription in a methylation-dependent manner.These data suggest that the suppression of myogenic differentiation by CIBZ is dependent,at least in part,on the regulation of Myog.Our data show that the methylation status of this proximal Myog promoter inversely correlates with Myog transcription in cells and tissues,and during postnatal growth of skeletal muscle.Notably,induction of Myog transcription by CIBZ suppression is independent of the demethylation of CpG sites in the Myog promoter.These observations provide the first reported molecular mechanism illustrating how Myog transcription is coordinately regulated by a methyI-CpG-binding protein and the methylation status of the proximal Myog promoter.

  7. TET2 promoter DNA methylation and expression analysis in pediatric B-cell acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Ewa Musialik

    2014-03-01

    Full Text Available TET2 is a novel tumor suppressor gene involved in several hematological malignancies of myeloid and lymphoid origin. Besides loss-of-function mutations and deletions, hypermethylation of the CpG island at the TET2 promoter was found in human cancer. Previous analysis revealed no TET2 mutations in acute lymphoblastic leukemia (ALL. Since the TET2 promoter methylation status in pediatric ALL has not been reported, the aim of the present study was to determine if promoter hypermethylation may be a mechanism of TET2 inactivation in a group of pediatric ALL cases. Methylation of TET2 promoter region in one (1/45 ALL B-common patient was detected by methylation specific polymerase chain reaction (PCR and subsequently analyzed by bisulfite sequencing. We found no correlation between promoter methylation and gene expression, measured by quantitative reverse transcriptase-PCR, however the level of TET2 expression in ALL group was significantly decreased compared to children’s normal peripheral blood mononuclear cells and isolated B-cells. TET2 promoter hypermethylation seems to have limited clinical relevance in childhood B-cell ALL due to its low frequency.

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

    Science.gov (United States)

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

    2015-10-01

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

  9. A crossed molecular beam and ab initio investigation of the exclusive methyl loss pathway in the gas phase reaction of boron monoxide (BO; X2Σ+) with dimethylacetylene (CH3CCCH3; X1A(1g)).

    Science.gov (United States)

    Kaiser, Ralf I; Maity, Surajit; Dangi, Beni B; Su, Yuan-Siang; Sun, B J; Chang, Agnes H H

    2014-01-21

    The crossed molecular beam reaction of boron monoxide ((11)BO; X(2)Σ(+)) with dimethylacetylene (CH3CCCH3; X(1)A(1g)) was investigated at a collision energy of 23.9 ± 1.5 kJ mol(-1). The scattering dynamics were suggested to be indirect (complex forming reaction) and were initiated by the addition of (11)BO(X(2)Σ(+)) with the radical center located at the boron atom to the π electron density at the acetylenic carbon-carbon triple bond without entrance barrier leading to cis-trans(11)BOC4H6 doublet radical intermediates. cis-(11)BOC4H6 underwent cis-trans isomerization followed by unimolecular decomposition via a methyl group (CH3) loss forming 1-propynyl boron monoxide (CH3CC(11)BO) in an overall exoergic reaction (experimental: -91 ± 22 kJ mol(-1); theoretical: -105 ± 9 kJ mol(-1); NIST: -104 ± 12 kJ mol(-1)) via a tight exit transition state; trans-(11)BOC4H6 was found to lose a methyl group instantaneously. Neither atomic nor molecular hydrogen loss pathways were detectable. The experimental finding of an exclusive methyl loss pathway gains full support from our computational study predicting a methyl group versus atomic hydrogen loss branching ratio of 99.99% to 0.01% forming 1-propynyl boron monoxide (CH3CC(11)BO) and 1-methyl-propadienyl boron monoxide (CH3((11)BO)CCCH2), respectively.

  10. Emerging Molecular and Biological Functions of MBD2, a Reader of DNA Methylation

    Directory of Open Access Journals (Sweden)

    Kathleen H Wood

    2016-05-01

    Full Text Available DNA methylation is an epigenetic mark that is essential for many biological processes and is linked to diseases such as cancer. Methylation is usually associated with transcriptional silencing, but new research has challenged this model. Both transcriptional activation and repression have recently been found to be associated with DNA methylation in a context-specific manner. How DNA methylation patterns are interpreted into different functional output remains poorly understood. One mechanism involves the protein ‘readers’ of methylation, which includes the methyl-CpG binding domain (MBD family of proteins. This review examines the molecular and biological functions of MBD2, which binds to CpG methylation and is an integral part of the nucleosome remodeling and histone deacetylation (NuRD complex. MBD2 has been linked to immune system function and tumorigenesis, yet little is known about its functions in vivo. Recent studies have found the MBD2 protein is ubiquitously expressed, with relatively high levels in the lung, liver and colon. Mbd2 null mice surprisingly show relatively mild phenotypes compared to mice with loss of function of other MBD proteins. This evidence has previously been interpreted as functional redundancy between the MBD proteins. Here we examine and contextualize research that suggests MBD2 has unique properties and functions among the MBD proteins. These functions translate to recently described roles in the development and differentiation of multiple cell lineages, including pluripotent stem cells and various cell types of the immune system, as well as in tumorigenesis. We also consider possible models for the dynamic interactions between MBD2 and NuRD in different tissues in vivo. The functions of MBD2 may have direct therapeutic implications for several areas of human disease, including autoimmune conditions and cancer, in addition to providing insights into the actions of NuRD and chromatin regulation.

  11. Genetic Effects on DNA Methylation and Its Potential Relevance for Obesity in Mexican Americans

    Science.gov (United States)

    Carless, Melanie A.; Kulkarni, Hemant; Kos, Mark Z.; Charlesworth, Jac; Peralta, Juan M.; Göring, Harald H. H.; Curran, Joanne E.; Almasy, Laura; Dyer, Thomas D.; Comuzzie, Anthony G.; Mahaney, Michael C.; Blangero, John

    2013-01-01

    Several studies have identified effects of genetic variation on DNA methylation patterns and associated heritability, with research primarily focused on Caucasian individuals. In this paper, we examine the evidence for genetic effects on DNA methylation in a Mexican American cohort, a population burdened by a high prevalence of obesity. Using an Illumina-based platform and following stringent quality control procedures, we assessed a total of 395 CpG sites in peripheral blood samples obtained from 183 Mexican American individuals for evidence of heritability, proximal genetic regulation and association with age, sex and obesity measures (i.e. waist circumference and body mass index). We identified 16 CpG sites (∼4%) that were significantly heritable after Bonferroni correction for multiple testing and 27 CpG sites (∼6.9%) that showed evidence of genetic effects. Six CpG sites (∼2%) were associated with age, primarily exhibiting positive relationships, including CpG sites in two genes that have been implicated in previous genome-wide methylation studies of age (FZD9 and MYOD1). In addition, we identified significant associations between three CpG sites (∼1%) and sex, including DNA methylation in CASP6, a gene that may respond to estradiol treatment, and in HSD17B12, which encodes a sex steroid hormone. Although we did not identify any significant associations between DNA methylation and the obesity measures, several nominally significant results were observed in genes related to adipogenesis, obesity, energy homeostasis and glucose homeostasis (ARHGAP9, CDKN2A, FRZB, HOXA5, JAK3, MEST, NPY, PEG3 and SMARCB1). In conclusion, we were able to replicate several findings from previous studies in our Mexican American cohort, supporting an important role for genetic effects on DNA methylation. In addition, we found a significant influence of age and sex on DNA methylation, and report on trend-level, novel associations between DNA methylation and measures of obesity

  12. DNA Methylation and Temperature Stress in an Antarctic Polychaete, Spiophanes tcherniai

    Directory of Open Access Journals (Sweden)

    Adam G. Marsh

    2014-05-01

    Full Text Available Epigenetic modifications of DNA and histones are a primary mechanism by which gene expression activities may be modified in response to environmental stimuli. Here we characterize patterns of methyl-cytosine composition in the marine polychaete emph{Spiophanes tcherniai} from McMurdo Sound, Antarctica. We cultured adult worms at two temperatures, -1.5 C (ambient control and +4 C (warm treatment, for four weeks. We observed a rapid capacity for emph{S. tcherniai} organismal respiration rates and underlying catalytic rates of citrate synthase to acclimate at +4 C and return to control levels. We profiled changes in the methylation states of CpG sites in these treatments using an NGS strategy to computationally reconstruct and quantify methylation status across the genome. In our analysis we recovered 120,000 CpG sites in assembled contigs from both treatments. Of those, we were able to align 28,000 CpG sites in common between the two sample groups. In comparing these aligned sites between treatments, only 3,000 (11% evidenced a change in methylation state, but over 85% of changes involved a gain of a 5-methyl group on a CpG site (net increase in methyation. The ability to score CpG sites as partially methylated among gDNA copies in a sample opens up a new avenue for assessing DNA methylation responses to changing environments. By quantitatively distinguishing a ``mixed'' population of copies of one CpG site, we can begin to identify dynamic, non-binary, continuous-response reactions in DNA methylation intensity or density that previously may have been overlooked as noise.

  13. Genetic effects on DNA methylation and its potential relevance for obesity in Mexican Americans.

    Directory of Open Access Journals (Sweden)

    Melanie A Carless

    Full Text Available Several studies have identified effects of genetic variation on DNA methylation patterns and associated heritability, with research primarily focused on Caucasian individuals. In this paper, we examine the evidence for genetic effects on DNA methylation in a Mexican American cohort, a population burdened by a high prevalence of obesity. Using an Illumina-based platform and following stringent quality control procedures, we assessed a total of 395 CpG sites in peripheral blood samples obtained from 183 Mexican American individuals for evidence of heritability, proximal genetic regulation and association with age, sex and obesity measures (i.e. waist circumference and body mass index. We identified 16 CpG sites (~4% that were significantly heritable after Bonferroni correction for multiple testing and 27 CpG sites (~6.9% that showed evidence of genetic effects. Six CpG sites (~2% were associated with age, primarily exhibiting positive relationships, including CpG sites in two genes that have been implicated in previous genome-wide methylation studies of age (FZD9 and MYOD1. In addition, we identified significant associations between three CpG sites (~1% and sex, including DNA methylation in CASP6, a gene that may respond to estradiol treatment, and in HSD17B12, which encodes a sex steroid hormone. Although we did not identify any significant associations between DNA methylation and the obesity measures, several nominally significant results were observed in genes related to adipogenesis, obesity, energy homeostasis and glucose homeostasis (ARHGAP9, CDKN2A, FRZB, HOXA5, JAK3, MEST, NPY, PEG3 and SMARCB1. In conclusion, we were able to replicate several findings from previous studies in our Mexican American cohort, supporting an important role for genetic effects on DNA methylation. In addition, we found a significant influence of age and sex on DNA methylation, and report on trend-level, novel associations between DNA methylation and measures of

  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. Methyl Gallate Inhibits Osteoclast Formation and Function by Suppressing Akt and Btk-PLCγ2-Ca2+ Signaling and Prevents Lipopolysaccharide-Induced Bone Loss

    Science.gov (United States)

    Baek, Jong Min; Kim, Ju-Young; Lee, Chang Hoon; Yoon, Kwon-Ha; Lee, Myeung Su

    2017-01-01

    In the field of bone research, various natural derivatives have emerged as candidates for osteoporosis treatment by targeting abnormally elevated osteoclastic activity. Methyl gallate, a plant-derived phenolic compound, is known to have numerous pharmacological effects against inflammation, oxidation, and cancer. Our purpose was to explore the relation between methyl gallate and bone metabolism. Herein, we performed screening using methyl gallate by tartrate resistant acid phosphatase (TRAP) staining and revealed intracellular mechanisms responsible for methyl gallate-mediated regulation of osteoclastogenesis by Western blotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). Furthermore, we assessed the effects of methyl gallate on the characteristics of mature osteoclasts. We found that methyl gallate significantly suppressed osteoclast formation through Akt and Btk-PLCγ2-Ca2+ signaling. The blockade of these pathways was confirmed through transduction of cells with a CA-Akt retrovirus and evaluation of Ca2+ influx intensity (staining with Fluo-3/AM). Indeed, methyl gallate downregulated the formation of actin ring-positive osteoclasts and resorption pit areas. In agreement with in vitro results, we found that administration of methyl gallate restored osteoporotic phenotype stimulated by acute systemic injection of lipopolysaccharide in vivo according to micro-computed tomography and histological analysis. Our data strongly indicate that methyl gallate may be useful for the development of a plant-based antiosteoporotic agent. PMID:28272351

  16. The Role of Histone Deacetylase and DNA Methylation in Estrogen a Expression in Breast Cancer

    Science.gov (United States)

    2001-06-01

    cancer cells is not a lethal event. Further, total genomic AP-2, E2F and NFiJB to DNA (Hermann & Doerfler 1991). methylated CpG content was-reduced by...MT & Galloway DA 1998 status of CpG islands. PNAS 93 9821-9826. Inactivation of p16 in human mammary epithelial cells by CpG Hermann R & Doerfler W...Development and Biology 10 215-225. PNAS 91 11733-11737. Muller K, Heller H & Doerfler W 2001 Foreign DNA integration: Lei H, Oh SP, Okano M, Juttermann R, Goss

  17. 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.

  18. p16 promoter methylation in Pb2+ -exposed individuals.

    Science.gov (United States)

    Kovatsi, Leda; Leda, Kovatsi; Georgiou, Elisavet; Elisavet, Georgiou; Ioannou, Antrea; Antrea, Ioannou; Haitoglou, Costas; Costas, Haitoglou; Tzimagiorgis, George; George, Tzimagiorgis; Tsoukali, Helen; Helen, Tsoukali; Kouidou, Sofia; Sofia, Kouidou

    2010-02-01

    One of the principle symptoms of lead poisoning is the development of neurological disorders. Neuronal response is closely related to DNA methylation changes. Aim. In this study, we estimated p16 methylation in nine individuals exposed to lead using methylation-specific polymerase chain reaction followed by analysis of the methylated cytosine content of the product by thermal denaturation. We found that, based on lead blood concentration, lead-exposed individuals were divided into two groups. Among highly exposed individuals (blood Pb(2+) concentration = 51-100 microg/dL), we observed complete CpG methylation, whereas for low Pb(2+) concentrations (blood Pb(2+) concentration = 6-11 microg/dL), we observed partial methylation. Our results show that among lead-overexposed individuals, p16 methylation is frequent and extensive, and suggest that DNA methylation could be involved in the mechanism by which lead induces neurotoxicity.

  19. CTCF Haploinsufficiency Destabilizes DNA Methylation and Predisposes to Cancer

    Directory of Open Access Journals (Sweden)

    Christopher J. Kemp

    2014-05-01

    Full Text Available Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf+/− tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/− tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.

  20. CpG oligodeoxyribonucleotides protect mice from Burholderia pseudomallei but not Francisella tularensis Schu 54 aersols

    Science.gov (United States)

    2010-01-01

    CpG ODN 10103 performs comparably in mice to CpG ODN 7909 (5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’), which was previously reported to protect the BALB/c mice...SHORT REPORT Open Access CpG oligodeoxyribonucleotides protect mice from Burkholderia pseudomallei but not Francisella tularensis Schu S4 aerosols...that CpG oligodeoxyribonucleotides (ODN) protect mice from various bacterial pathogens, including Burkholderia pseudomallei and Francisella tularensis

  1. 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.

  2. Arylesterase activity is associated with antioxidant intake and paraoxonase-1 (PON1) gene methylation in metabolic syndrome patients following an energy restricted diet.

    Science.gov (United States)

    de la Iglesia, Rocio; Mansego, Maria L; Sánchez-Muniz, Francisco J; Zulet, M Angeles; Martinez, J Alfredo

    2014-01-01

    The arylesterase (ARE) activity linked to the paraoxonase-1 (PON1) gene is known to protect lipoproteins from oxidation and provide defense against metabolic syndrome (MetS) and cardiovascular diseases. The epigenetic regulation of enzymatic activities is gaining importance nowadays. This research aimed to assess the potential relationships between the ARE activity with the methylation levels of the PON1 gene transcriptional regulatory region, anthropometrics, biochemical markers and antioxidant dietary components. Forty-seven subjects (47 ± 10 y.o; BMI 36.2 ± 3.8 kg/m(2); 46.8 % female) with MetS features, who followed a six-month energy-restricted dietary weight-loss intervention, were included in this study (www.clinicaltrials.gov; NCT01087086). Anthropometric, biochemical, enzymatic and dietary data were assessed using validated procedures. PON1 transcriptional regulatory region methylation was analyzed by a microarray technical approach. Volunteers reduced ARE activity in parallel with body weight (p = 0.005), BMI (p = 0.006), total fat mass (p = 0.020), diastolic blood pressure (p = 0.018), mean blood pressure (p = 0.022) and triglycerides (p = 0.014). Methylation levels of some CpG sites of the PON1 gene correlated negatively with ARE activity (p < 0.05). Interestingly, dietary vitamin C (p = 0.001), tocopherols (p = 0.009) and lycopene (p = 0.038) were positively associated with ARE activity and showed an inverse correlation (p = 0.004, p = 0.029 and p = 0.021, respectively) with the methylation of some selected CpG sites of the PON1 gene. In conclusion, ARE activity decreased in parallel with MetS-related markers associated to the energy restriction, while dietary antioxidants might enhance the ARE activity by lowering the PON1 gene methylation in patients with MetS features.

  3. Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-7) gene promoter and its effect on IFN-7 expression in chronic hepatitis B. Method The authors recruited 30 patients with UBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequeneing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expression of IFN-γ was analyzed by real-time RT-PCR and ELISA. HBV DNA in PBMCs was detected by nested PCR. Results The methylation level at CpG site -55 in the IFN-γ gene promoter was significantly increased, resulting in subsequent down-regulation of the expression of this cytoldne in CHB. The methylation level at CpG site -55 was significantly higher in HBeAg-positive patients than in HBeAg-negative ones (P<0.01) and was also significantly higher in PBMCs from HBV DNA-positive patients than from HBV DNA-negative ones (P<0.01) ; the methylation level at CpG site -55 was positively correlated with the amount of HBV DNA in serum (P<0.01). Oonclusion IFN-γ gene expression appears to be regulated by methylation of the IFN-γ gene promoter in CHB; the methylation level at CpG site -55 is associated with HBV infection.

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

    Science.gov (United States)

    Xie, Hao; Tubbs, Raymond; Yang, Bin

    2015-01-01

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

  5. DNA Methylation Pyrosequencing Assay Is Applicable for the Assessment of Epigenetic Active Environmental or Clinical Relevant Chemicals

    Directory of Open Access Journals (Sweden)

    Ana-Maria Florea

    2013-01-01

    Full Text Available Exposure of cells and organisms to stressors might result in epigenetic changes. Here it is shown that investigation of DNA methylation using pyrosequencing is an alternative for in vitro and in vivo toxicological testing of epigenetic effects induced by chemicals and drugs. An in vitro evaluation of global and CpG site specific DNA methylation upon treatment of cells with chemicals/drugs is shown. Bisulfite genomic sequencing of methylation controls showed high methylation of LINE1 in methylation positive control and low methylation in the negative controls. The CpG sites within the LINE1 element are methylated at different levels. In vitro cell cultures show a methylation level ranging from 56% to 49%. Cultures of drug resistant tumor cells show significant hypomethylation as compared with the originating nonresistant tumor cells. The in vitro testing of epigenetically active chemicals (5-methyl-2’-deoxycytidine and trichostatin A revealed a significant change of LINE1 methylation status upon treatment, while specific CpG sites were more prone to demethylation than others (focal methylation. In conclusion, DNA methylation using pyrosequencing might be used not only for testing epigenetic toxins/drugs but also in risk assessment of drugs, food, and environmental relevant pollutants.

  6. Methyl-CpG binding proteins in the nervous system

    Institute of Scientific and Technical Information of China (English)

    Guoping FAN; Leah HUTNICK

    2005-01-01

    Classical methyl-CpG binding proteins contain the conserved DNA binding motif methyl-cytosine binding domain (MBD), which preferentially binds to methylated CpG dinucleotides. These proteins serve as transcriptional repressors,mediating gene silencing via DNA cytosine methylation. Mutations in methyl-CpG binding protein 2 (MeCP2) have been linked to the human mental retardation disorder Rett syndrome, suggesting an important role for methyl-CpG binding proteins in brain development and function. This mini-review summarizes the recent advances in studying the diverse functions of MeCP2 as a prototype for other methyl-CpG binding proteins in the development and function of the vertebrate nervous system.

  7. Effect of amino groups of mesoporous silica nanoparticles on CpG oligodexynucleotide delivery

    Science.gov (United States)

    Xu, Yi; Claiden, Peter; Zhu, Yufang; Morita, Hiromi; Hanagata, Nobutaka

    2015-08-01

    In this study, we proposed to modify mesoporous silica nanoparticles (MSNs) with 3-aminopropyltriethoxysilane (NH2-TES), aminoethylaminopropyltriethoxysilane (2NH2-TES) and 3-[2-(2-aminoethylamino)ethylamino] propyl-trimethoxysilane (3NH2-TES) for binding of cytosine-phosphate-guanosine oligodexynucleotides (CpG ODN), and investigated the effect of different amino groups of MSNs on the CpG ODN delivery. Serum stability, in vitro cytotoxicity, and cytokine interleukin-6 (IL-6) induction by MSN-NH2/CpG, MSN-2NH2/CpG and MSN-3NH2/CpG complexes were investigated in detail. The results showed that three kinds of aminated-MSN-based CpG ODN delivery systems had no cytotoxicity to RAW264.7 cells, and binding of CpG ODN to MSN-NH2, MSN-2NH2 and MSN-3NH2 nanoparticles enhanced the serum stability of CpG ODN due to protection by the nanoparticles. However, three aminated MSN-based CpG ODN delivery systems exhibited different CpG ODN delivery efficiency, and MSN-NH2/CpG complexes had the highest ability to induce IL-6 secretion.

  8. Conserved DNA methylation patterns in healthy blood cells and extensive changes in leukemia measured by a new quantitative technique

    OpenAIRE

    Jelinek, Jaroslav; Liang, Shoudan; Lu, Yue; He, Rong; Ramagli, Louis S.; Shpall, Elizabeth J; Estecio, Marcos R.H.; Issa, Jean-Pierre J.

    2012-01-01

    Genome wide analysis of DNA methylation provides important information in a variety of diseases, including cancer. Here, we describe a simple method, Digital Restriction Enzyme Analysis of Methylation (DREAM), based on next generation sequencing analysis of methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes. DREAM provides information on 150,000 unique CpG sites, of which 39,000 are in CpG islands and 30,000 are at transcription start sit...

  9. Comprehensive DNA methylation analysis of human neuroblastoma cells treated with blonanserin.

    Science.gov (United States)

    Murata, Yui; Nishioka, Masaki; Bundo, Miki; Sunaga, Fumiko; Kasai, Kiyoto; Iwamoto, Kazuya

    2014-03-20

    Blonanserin is a second-generation antipsychotic drug for schizophrenia. The pharmacological actions of blonanserin are shown to be the antagonism of dopamine receptor 2 and serotonin receptors. However, its molecular mechanisms in brain cells have not been fully characterized. Accumulating evidence suggests that antipsychotic drugs and mood stabilizers show epigenetic effects on a wide range of genes in animal and cellular models. We performed genome-wide DNA methylation analysis targeting 479,814 CpG sites of cultured human neuroblastoma cells administered with blonanserin. We found that 3,057 CpG sites showed statistically significant changes in DNA methylation at two different doses of blonanserin (1.36 nM and 13.6 nM). These included hypermethylated CpG sites that were enriched in genes related to axonogenesis and cell morphogenesis involved in neuron differentiation. We also showed that the global effect on DNA methylome depends on the concentration of the drug. With a high dose of blonanserin, the overall methylation levels across all CpG sites significantly increased. These increases in DNA methylation were prominent in the CpG sites distant from promoter regions. We further examined DNA methylation changes in specific genes implicated for the actions of antipsychotic drugs, such as the dopamine receptor 2 (DRD2) gene and the serotonin receptor 2A (HTR2A) gene. We observed that CpG sites that were located within DRD2 and HTR2A genes were significantly hypermethylated by blonanserin. The DNA methylation changes induced by the treatment with blonanserin will be useful for understanding its pharmacological actions at the cellular level. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Methylation plotter: a web tool for dynamic visualization of DNA methylation data.

    Science.gov (United States)

    Mallona, Izaskun; Díez-Villanueva, Anna; Peinado, Miguel A

    2014-01-01

    Methylation plotter is a Web tool that allows the visualization of methylation data in a user-friendly manner and with publication-ready quality. The user is asked to introduce a file containing the methylation status of a genomic region. This file can contain up to 100 samples and 100 CpGs. Optionally, the user can assign a group for each sample (i.e. whether a sample is a tumoral or normal tissue). After the data upload, the tool produces different graphical representations of the results following the most commonly used styles to display this type of data. They include an interactive plot that summarizes the status of every CpG site and for every sample in lollipop or grid styles. Methylation values ranging from 0 (unmethylated) to 1 (fully methylated) are represented using a gray color gradient. A practical feature of the tool allows the user to choose from different types of arrangement of the samples in the display: for instance, sorting by overall methylation level, by group, by unsupervised clustering or just following the order in which data were entered. In addition to the detailed plot, Methylation plotter produces a methylation profile plot that summarizes the status of the scrutinized region, a boxplot that sums up the differences between groups (if any) and a dendrogram that classifies the data by unsupervised clustering. Coupled with this analysis, descriptive statistics and testing for differences at both CpG and group levels are provided. The implementation is based in R/shiny, providing a highly dynamic user interface that generates quality graphics without the need of writing R code. Methylation plotter is freely available at http://gattaca.imppc.org:3838/methylation_plotter/.

  11. 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.

  12. 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.

  13. Colorectal Cancer "Methylator Phenotype": Fact or Artifact?

    Directory of Open Access Journals (Sweden)

    Charles Anacleto

    2005-04-01

    Full Text Available It has been proposed that human colorectal tumors can be classified into two groups: one in which methylation is rare, and another with methylation of several loci associated with a "CpG island methylated phenotype (CIMP," characterized by preferential proximal location in the colon, but otherwise poorly defined. There is considerable overlap between this putative methylator phenotype and the well-known mutator phenotype associated with microsatellite instability (MSI. We have examined hypermethylation of the promoter region of five genes (DAPK, MGMT, hMLH1, p16INK4a, and p14ARF in 106 primary colorectal cancers. A graph depicting the frequency of methylated loci in the series of tumors showed a continuous, monotonically decreasing distribution quite different from the previously claimed discontinuity. We observed a significant association between the presence of three or more methylated loci and the proximal location of the tumors. However, if we remove from analysis the tumors with hMLH1 methylation or those with MSI, the significance vanishes, suggesting that the association between multiple methylations and proximal location was indirect due to the correlation with MSI. Thus, our data do not support the independent existence of the so-called methylator phenotype and suggest that it rather may represent a statistical artifact caused by confounding of associations.

  14. Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

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    Down Thomas A

    2010-09-01

    Full Text Available Abstract Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS" but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq not to be biological transcription factor binding sites ("empirical TFBS". We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Identification of 5 novel genes methylated in breast and other epithelial cancers.

    Science.gov (United States)

    Hill, Victoria K; Hesson, Luke B; Dansranjavin, Temuujin; Dallol, Ashraf; Bieche, Ivan; Vacher, Sophie; Tommasi, Stella; Dobbins, Timothy; Gentle, Dean; Euhus, David; Lewis, Cheryl; Dammann, Reinhard; Ward, Robyn L; Minna, John; Maher, Eammon R; Pfeifer, Gerd P; Latif, Farida

    2010-03-05

    There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer. Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers. The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.

  17. The potential role of DNA methylation in the pathogenesis of abdominal aortic aneurysm.

    Science.gov (United States)

    Toghill, Bradley J; Saratzis, Athanasios; Harrison, Seamus C; Verissimo, Ana R; Mallon, Eamonn B; Bown, Matthew J

    2015-07-01

    Abdominal aortic aneurysm (AAA) is characterised by the chronic degradation and gradual, irreversible dilation of the abdominal aorta. Smoking, genetics, male sex and increased age are major factors associated with developing AAA. Rupture contributes to around 2% of deaths in all Caucasians over 65, and there is no pharmaco-therapeutic treatment. Methylation is an epigenetic modification to DNA, where a methyl group is added to a cytosine base 5' to a guanine (CpG dinucleotide). Methylation patterns are long term, inherited signatures that can induce changes in gene transcription, and can be affected by both genetic and environmental factors. Methylation changes are involved in hypertension and atherosclerosis, both of which are risk factors of, and often coexist with AAA. Extra-cellular matrix degradation and inflammation, both important pathological hallmarks of AAA, are also promoted by changes in CpG methylation in other diseases. Additionally, the adverse effects of smoking and ageing take place largely through epigenetic manipulation of the genome. Every factor associated with AAA appears to be associated with DNA methylation, yet no direct evidence confirms this. Future work to identify a link between global methylation and AAA, and differentially methylated regions may reveal valuable insight. The identification of a common epigenetic switching process may also signify a promising future for AAA pharmaco-therapeutic strategies. Epigenetic therapies are being designed to target pathogenic CpG methylation changes in other diseases, and it is feasible that these therapies may also be applicable to AAA in the future.

  18. Dietary Methionine Affect Meat Qulity and Myostatin Gene Exon 1 Region Methylation in Skeletal Muscle Tissues of Broilers

    Institute of Scientific and Technical Information of China (English)

    LIU Guo-qing; ZONG Kai; ZHANG Li-li; CAO Shu-qing

    2010-01-01

    Dietary amino acids imbalance will result in stunted broiler performance and deteriorated meat quality,which are involved in various biochemical cycles in vivo.In this study,the effects of dietary methionine on meat quality and methylation of myostatin exon 1 were investigated.Drip loss of the broilers fed with diet of high methionine levels(0.2%)increased from(6.3±0.1)%(control group)to(10.1±1.0)%,and the muscle shearing force increased from(22.8±1.9)N(control group)to(26.3±2.3)N.Moreover,many CpG sites were found at the myostatin exon 1 region(nucleotides 2360-2540 bp).To further understand the regulation of broiler myostatin expression,the methylation status of broiler myostatin exon 1 and its mRNA expression were analyzed.At the myostatin exon 1 region where CG enriches(nucleotides 2360-2540 bp),the percentages of methylation were 46 and 84% in low Met and high Met content groups after 55-d feeding,respectively.In skeletal muscle tissues,the exon 1 hypermethylation status of myostatin gene was found to be negatively correlated with the gene expression.These results suggested that methylation of this gene is a dynamic process,which plays a dominant role in regulating gene expression for development of individuals.

  19. Structure-dependent immunostimulatory effect of CpG oligodeoxynucleotides and their delivery system

    Directory of Open Access Journals (Sweden)

    Hanagata N

    2012-04-01

    Full Text Available Nobutaka HanagataNanotechnology Innovation Station, National Institute for Materials Science, Tsukuba, Ibaraki, and Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo, JapanAbstract: Unmethylated cytosine-phosphate-guanosine (CpG oligodeoxynucleotides (ODNs are recognized by Toll-like receptor 9 (TLR9 found in antigen-presenting cells and B cells and can activate the immune system. Using CpG ODNs as an adjuvant has been found to be effective for treating infectious diseases, cancers, and allergies. Because natural ODNs with only a phosphodiester backbone are easily degraded by nuclease (deoxyribonuclease [DNase] in serum, CpG ODNs with a phosphorothioate backbone have been studied for clinical application. CpG ODNs with a phosphorothioate backbone have raised concern regarding undesirable side effects; however, several CpG ODNs with only a phosphodiester backbone have been reported to be stable in serum and to show an immunostimulatory effect. In recent years, research has been conducted on delivery systems for CpG ODNs using nanoparticles (NPs. The advantages of NP-based delivery of CpG ODN include (1 it can protect CpG ODN from DNase, (2 it can retain CpG ODN inside the body for a long period of time, (3 it can improve the cellular uptake efficiency of CpG ODN, and (4 it can deliver CpG ODN to the target tissues. Because the target cells of CpG ODN are cells of the immune system and TLR9, the receptor of CpG ODN is localized in endolysosomes, CpG ODN delivery systems are required to have qualities different from other nucleic acid drugs such as antisense DNA and small interfering RNA. Studies until now have reported various NPs as carriers for CpG ODN delivery. This review presents DNase-resistant CpG ODNs with various structures and their immunostimulatory effects and also focuses on delivery systems of CpG ODNs that utilize NPs. Because CpG ODNs interact with TLR9 and activate both the innate and the adaptive immune

  20. The Dnmt3L ADD Domain Controls Cytosine Methylation Establishment during Spermatogenesis

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    Georgios Vlachogiannis

    2015-02-01

    Full Text Available A critical aspect of mammalian gametogenesis is the reprogramming of genomic DNA methylation. The catalytically inactive adaptor Dnmt3L is essential to ensuring this occurs correctly, but the mechanism by which it functions is unclear. Using gene targeting to engineer a single-amino-acid mutation, we show that the Dnmt3L histone H3 binding domain (ADD is necessary for spermatogenesis. Genome-wide single-base-resolution DNA methylome analysis of mutant germ cells revealed overall reductions in CG methylation at repetitive sequences and non-promoter CpG islands. Strikingly, we also observe an even more severe loss of non-CG methylation, suggesting an unexpected role for the ADD in this process. These epigenetic deficiencies were coupled with defects in spermatogonia, with mutant cells displaying marked changes in gene expression and reactivation of retrotransposons. Our results demonstrate that the Dnmt3L ADD is necessary for Dnmt3L function and full reproductive fitness.

  1. Methylation-Associated Gene Silencing of RARB in Areca Carcinogens Induced Mouse Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Zi-Lun Lai

    2014-01-01

    Full Text Available Regarding oral squamous cell carcinoma (OSCC development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO, or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis.

  2. Methylation-Associated Gene Silencing of RARB in Areca Carcinogens Induced Mouse Oral Squamous Cell Carcinoma

    Science.gov (United States)

    Tsou, Yung-An; Fan, Shin-Ru; Tsai, Ming-Hsui; Chen, Hsiao-Ling; Chang, Nai-Wen; Cheng, Ju-Chien

    2014-01-01

    Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis. PMID:25197641

  3. CPG Control for Biped Hopping Robot in Unpredictable Environment

    Institute of Scientific and Technical Information of China (English)

    Tingting Wang; Wei Guo; Mantian Li; Fusheng Zha; Lining Sun

    2012-01-01

    A CPG control mechanism is proposed for hopping motion control of biped robot in unpredictable environment.Based on analysis of robot motion and biological observation of animal's control mechanism,the motion control task is divided into two simple parts:motion sequence control and output force control.Inspired by a two-level CPG model,a two-level CPG control mechanism is constructed to coordinate the drivers of robot joint,while various feedback information are introduced into the control mechanism.Interneurons within the control mechanism are modeled to generate motion rhythm and pattern promptly for motion sequence control; motoneurons are modeled to control output forces of joint drivers in real time according to feedbacks.The control system can perceive changes caused by unknown perturbations and environment changes according to feedback information,and adapt to unpredictable environment by adjusting outputs of neurons.The control mechanism is applied to a biped hopping robot in unpredictable environment on simulation platform,and stable adaptive motions are obtained.

  4. Electrophysiological Representation of Scratching CPG Activity in the Cerebellum

    Science.gov (United States)

    Martínez-Silva, Lourdes; Manjarrez, Elias; Gutiérrez-Ospina, Gabriel; Quevedo, Jorge N.

    2014-01-01

    We analyzed the electrical activity of neuronal populations in the cerebellum and the lumbar spinal cord during fictive scratching in adult decerebrate cats before and after selective sections of the Spino-Reticulo Cerebellar Pathway (SRCP) and the Ventral-Spino Cerebellar Tract (VSCT). During fictive scratching, we found a conspicuous sinusoidal electrical activity, called Sinusoidal Cerebellar Potentials (SCPs), in the cerebellar vermis, which exhibited smaller amplitude in the paravermal and hemisphere cortices. There was also a significant spino-cerebellar coherence between these SCPs and the lumbar sinusoidal cord dorsum potentials (SCDPs). However, during spontaneous activity such spino-cerebellar coherence between spontaneous potentials recorded in the same regions decreased. We found that the section of the SRCP and the VSCT did not abolish the amplitude of the SCPs, suggesting that there are additional pathways conveying information from the spinal CPG to the cerebellum. This is the first evidence that the sinusoidal activity associated to the spinal CPG circuitry for scratching has a broad representation in the cerebellum beyond the sensory representation from hindlimbs previously described. Furthermore, the SCPs represent the global electrical activity of the spinal CPG for scratching in the cerebellar cortex. PMID:25350378

  5. Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-γ) gene promoter and its effect on IFN-γ expression in chronic hepatitis B. Method The authors recruited 30 patients with HBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequencing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expres...

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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

  9. 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

  10. Genome-wide methylation analyses in glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Rose K Lai

    Full Text Available Few studies had investigated genome-wide methylation in glioblastoma multiforme (GBM. Our goals were to study differential methylation across the genome in gene promoters using an array-based method, as well as repetitive elements using surrogate global methylation markers. The discovery sample set for this study consisted of 54 GBM from Columbia University and Case Western Reserve University, and 24 brain controls from the New York Brain Bank. We assembled a validation dataset using methylation data of 162 TCGA GBM and 140 brain controls from dbGAP. HumanMethylation27 Analysis Bead-Chips (Illumina were used to interrogate 26,486 informative CpG sites in both the discovery and validation datasets. Global methylation levels were assessed by analysis of L1 retrotransposon (LINE1, 5 methyl-deoxycytidine (5m-dC and 5 hydroxylmethyl-deoxycytidine (5hm-dC in the discovery dataset. We validated a total of 1548 CpG sites (1307 genes that were differentially methylated in GBM compared to controls. There were more than twice as many hypomethylated genes as hypermethylated ones. Both the discovery and validation datasets found 5 tumor methylation classes. Pathway analyses showed that the top ten pathways in hypomethylated genes were all related to functions of innate and acquired immunities. Among hypermethylated pathways, transcriptional regulatory network in embryonic stem cells was the most significant. In the study of global methylation markers, 5m-dC level was the best discriminant among methylation classes, whereas in survival analyses, high level of LINE1 methylation was an independent, favorable prognostic factor in the discovery dataset. Based on a pathway approach, hypermethylation in genes that control stem cell differentiation were significant, poor prognostic factors of overall survival in both the discovery and validation datasets. Approaches that targeted these methylated genes may be a future therapeutic goal.

  11. 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.

  12. DNA methylome profiling of human tissues identifies global and tissue-specific methylation patterns.

    Science.gov (United States)

    Lokk, Kaie; Modhukur, Vijayachitra; Rajashekar, Balaji; Märtens, Kaspar; Mägi, Reedik; Kolde, Raivo; Koltšina, Marina; Nilsson, Torbjörn K; Vilo, Jaak; Salumets, Andres; Tõnisson, Neeme

    2014-04-01

    DNA epigenetic modifications, such as methylation, are important regulators of tissue differentiation, contributing to processes of both development and cancer. Profiling the tissue-specific DNA methylome patterns will provide novel insights into normal and pathogenic mechanisms, as well as help in future epigenetic therapies. In this study, 17 somatic tissues from four autopsied humans were subjected to functional genome analysis using the Illumina Infinium HumanMethylation450 BeadChip, covering 486 428 CpG sites. Only 2% of the CpGs analyzed are hypermethylated in all 17 tissue specimens; these permanently methylated CpG sites are located predominantly in gene-body regions. In contrast, 15% of the CpGs are hypomethylated in all specimens and are primarily located in regions proximal to transcription start sites. A vast number of tissue-specific differentially methylated regions are identified and considered likely mediators of tissue-specific gene regulatory mechanisms since the hypomethylated regions are closely related to known functions of the corresponding tissue. Finally, a clear inverse correlation is observed between promoter methylation within CpG islands and gene expression data obtained from publicly available databases. This genome-wide methylation profiling study identified tissue-specific differentially methylated regions in 17 human somatic tissues. Many of the genes corresponding to these differentially methylated regions contribute to tissue-specific functions. Future studies may use these data as a reference to identify markers of perturbed differentiation and disease-related pathogenic mechanisms.

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

    Science.gov (United States)

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

    2015-05-01

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

  14. Differential DNA Methylation in Relation to Age and Health Risks of Obesity

    Directory of Open Access Journals (Sweden)

    María Luisa Mansego

    2015-07-01

    Full Text Available The aim of this study was to evaluate whether genome-wide levels of DNA methylation are associated with age and the health risks of obesity (HRO; defined according to BMI categories as “Low HRO” (overweight and class 1 obesity versus “High HRO” (class 2 and class 3 obesity. Anthropometric measurements were assessed in a subsample of 48 volunteers from the Metabolic Syndrome Reduction in Navarra (RESMENA study and 24 women from another independent study, Effects of Lipoic Acid and Eicosapentaenoic Acid in Human Obesity (OBEPALIP study. In the pooled population; the methylation levels of 55 CpG sites were significantly associated with age after Benjamini-Hochberg correction. In addition, DNA methylation of three CpG sites located in ELOVL2; HOXC4 and PI4KB were further negatively associated with their mRNA levels. Although no differentially methylated CpG sites were identified in relation to HRO after multiple testing correction; several nominally significant CpG sites were identified in genes related to insulin signaling; energy and lipid metabolism. Moreover, statistically significant associations between BMI or mRNA levels and two HRO-related CpG sites located in GPR133 and ITGB5 are reported. As a conclusion, these findings from two Spanish cohorts add knowledge about the important role of DNA methylation in the age-related regulation of gene expression. In addition; a relevant influence of age on DNA methylation in white blood cells was found, as well as, on a trend level, novel associations between DNA methylation and obesity.

  15. Loss of CDH1 (E-cadherin) expression is associated with infiltrative tumour growth and lymph node metastasis.

    Science.gov (United States)

    Kim, Sun A; Inamura, Kentaro; Yamauchi, Mai; Nishihara, Reiko; Mima, Kosuke; Sukawa, Yasutaka; Li, Tingting; Yasunari, Mika; Morikawa, Teppei; Fitzgerald, Kathryn C; Fuchs, Charles S; Wu, Kana; Chan, Andrew T; Zhang, Xuehong; Ogino, Shuji; Qian, Zhi Rong

    2016-01-19

    Loss of CDH1 (E-cadherin) expression in cancer cells may promote cell migration and invasion. Therefore, we hypothesised that loss of CDH1 expression in colorectal carcinoma might be associated with aggressive features and clinical outcome. Utilising molecular pathological epidemiology database of 689 rectal and colon cancer cases in the Nurses' Health Study and the Health Professionals Follow-up Study, we assessed tumour CDH1 expression by immunohistochemistry. Multivariate logistic regression analysis was conducted to assess association of CDH1 loss with tumour growth pattern (expansile-intermediate vs infiltrative) and lymph node metastasis and distant metastasis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and PIK3CA, BRAF and KRAS mutations. Mortality according to CDH1 status was assessed using Cox proportional hazards model. Loss of tumour CDH1 expression was observed in 356 cases (52%), and associated with infiltrative tumour growth pattern (odds ratio (OR), 2.02; 95% confidence interval (CI), 1.23-3.34; P=0.006) and higher pN stage (OR, 1.73; 95% CI, 1.23-2.43; P=0.001). Tumour CDH1 expression was not significantly associated with distant metastasis or prognosis. Loss of CDH1 expression in colorectal cancer is associated with infiltrative tumour growth pattern and lymph node metastasis.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Silja Vorjohann

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

  19. Characterization of a CpG island at the 3{prime} end of the myotonic dystrophy protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Boucher, C.A.; Carey, N.; Rahman, S. [Charing Cross and Westminster Medical School, London (United Kingdom)] [and others

    1994-09-01

    Myotonic dystrophy (DM) is a multisystemic disorder characterized by a very variable presentation. The mutation underlying DM is an expansion of a CTG repeat in the 3{prime} untranslated region of the DM protein kinase (DMPK). However it is not yet clear how this expansion leads to the observed phenotype. Published data on the effects of the expansion on the expression of DMPK are equivocal, and do not yet explain how changes in expression of this gene lead to the broad range of symptoms found in this disorder. The region of chromosome 19 in which DMPK lies is extremely gene-rich, and it has been postulated that the expression of other genes may also be affected by the CTG expansion. We present sequencing and methylation data which show the full extent of a large CpG island which covers the 3{prime} end of DMPK (encompassing the CTG repeat) and extends downstream beyond this. As CpG islands are mostly found at the 5{prime} end of genes, this may indicate that there is another gene immediately downstream of the repeat. We are currently using a single-copy probe which we have isolated from this region to screen cDNA libraries in an attempt to find such a gene.

  20. Enhancement of the Anthrax AVA Vaccine with CpG ODN’s

    Science.gov (United States)

    2005-08-28

    NUMBER OF PAGES 20. LIMITATION OF ABSTRACT UL - 28-Aug-2005 Final Report on ACCELERATE ANTHRAX: CpG 7909 Vaccine Adjuvant Program Report Title...Vaccine Adsorbed (BioThrax?) Combined with CPG 7909 in Normal Volunteers” was completed and presented at the 2005 Interscience Conference on Antimicrobial...Agents and Chemotherapy in a poster entitled “Marked Enhancement Of Antibody Response To Anthrax Vaccine Adsorbed With CPG 7909 In Healthy

  1. The impact of P2Y12 promoter DNA methylation on the recurrence of ischemic events in Chinese patients with ischemic cerebrovascular disease

    Science.gov (United States)

    Li, Xin-Gang; Ma, Ning; Wang, Bo; Li, Xiao-Qing; Mei, Sheng-Hui; Zhao, Kun; Wang, Yong-Jun; Li, Wei; Zhao, Zhi-Gang; Sun, Shu-Sen; Miao, Zhong-Rong

    2016-01-01

    The primary mechanism of clopidogrel resistance is still unclear. We aimed to investigate whether the methylation status of the P2Y12 promoter has effects on platelet function and clinical ischemic events. Patients with ischemic cerebrovascular disease were enrolled into our study. Venous blood samples were drawn for thrombelastograpy (TEG) and active metabolite assay. Patients were divided into a case- or control-group based on the occurrence of ischemic events during a one year follow-up. Two TEG parameters between the case and control groups were statistically significant [ADP inhibition rate (ADP%): P = 0.018; ADP-induced platelet-fibrin clot strength (MAADP): P = 0.030]. The concentrations of clopidogrel active metabolite had no significant difference (P = 0.281). Sixteen CpG dinucleotides on P2Y12 promoter were tested. Three CpG sites (CpG11 and CpG12 + 13) showed lower methylation status, which correlated with a strong association with increased risk of clinical events. Changes of MAADP and ADP% were also associated with methylation levels of CpG 11 and CpG 12 + 13. Hypomethylation of the P2Y12 promoter is associated with a higher platelet reactivity and increased risk of ischemic events in our patients. Methylation analysis of peripheral blood samples might be a novel molecular marker to help early identification of patients at high risk for clinical ischemic events. PMID:27686864

  2. Epigenetic loss of the PIWI/piRNA machinery in human testicular tumorigenesis.

    Science.gov (United States)

    Ferreira, Humberto J; Heyn, Holger; Garcia del Muro, Xavier; Vidal, August; Larriba, Sara; Muñoz, Clara; Villanueva, Alberto; Esteller, Manel

    2014-01-01

    Although most cancer research has focused in mRNA, non-coding RNAs are also an essential player in tumorigenesis. In addition to the well-recognized microRNAs, recent studies have also shown that epigenetic silencing by CpG island hypermethylation of other classes of non-coding RNAs, such as transcribed ultraconserved regions (T-UCRs) or small nucleolar RNAs (snoRNAs), also occur in human neoplasia. Herein we have studied the putative existence of epigenetic aberrations in the activity of PIWI proteins, an Argonaute family protein subclass, and the small regulatory PIWI-interacting RNAs (piRNAs) in testicular cancer, as the PIWI/piRNA pathway plays a critical role in male germline development. We have observed the existence of promoter CpG island hypermethylation-associated silencing of PIWIL1, PIWIL2, PIWIL4, and TDRD1 in primary seminoma and non-seminoma testicular tumors, in addition to testicular germ cell tumor cell lines. Most importantly, these epigenetic lesions occur in a context of piRNA downregulation and loss of DNA methylation of the LINE-1 repetitive sequences, one of the target genomic loci where the PIWI/piRNA machinery acts as a caretaker in non-transformed cells.

  3. Consolidated Recovered Materials Advisory Notice (RMAN) for the Comprehensive Procurement Guideline (CPG)

    Data.gov (United States)

    U.S. Environmental Protection Agency — EPA's Comprehensive Procurement Guideline (CPG) designates recycled content products that government agencies should buy. EPA publishes purchasing guidance and...

  4. Loss of methylation at the IFNG promoter and CNS-1 is associated with the development of functional IFN-γ memory in human CD4(+) T lymphocytes.

    Science.gov (United States)

    Dong, Jun; Chang, Hyun-Dong; Ivascu, Claudia; Qian, Yu; Rezai, Soheila; Okhrimenko, Anna; Cosmi, Lorenzo; Maggi, Laura; Eckhardt, Florian; Wu, Peihua; Sieper, Joachim; Alexander, Tobias; Annunziato, Francesco; Gossen, Manfred; Li, Jun; Radbruch, Andreas; Thiel, Andreas

    2013-03-01

    Cytokine memory for IFN-γ production by effector/memory Th1 cells plays a key role in both protective and pathological immune responses. To understand the epigenetic mechanism determining the ontogeny of effector/memory Th1 cells characterized by stable effector functions, we identified a T-cell-specific methylation pattern at the IFNG promoter and CNS-1 in ex vivo effector/memory Th1 cells, and investigated methylation dynamics of these regions during the development of effector/memory Th1 cells. During Th1 differentiation, demethylation occurred at both the promoter and CNS-1 regions of IFNG as early as 16 h, and this process was independent of cell proliferation and DNA synthesis. Using an IFN-γ capture assay, we found early IFN-γ-producing cells from 2-day differentiating cultures acquired "permissive" levels of demethylation and developed into effector/memory Th1 cells undergoing progressive demethylation at the IFNG promoter and CNS-1 when induced by IL-12. Methylation levels of these regions in effector/memory Th1 cells of peripheral blood from rheumatoid arthritis patients correlated inversely with reduced frequencies of IFN-γ-producers, coincident with recruitment of effector/memory Th1 cells to the site of inflammation. Thus, after termination of TCR stimulation, IL-12 signaling potentiates the stable functional IFN-γ memory in effector/memory Th1 cells characterized by hypomethylation at the IFNG promoter and CNS-1.

  5. Genome-wide peripheral blood leukocyte DNA methylation microarrays identified a single association with inflammatory bowel diseases

    DEFF Research Database (Denmark)

    Harris, R Alan; Nagy-Szakal, Dorottya; Pedersen, Natalia

    2012-01-01

    Crohn's disease (CD) and ulcerative colitis (UC) are common forms of inflammatory bowel disease (IBD). Monozygotic (MZ) twin discordance rates and epidemiologic data implicate that environmental changes and epigenetic factors may play a pathogenic role in IBD. DNA methylation (the methylation...... of cytosines within CpG dinucleotides) is an epigenetic modification, which can respond to environmental influences. We investigated whether DNA methylation might be connected with IBD in peripheral blood leukocyte (PBL) DNA by utilizing genome-wide microarrays....

  6. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA - Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis

    DEFF Research Database (Denmark)

    Hatt, Lotte; Aagaard, Mads M; Graakjaer, Jesper

    2015-01-01

    of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation...... profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated Cp...

  7. 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.

  8. DNA methylation and healthy human aging.

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

    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.

  10. VEZF1 elements mediate protection from DNA methylation.

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    Jacqueline Dickson

    2010-01-01

    Full Text Available There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm beta-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.

  11. Demethylation of Cancer/Testis Antigens and CpG ODN Stimulation Enhance Dendritic Cell and Cytotoxic T Lymphocyte Function in a Mouse Mammary Model

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    Jun-Zhong Sun

    2013-01-01

    Full Text Available Background. Cancer/testis antigens (CTAs are ideal targets for cancer immunotherapy in virtue of their restricted expression profile in normal tissues. However, CTA-targeted immunotherapy has been rather disappointing clinical setting for CTAs are downregulated by cytosine-phosphate-guanosine (CpG methylation in their promoter regions, so that tumor cells have low immunogenicity. Methods. We reinduced mouse CTA P1A through demethylation process and generated P1A-specific cytotoxic lymphocytes (CTLs by immunizing BALB/c (H-2d mice with dendritic cells pulsed with a P1A-specific peptide and CpG oligodeoxynucleotide (ODN immune adjuvant. Results. We found that demethylation and CpG ODN immune adjuvant stimulation facilitated DC maturation and enhanced the allogenic capacity of P1A-specific CTLs against target cells both in vitro and in vivo. Conclusions. Our results suggested that CTA induction and immune adjuvant stimulation is a feasible strategy in cancer immunotherapy.

  12. Tumor-associated methylation of the putative tumor suppressor AJAP1 gene and association between decreased AJAP1 expression and shorter survival in patients with glioma

    Institute of Scientific and Technical Information of China (English)

    David Cogdell; Woonbok Chung; Yuexin Liu; Matthew McDonald; Kenneth Aldape; Jean-Pierre J. Issa; Gregory N. Fuller; Wei Zhang

    2011-01-01

    Allelic loss of the short arm of chromosome 1 has been observed frequently in a wide spectrum of cancers, most frequently in oligodendroglioma. In our previous studies, we evaluated 177 oligodendroglial tumor samples and identified the AJAP1 gene (formerly Shrew1) in the consensus region of deletion.AJAP1 is a transmembrane protein found in adheren junctions and functions to inhibit glioma cell adhesion and migration. Whereas a putative tumor suppressor gene, we did not detect AJAP1 gene mutations. In subsequent studies, we found that AJAP1 was underexpressed in oligodendrogliomas relative to normal brain tissues. Bioinformatic analysis revealed the presence of CpG islands in the promoter of AJAP1.Methylation analysis of the AJAP1 promoter identified hypermethylation in 21% of oligodendrogliomas (n = 27), and the degree of methylation correlated with Iow levels of AJAP1 expression (P = 0.045). The AJAP1 promoter was also highly methylated in a wide spectrum of cell lines (n = 22), including cell lines of glioblastoma. Analysis of the National Cancer Institute's REMBRANDT dataset, which contains 343 glioma samples, indicated that Iow AJAP1 gene expression was associated with decreased survival. Thus,both genetic (gene deletion) and epigenetic alterations (promoter methylation) are likely mechanisms that inactivate the putative tumor suppressor AJAP1 in gliomas, which contributes to poor prognosis.

  13. Tumor-associated methylation of the putative tumor suppressor AJAP1 gene and association between decreased AJAP1 expression and shorter survival in patients with glioma.

    Science.gov (United States)

    Cogdell, David; Chung, Woonbok; Liu, Yuexin; McDonald, J Matthew; Aldape, Kenneth; Issa, Jean-Pierre J; Fuller, Gregory N; Zhang, Wei

    2011-04-01

    Allelic loss of the short arm of chromosome 1 has been observed frequently in a wide spectrum of cancers, most frequently in oligodendroglioma. In our previous studies, we evaluated 177 oligodendroglial tumor samples and identified the AJAP1 gene (formerly Shrew1) in the consensus region of deletion. AJAP1 is a transmembrane protein found in adheren junctions and functions to inhibit glioma cell adhesion and migration. Whereas a putative tumor suppressor gene, we did not detect AJAP1 gene mutations. In subsequent studies, we found that AJAP1 was underexpressed in oligodendrogliomas relative to normal brain tissues. Bioinformatic analysis revealed the presence of CpG islands in the promoter of AJAP1. Methylation analysis of the AJAP1 promoter identified hypermethylation in 21% of oligodendrogliomas (n =27), and the degree of methylation correlated with low levels of AJAP1 expression (P = 0.045). The AJAP1 promoter was also highly methylated in a wide spectrum of cell lines (n = 22), including cell lines of glioblastoma. Analysis of the National Cancer Institute's REMBRANDT dataset, which contains 343 glioma samples, indicated that low AJAP1 gene expression was associated with decreased survival. Thus, both genetic (gene deletion) and epigenetic alterations (promoter methylation) are likely mechanisms that inactivate the putative tumor suppressor AJAP1 in gliomas, which contributes to poor prognosis.

  14. p16/MTS1 inactivation in ovarian carcinomas: high frequency of reduced protein expression associated with hyper-methylation or mutation in endometrioid and mucinous tumors.

    Science.gov (United States)

    Milde-Langosch, K; Ocon, E; Becker, G; Löning, T

    1998-02-20

    Inactivation of the tumor-suppressor gene p16 (MTS1/ CDKN2/INK4a) has been described in various human malignancies. Although p16 deletion has been found in various ovarian tumor cell lines, p16 inactivation by homozygous deletion or mutation has been reported only sporadically in primary ovarian carcinomas. In a comprehensive study, we analyzed p16 protein expression by immuno-histochemistry (IHC) on paraffin sections of 94 primary ovarian carcinomas of different histological subtype. Loss of expression was detected in 19 primary tumors (20%), mainly mucinous and endometrioid carcinomas. To reveal the cause of suppressed expression, we performed (i) analysis of homozygous deletions by comparative PCR after micro-dissection, (ii) mutation analysis by single-strand conformation polymorphism analysis and subsequent direct sequencing and (iii) methylation-specific PCR to determine the methylation status of 5'-CpG islands. Loss of or weak p16 expression was caused by hyper-methylation (12/19 IHC-negative cases), somatic mutation (10 tumors) or homozygous deletion (1 case). Aberrant p 16 results by one of these methods were detected in 71-79% of endometrioid and mucinous, but in only 10% of serous-papillary, carcinomas. Our data suggest that p16 inactivation is a typical feature of certain subtypes of ovarian carcinoma.

  15. High-throughput DNA methylation analysis in colorectal cancer and childhood leukemia

    NARCIS (Netherlands)

    Roon, Eddy Herman Jasper van

    2012-01-01

    The first study described the colon tumor-specific methylation of a low CG-dense CpG island that is located in the first intron of the PTPRG gene (PTPRGint1). High levels of specificity and sensitivity of this region were observed in sporadic and familial colon cancer which makes this region interes

  16. High-throughput DNA methylation analysis in colorectal cancer and childhood leukemia

    NARCIS (Netherlands)

    Roon, Eddy Herman Jasper van

    2012-01-01

    The first study described the colon tumor-specific methylation of a low CG-dense CpG island that is located in the first intron of the PTPRG gene (PTPRGint1). High levels of specificity and sensitivity of this region were observed in sporadic and familial colon cancer which makes this region interes

  17. Promoter methylation of p16, Runx3, DAPK and CHFR genes is frequent in gastric carcinoma.

    Science.gov (United States)

    Hu, Shi-Lian; Kong, Xiang-Yong; Cheng, Zhao-Dong; Sun, Yu-Bei; Shen, Gan; Xu, Wei-Ping; Wu, Lei; Xu, Xiu-Cai; Jiang, Xiao-Dong; Huang, Da-Bing

    2010-01-01

    Transcriptional silencing induced by hypermethylation of CpG islands in the promoter regions of genes is believed to be an important mechanism of carcinogenesis in human cancers including gastric cancer. A number of reports on methylation of various genes in gastric cancer have been published, but most of these studies focused on cancer tissues or only a single gene. In this study, we determined the promoter hypermethylation status and mRNA expression of 4 genes: p16, Runx3, DAPK and CHFR. Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of p16, Runx3, DAPK and CHFR gene promoters in cancer and adjacent normal gastric mucosa specimens from 70 patients with gastric cancer, as well as normal gastric biopsy samples from 30 people without cancer serving as controls. In addition, the mRNA expression of p16, Runx3, DAPK and CHFR was investigated in 34 gastric cancer patients by RT-PCR. Bisulfite DNA sequence analysis was applied to check the positive samples detected by MSP. When carcinoma specimens were compared with adjacent normal gastric mucosa samples, a significant increase in promoter methylation of p16, Runx3, DAPK and CHFR was observed, while all 30 histologically normal gastric specimens were methylation free for all 4 genes. The methylation rate of the 4 genes increased from normal stomach tissue to tumor-adjacent gastric mucosa to gastric cancer tissue. Concurrent methylation in 2 or more genes was found in 22.9% of tumor-adjacent normal gastric mucosa and 75.7% of cancer tissues. No correlation was found between hypermethylation and other clinicopathological parameters such as sex, age, and tumor location. However, the frequency of DAPK and CHFR methylation in cancer tissues was significantly associated with the extent of differentiation and lymph node metastasis (P p16, Runx3, DAPK and CHFR is frequent in gastric cancer. DAPK and CHFR promoter hypermethylation may be an important help in evaluating the

  18. A genome-wide methylation study on obesity: differential variability and differential methylation.

    Science.gov (United States)

    Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Hidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

    2013-05-01

    Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common complex disease. We analyzed genome-wide methylation profiles of over 470,000 CpGs in peripheral blood samples from 48 obese and 48 lean African-American youth aged 14-20 y old. A substantial number of differentially variable CpG sites (DVCs), using statistics based on variances, as well as a substantial number of differentially methylated CpG sites (DMCs), using statistics based on means, were identified. Similar to the findings in cancers, DVCs generally exhibited an outlier structure and were more variable in cases than in controls. By randomly splitting the current sample into a discovery and validation set, we observed that both the DVCs and DMCs identified from the first set could independently predict obesity status in the second set. Furthermore, both the genes harboring DMCs and the genes harboring DVCs showed significant enrichment of genes identified by genome-wide association studies on obesity and related diseases, such as hypertension, dyslipidemia, type 2 diabetes and certain types of cancers, supporting their roles in the etiology and pathogenesis of obesity. We generalized the recent finding on methylation variability in cancer research to obesity and demonstrated that differential variability is also an important feature of obesity-related methylation changes. Future studies on the epigenetics of obesity will benefit from both statistics based on means and statistics based on variances.

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

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    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.

  20. Statistical methods for detecting differentially methylated loci and regions

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    Mark D Robinson

    2014-09-01

    Full Text Available DNA methylation, the reversible addition of methyl groups at CpG dinucleotides, represents an important regulatory layer associated with gene expression. Changed methylation status has been noted across diverse pathological states, including cancer. The rapid development and uptake of microarrays and large scale DNA sequencing has prompted an explosion of data analytic methods for processing and discovering changes in DNA methylation across varied data types. In this mini-review, we present a compact and accessible discussion of many of the salient challenges, such as experimental design, statistical methods for differential methylation detection, critical considerations such as cell type composition and the potential confounding that can arise from batch effects. From a statistical perspective, our main interests include the use of empirical Bayes or hierarchical models, which have proved immensely powerful in genomics, and the procedures by which false discovery control is achieved.

  1. Neocortical RELN promoter methylation increases significantly after puberty.

    Science.gov (United States)

    Lintas, Carla; Persico, Antonio M

    2010-01-27

    Reelin plays a pivotal role in neurodevelopment. Excessive RELN promoter methylation and/or decreased RELN gene expression have been described in schizophrenia and autism. We assessed RELN promoter methylation in post-mortem temporocortical tissue (Brodmann Area 41/42) of three prepuberal and six postpuberal normal individuals. The former display very little or no methylation, whereas most postpuberal individuals are heavily methylated, especially at CpG positions located between -131 and -98 bp (prepuberal vs. postpuberal, P<0.05). Sex hormones thus seemingly boost DNA methylation at the RELN promoter. This physiological change could significantly contribute to the onset of schizophrenia and the worsening of autistic behaviors, both typically occurring at puberty.

  2. Genome-wide methylation and expression profiling identifies promoter characteristics affecting demethylation-induced gene up-regulation in melanoma

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    Halaban Ruth

    2010-02-01

    Full Text Available Abstract Background Abberant DNA methylation at CpG dinucleotides represents a common mechanism of transcriptional silencing in cancer. Since CpG methylation is a reversible event, tumor supressor genes that have undergone silencing through this mechanism represent promising targets for epigenetically active anti-cancer therapy. The cytosine analog 5-aza-2'-deoxycytidine (decitabine induces genomic hypomethylation by inhibiting DNA methyltransferase, and is an example of an epigenetic agent that is thought to act by up-regulating silenced genes. Methods It is unclear why decitabine causes some silenced loci to re-express, while others remain inactive. By applying data-mining techniques to large-scale datasets, we attempted to elucidate the qualities of promoter regions that define susceptibility to the drug's action. Our experimental data, derived from melanoma cell strains, consist of genome-wide gene expression data before and after treatment with decitabine, as well as genome-wide data on un-treated promoter methylation status, and validation of specific genes by bisulfite sequencing. Results We show that the combination of promoter CpG content and methylation level informs the ability of decitabine treatment to up-regulate gene expression. Promoters with high methylation levels and intermediate CpG content appear most susceptible to up-regulation by decitabine, whereas few of those highly methylated promoters with high CpG content are up-regulated. For promoters with low methylation levels, those with high CpG content are more likely to be up-regulated, whereas those with low CpG content are underrepresented among up-regulated genes. Conclusions Clinically, elucidating the patterns of action of decitabine could aid in predicting the likelihood of up-regulating epigenetically silenced tumor suppressor genes and others from pathways involved with tumor biology. As a first step toward an eventual translational application, we build a classifier

  3. Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis

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    Kibbe Warren A

    2010-11-01

    Full Text Available Abstract Background High-throughput profiling of DNA methylation status of CpG islands is crucial to understand the epigenetic regulation of genes. The microarray-based Infinium methylation assay by Illumina is one platform for low-cost high-throughput methylation profiling. Both Beta-value and M-value statistics have been used as metrics to measure methylation levels. However, there are no detailed studies of their relations and their strengths and limitations. Results We demonstrate that the relationship between the Beta-value and M-value methods is a Logit transformation, and show that the Beta-value method has severe heteroscedasticity for highly methylated or unmethylated CpG sites. In order to evaluate the performance of the Beta-value and M-value methods for identifying differentially methylated CpG sites, we designed a methylation titration experiment. The evaluation results show that the M-value method provides much better performance in terms of Detection Rate (DR and True Positive Rate (TPR for both highly methylated and unmethylated CpG sites. Imposing a minimum threshold of difference can improve the performance of the M-value method but not the Beta-value method. We also provide guidance for how to select the threshold of methylation differences. Conclusions The Beta-value has a more intuitive biological interpretation, but the M-value is more statistically valid for the differential analysis of methylation levels. Therefore, we recommend using the M-value method for conducting differential methylation analysis and including the Beta-value statistics when reporting the results to investigators.

  4. Genomic distribution and inter-sample variation of non-CpG methylation across human cell types.

    Directory of Open Access Journals (Sweden)

    Michael J Ziller

    2011-12-01

    Full Text Available DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set.

  5. Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells.

    Science.gov (United States)

    Mariati; Yeo, Jessna H M; Koh, Esther Y C; Ho, Steven C L; Yang, Yuansheng

    2014-01-01

    The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild-type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation. © 2014 American Institute of Chemical Engineers.

  6. Expression of ECRG4, a novel esophageal cancer-related gene,downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Chun-Mei Yue; Da-Jun Deng; Mei-Xia Bi; Li-Ping Guo; Shih-Hsin Lu

    2003-01-01

    AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293)and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR. RESULTS: The expression level of ECRG 4 gene was varied.Of 20 esophageal cancer tissues, nine were unexpressed,six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed)of the 15 (80%) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing. CONCLUSION: These results indicate that the inactivation of ECRG 4gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.

  7. Suppression of HIV replication in vitro by CpG and CpG conjugated to the non toxic B subunit of cholera toxin.

    Science.gov (United States)

    Nowroozalizadeh, Salma; Jansson, Marianne; Adamsson, Jenni; Lindblad, Marianne; Fenyö, Eva-Maria; Holmgren, Jan; Harandi, Ali M

    2008-05-01

    Administration of oligodeoxynucleotides (ODNs) containing CpG motifs generates a rapid and potent response of CC-chemokines, known as ligands of the HIV-1 co-receptor CCR5, in the murine female genital tract. The present study explored the potential HIV inhibitory activities of different human CpG prototypes either alone or conjugated to the non-toxic subunit of cholera toxin (CTB). Results showed that in vitro replication of both HIV-1 and HIV-2 can be suppressed by different human CpG prototypes. Importantly, the conjugation of CpG ODN to CTB (CTB-CpG) enhanced the antiviral activity of CpG against primary HIV-1 isolates of both R5 and X4 phenotypes in peripheral blood mononuclear cells (PBMC) as well as U87.CD4 co-receptor indicator cells. CTB-CpGs triggered higher amounts of MIP-1alpha, and MIP-1beta in PBMC than the corresponding CpG ODNs, which may explain the superior antiviral effect of CTB-CpG against R5 virus in PBMC. Incubation of PBMC with CpG ODN and CTB-CpG did not alter surface expression of HIV-1 receptors indicating that the observed anti-HIV-1 effect is not mediated through down regulation of HIV-1 receptors on target cells. Further, the enhanced antiviral effect of CTB-CpG was dependent on the presence of phosphorothioate backbone in the ODN, whereas the presence of CpG motif in ODNs was dispensable. These results have implications for the development of novel intervention strategies to prevent HIV infection.

  8. Epigenetic Changes in the Methylation Patterns of KCNQ1 and WT1 after a Weight Loss Intervention Program in Obese Stroke Patients.

    Science.gov (United States)

    Abete, Itziar; Gómez-Úriz, Ana M; Mansego, María L; De Arce, Ana; Goyenechea, Estíbaliz; Blázquez, Vanessa; Martínez-Zabaleta, Maria T; González-Muniesa, Pedro; López De Munain, Adolfo; Martínez, J Alfredo; Campión, Javier; Milagro, Fermin I

    2015-01-01

    Ischemic stroke patients often show high concentrations of circulating inflammatory markers that are associated with increased risk of recurrence. Epigenetic mechanisms could be involved in obesity, inflammation and stroke. The objective of this research was to investigate, in obese patients suffering a previous stroke, the effects of a nutritional program on anthropometric and biochemical variables, and on the methylation patterns of two stroke-related genes (KCNQ1: potassium channel, voltage gated KQT-like subfamily Q, member 1; and WT1: Wilms tumor 1). Twenty-two ischemic stroke patients were compared with a control group composed of eighteen obese subjects with similar age and body mass index ranges. Both groups followed a 20-week nutritional program based on an energy-restricted balanced diet with high adherence to the Mediterranean dietary pattern. The intervention significantly improved anthropometric and metabolic variables, such as the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and C-reactive protein concentration, in ischemic stroke patients, and was accompanied by changes in the methylation patterns of both stroke-related genes, which correlated with anthropometric and biochemical variables.

  9. Effects of systemic pretreatment with CpG oligodeoxynucleotides on skin wound healing in mice.

    Science.gov (United States)

    Hergert, Bettina; Grambow, Eberhard; Butschkau, Antje; Vollmar, Brigitte

    2013-01-01

    Unmethylated CpG oligodeoxynucleotides (ODN) bind to the Toll-like receptor 9, thus stimulating the immune system. To study the effects of systemic pretreatment with CpG ODN on dermal regeneration, C57BL6/J Tyr mice were treated with CpG or control ODN 6 days prior to implantation of a dorsal skinfold chamber and skin wounding. Wound epithelialization was analyzed by planimetric microscopy. On day 18, wound tissues were taken for (immuno)histochemical staining. CpG ODN increased epithelialization compared with control ODN treatment. Histological analysis revealed reduced capillary density, reduced wound cellularity, and reduced numbers of infiltrating leukocytes, as well as reduced F4/80-positive macrophages, but increased numbers of RELM-α-positive M2 macrophages after CpG ODN treatment, reflecting a better quality of wound healing on day 18 compared with control ODN treatment. Reverse transcription-polymerase chain reaction analysis of Toll-like receptor 9 showed the receptor expression on both fibroblasts and keratinocytes. Fibroblasts showed an increase of migration upon increasing dosages of CpG and not control ODN, reaching ∼50% of the response of basic fibroblast growth factor-exposed cells. Keratinocytes dose-dependently responded to both CpG and control ODN up to values found in keratinocyte growth factor-exposed cells. In summary, CpG ODN support late tissue-remodeling processes that contribute to resolution of inflammation and solid wounds during skin regeneration.

  10. Modulation of CpG oligodeoxynucleotide-mediated immune stimulation by locked nucleic acid (LNA)

    DEFF Research Database (Denmark)

    Vollmer, Jörg; Jepsen, Jan Stenvang; Uhlmann, Eugen

    2004-01-01

    Locked nucleic acid (LNA) is an RNA derivative that when introduced into oligodeoxynucleotides (ODN), mediates high efficacy and stability. CpG ODNs are potent immune stimulators and are recognized by toll-like receptor-9 (TLR9). Some phosphorothioate antisense ODNs bearing CpG dinucleotides have...

  11. P16 METHYLATION OF THE COLORECTAL CANCER AND ASSOCIATION WITH DUKES STAGES

    Institute of Scientific and Technical Information of China (English)

    王志伟; 易静; 仓辉; 邹鸿志; 郁宝铭; 汤雪明

    2001-01-01

    To explore whether methylation of the CpG island in the promoter of the p16 tumor suppressor gene was associated with clinicopathological characteristics of the colorectal cancer patients. Methods: Methylation- specific PCR (MSP) was used to detect p16 methylation of the colorectal cancer patients. Results: In 58 sporadic colorectal cancer, 43.1% of the tumors had detectable p16 methylation. Dukes' stage was associated with p16 methylation status. Dukes C, D patients (75%) were more likely to contain methylated p16 compared with Dukes A, B patients (13.3%). Conclusion: p16 methylation plays a role in the carcinogenesis of a subset of colorectal cancer. P16 methylation might be considered as a prognostic indicator.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Development of a Diagnostic Tool to Detect DNA Methylation Biomarkers for Early-Stage Lung Cancer

    Science.gov (United States)

    2015-02-01

    Lung Cancer PRINCIPAL INVESTIGATOR: Chongli Yuan CONTRACTING ORGANIZATION: Purdue University West Lafayette, IN 47907 REPORT DATE: February...DNA methylation with high spatial resolution. Task 2.1 Design a library of DNA constructs in which the location of the single methylated CpG site is...the impact on the development of the principal discipline of the project? This proposal aims to design and demonstrate proof-of-concept for these

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

    DEFF Research Database (Denmark)

    Rönnerblad, Michelle; Andersson, Robin; Olofsson, Tor

    2014-01-01

    at specific differentiation stages and overlap with changes in transcription and activity of key hematopoietic transcription factors. DMSs were preferentially located in areas distal to CpG islands and shores. Also, DMSs were overrepresented in enhancer elements and enriched in enhancers that become active...... during granulopoiesis using 4 distinct cell populations ranging from the oligopotent common myeloid progenitor stage to terminally differentiated neutrophils. We observed that differentially methylated sites (DMSs) generally show decreased methylation during granulopoiesis. Methylation appears to change...... during differentiation. Overall, this study depicts in detail the epigenetic and transcriptional changes that occur during granulopoiesis and supports the role of DNA methylation as a regulatory mechanism in blood cell differentiation....

  15. Epigenetic silencing of the XAF1 gene is mediated by the loss of CTCF binding

    Science.gov (United States)

    Victoria-Acosta, Georgina; Vazquez-Santillan, Karla; Jimenez-Hernandez, Luis; Muñoz-Galindo, Laura; Maldonado, Vilma; Martinez-Ruiz, Gustavo Ulises; Melendez-Zajgla, Jorge

    2015-01-01

    XAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding. PMID:26443201

  16. Altered Mitochondrial DNA Methylation Pattern in Alzheimer Disease-Related Pathology and in Parkinson Disease.

    Science.gov (United States)

    Blanch, Marta; Mosquera, Jose Luis; Ansoleaga, Belén; Ferrer, Isidre; Barrachina, Marta

    2016-02-01

    Mitochondrial dysfunction is linked with the etiopathogenesis of Alzheimer disease and Parkinson disease. Mitochondria are intracellular organelles essential for cell viability and are characterized by the presence of the mitochondrial (mt)DNA. DNA methylation is a well-known epigenetic mechanism that regulates nuclear gene transcription. However, mtDNA methylation is not the subject of the same research attention. The present study shows the presence of mitochondrial 5-methylcytosine in CpG and non-CpG sites in the entorhinal cortex and substantia nigra of control human postmortem brains, using the 454 GS FLX Titanium pyrosequencer. Moreover, increased mitochondrial 5-methylcytosine levels are found in the D-loop region of mtDNA in the entorhinal cortex in brain samples with Alzheimer disease-related pathology (stages I to II and stages III to IV of Braak and Braak; n = 8) with respect to control cases. Interestingly, this region shows a dynamic pattern in the content of mitochondrial 5-methylcytosine in amyloid precursor protein/presenilin 1 mice along with Alzheimer disease pathology progression (3, 6, and 12 months of age). Finally, a loss of mitochondrial 5-methylcytosine levels in the D-loop region is found in the substantia nigra in Parkinson disease (n = 10) with respect to control cases. In summary, the present findings suggest mtDNA epigenetic modulation in human brain is vulnerable to neurodegenerative disease states.

  17. Genome-wide divergence of DNA methylation marks in cerebral and cerebellar cortices.

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    Yurong Xin

    Full Text Available BACKGROUND: Emerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS. Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function. METHODOLOGY/SIGNIFICANT FINDINGS: Using methylation-sensitive SNP chip analysis (MSNP, we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum. CONCLUSIONS: These findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions.

  18. Carpel, a new Arabidopsis epi-mutant of the SUPERMAN gene: phenotypic analysis and DNA methylation status.

    Science.gov (United States)

    Rohde, A; Grunau, C; De Beck, L; Van Montagu, M; Rosenthal, A; Boerjan, W

    1999-09-01

    The carpel (car) mutation affects the morphology of reproductive organs in Arabidopsis thaliana. car flowers have an increased number of carpels, on average 2.7 +/- 0.8 instead of two in the wild type. Through allelism test with fon1-3 and analysis of the methylation state of the SUPERMAN (SUP) gene in car mutants, we show that car is an epi-mutation of SUP. The methylation pattern of car is clearly distinct from that of fon1-3, another epi-mutation of the SUP gene. Methylation was found predominantly in Cp(A/T)p(A/G) triplets and in CpG pairs. We suggest that the extensive SUP methylation in car has arisen from an abundant methylation of a single CpG site that was already present in abscisic acid-insensitive (abi3-4) mutants, from which car was segregating.

  19. Multiple sporadic colorectal cancers display a unique methylation phenotype.

    Science.gov (United States)

    Gonzalo, Victoria; Lozano, Juan Jose; Alonso-Espinaco, Virginia; Moreira, Leticia; Muñoz, Jenifer; Pellisé, Maria; Castellví-Bel, Sergi; Bessa, Xavier; Andreu, Montserrat; Xicola, Rosa M; Llor, Xavier; Ruiz-Ponte, Clara; Carracedo, Angel; Jover, Rodrigo; Castells, Antoni; Balaguer, Francesc

    2014-01-01

    Epigenetics are thought to play a major role in the carcinogenesis of multiple sporadic colorectal cancers (CRC). Previous studies have suggested concordant DNA hypermethylation between tumor pairs. However, only a few methylation markers have been analyzed. This study was aimed at describing the epigenetic signature of multiple CRC using a genome-scale DNA methylation profiling. We analyzed 12 patients with synchronous CRC and 29 age-, sex-, and tumor location-paired patients with solitary tumors from the EPICOLON II cohort. DNA methylation profiling was performed using the Illumina Infinium HM27 DNA methylation assay. The most significant results were validated by Methylight. Tumors samples were also analyzed for the CpG Island Methylator Phenotype (CIMP); KRAS and BRAF mutations and mismatch repair deficiency status. Functional annotation clustering was performed. We identified 102 CpG sites that showed significant DNA hypermethylation in multiple tumors with respect to the solitary counterparts (difference in β value ≥0.1). Methylight assays validated the results for 4 selected genes (p = 0.0002). Eight out of 12(66.6%) multiple tumors were classified as CIMP-high, as compared to 5 out of 29(17.2%) solitary tumors (p = 0.004). Interestingly, 76 out of the 102 (74.5%) hypermethylated CpG sites found in multiple tumors were also seen in CIMP-high tumors. Functional analysis of hypermethylated genes found in multiple tumors showed enrichment of genes involved in different tumorigenic functions. In conclusion, multiple CRC are associated with a distinct methylation phenotype, with a close association between tumor multiplicity and CIMP-high. Our results may be important to unravel the underlying mechanism of tumor multiplicity.

  20. A crossed molecular beam and ab-initio investigation of the reaction of boron monoxide (BO; X2Σ+) with methylacetylene (CH3CCH; X1A1): competing atomic hydrogen and methyl loss pathways.

    Science.gov (United States)

    Maity, Surajit; Parker, Dorian S N; Dangi, Beni B; Kaiser, Ralf I; Fau, Stefan; Perera, Ajith; Bartlett, Rodney J

    2013-11-21

    The gas-phase reaction of boron monoxide ((11)BO; X(2)Σ(+)) with methylacetylene (CH3CCH; X(1)A1) was investigated experimentally using crossed molecular beam technique at a collision energy of 22.7 kJ mol(-1) and theoretically using state of the art electronic structure calculation, for the first time. The scattering dynamics were found to be indirect (complex forming reaction) and the reaction proceeded through the barrier-less formation of a van-der-Waals complex ((11)BOC3H4) followed by isomerization via the addition of (11)BO(X(2)Σ(+)) to the C1 and/or C2 carbon atom of methylacetylene through submerged barriers. The resulting (11)BOC3H4 doublet radical intermediates underwent unimolecular decomposition involving three competing reaction mechanisms via two distinct atomic hydrogen losses and a methyl group elimination. Utilizing partially deuterated methylacetylene reactants (CD3CCH; CH3CCD), we revealed that the initial addition of (11)BO(X(2)Σ(+)) to the C1 carbon atom of methylacetylene was followed by hydrogen loss from the acetylenic carbon atom (C1) and from the methyl group (C3) leading to 1-propynyl boron monoxide (CH3CC(11)BO) and propadienyl boron monoxide (CH2CCH(11)BO), respectively. Addition of (11)BO(X(2)Σ(+)) to the C1 of methylacetylene followed by the migration of the boronyl group to the C2 carbon atom and/or an initial addition of (11)BO(X(2)Σ(+)) to the sterically less accessible C2 carbon atom of methylacetylene was followed by loss of a methyl group leading to the ethynyl boron monoxide product (HCC(11)BO) in an overall exoergic reaction (78 ± 23 kJ mol(-1)). The branching ratios of these channels forming CH2CCH(11)BO, CH3CC(11)BO, and HCC(11)BO were derived to be 4 ± 3%, 40 ± 5%, and 56 ± 15%, respectively; these data are in excellent agreement with the calculated branching ratios using statistical RRKM theory yielding 1%, 38%, and 61%, respectively.

  1. Up-regulation of expression and lack of 5' CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas

    Directory of Open Access Journals (Sweden)

    Frank Georgy A

    2007-03-01

    Full Text Available Abstract Background High risk type human papilloma viruses (HR-HPV induce carcinomas of the uterine cervix by expressing viral oncogenes E6 and E7. Oncogene E7 of HR-HPV disrupts the pRb/E2F interaction, which negatively regulates the S phase entry. Expression of tumor suppressor p16ink4a drastically increases in majority of HR-HPV associated carcinomas due to removal of pRb repression. The p16ink4a overexpression is an indicator of an aberrant expression of viral oncogenes and may serve as a marker for early diagnostic of cervical cancer. On the other hand, in 25–57% of cervical carcinomas hypermethylation of the p16 INK4a promoter has been demonstrated using a methylation-specific PCR, MSP. To evaluate a potential usage of the p16 INK4a 5' CpG island hypermethylation as an indicator of tumor cell along with p16ink4a overexpression, we analyzed the methylation status of p16 INK4a in cervical carcinomas Methods Methylation status of p16 INK4a was analyzed by MSP and by bisulfite-modified DNA sequencing. The expression of p16ink4a was analyzed by RT-PCR and by immunohistochemical technique. Results The extensive methylation within p16 INK4a 5' CpG island was not detected either in 13 primary cervical carcinomas or in 5 cancer cell lines by bisulfite-modified DNA sequencing (including those that were positive by MSP in our hands. The number and distribution of rare partially methylated CpG sites did not differ considerably in tumors and adjacent normal tissues. The levels of the p16 INK4a mRNA were increased in carcinomas compared to the normal tissues independently of the number of partially methylated CpGs within 5'CpG island. The transcriptional activation of p16 INK4a was accompanied by p16ink4a cytoplasmic immunoreactivity in the majority of tumor cells and presence of a varied number of the p16 positive nuclei in different tumors. Conclusion Hypermethylaion of the p16INK4a 5' CpG island is not a frequent event in HR-HPV-positive cervical

  2. Improved Prediction of Non-methylated Islands in Vertebrates Highlights Different Characteristic Sequence Patterns

    Science.gov (United States)

    Vingron, Martin

    2016-01-01

    Non-methylated islands (NMIs) of DNA are genomic regions that are important for gene regulation and development. A recent study of genome-wide non-methylation data in vertebrates by Long et al. (eLife 2013;2:e00348) has shown that many experimentally identified non-methylated regions do not overlap with classically defined CpG islands which are computationally predicted using simple DNA sequence features. This is especially true in cold-blooded vertebrates such as Danio rerio (zebrafish). In order to investigate how predictive DNA sequence is of a region’s methylation status, we applied a supervised learning approach using a spectrum kernel support vector machine, to see if a more complex model and supervised learning can be used to improve non-methylated island prediction and to understand the sequence properties of these regions. We demonstrate that DNA sequence is highly predictive of methylation status, and that in contrast to existing CpG island prediction methods our method is able to provide more useful predictions of NMIs genome-wide in all vertebrate organisms that were studied. Our results also show that in cold-blooded vertebrates (Anolis carolinensis, Xenopus tropicalis and Danio rerio) where genome-wide classical CpG island predictions consist primarily of false positives, longer primarily AT-rich DNA sequence features are able to identify these regions much more accurately. PMID:27984582

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

    Science.gov (United States)

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

    2016-06-10

    As a member of the neurotrophic factor family, brain derived neurotrophic factor (BDNF) plays an important role in the survival and differentiation of neurons. The aim of our work was to evaluate the role of BDNF promoter methylation in drug addiction. A total of 60 drug abusers (30 heroin and 30 methylamphetamine addicts) and 52 healthy age- and gender-matched controls were recruited for the current case control study. Bisulfite pyrosequencing technology was used to determine the methylation levels of five CpGs (CpG1-5) on the BDNF promoter. Among the five CpGs, CpG5 methylation was significantly lower in drug abusers than controls. Moreover, significant associations were found between CpG5 methylation and addictive phenotypes including tension-anxiety, anger-hostility, fatigue-inertia, and depression-dejection. In addition, luciferase assay showed that the DNA fragment of BDNF promoter played a key role in the regulation of gene expression. Our results suggest that BDNF promoter methylation is associated with drug addiction, although further studies are needed to understand the mechanisms by which BDNF promoter methylation contributes to the pathophysiology of drug addiction. Copyright © 2016. Published by Elsevier B.V.

  4. Tissue-specific variation in DNA methylation levels along human chromosome 1

    Directory of Open Access Journals (Sweden)

    De Bustos Cecilia

    2009-06-01

    Full Text Available Abstract Background DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. Results Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined. Conclusion The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.

  5. Sex differences in DNA methylation of the cord blood are related to sex-bias psychiatric diseases

    Science.gov (United States)

    Maschietto, Mariana; Bastos, Laura Caroline; Tahira, Ana Carolina; Bastos, Elen Pereira; Euclydes, Veronica Luiza Vale; Brentani, Alexandra; Fink, Günther; de Baumont, Angelica; Felipe-Silva, Aloísio; Francisco, Rossana Pulcineli Vieira; Gouveia, Gisele; Grisi, Sandra Josefina Ferraz Ellero; Escobar, Ana Maria Ulhoa; Moreira-Filho, Carlos Alberto; Polanczyk, Guilherme Vanoni; Miguel, Euripedes Constantino; Brentani, Helena

    2017-03-01

    Sex differences in the prevalence of psychiatric disorders are well documented, with exposure to stress during gestation differentially impacting females and males. We explored sex-specific DNA methylation in the cord blood of 39 females and 32 males born at term and with appropriate weight at birth regarding their potential connection to psychiatric outcomes. Mothers were interviewed to gather information about environmental factors (gestational exposure) that could interfere with the methylation profiles in the newborns. Bisulphite converted DNA was hybridized to Illumina HumanMethylation450 BeadChips. Excluding XYS probes, there were 2,332 differentially methylated CpG sites (DMSs) between sexes, which were enriched within brain modules of co-methylated CpGs during brain development and also differentially methylated in the brains of boys and girls. Genes associated with the DMSs were enriched for neurodevelopmental disorders, particularly for CpG sites found differentially methylated in brain tissue between patients with schizophrenia and controls. Moreover, the DMS had an overlap of 890 (38%) CpG sites with a cohort submitted to toxic exposition during gestation. This study supports the evidences that sex differences in DNA methylation of autosomes act as a primary driver of sex differences that are found in psychiatric outcomes.

  6. Genome-wide methylation profiling and a multiplex construction for the identification of body fluids using epigenetic markers.

    Science.gov (United States)

    Lee, Hwan Young; An, Ja Hyun; Jung, Sang-Eun; Oh, Yu Na; Lee, Eun Young; Choi, Ajin; Yang, Woo Ick; Shin, Kyoung-Jin

    2015-07-01

    The identification of body fluids found at crime scenes can contribute to solving crimes by providing important insights into crime scene reconstruction. In the present study, body fluid-specific epigenetic marker candidates were identified from genome-wide DNA methylation profiling of 42 body fluid samples including blood, saliva, semen, vaginal fluid and menstrual blood using the Illumina Infinium HumanMethylation450 BeadChip array. A total of 64 CpG sites were selected as body fluid-specific marker candidates by having more than 20% discrepancy in DNA methylation status between a certain type of body fluid and other types of body fluids and to have methylation or unmethylation pattern only in a particular type of body fluid. From further locus-specific methylation analysis in additional samples, 1 to 3 CpG sites were selected for each body fluid. Then, a multiplex methylation SNaPshot reaction was constructed to analyze methylation status of 8 body fluid-specific CpG sites. The developed multiplex reaction positively identifies blood, saliva, semen and the body fluid which originates from female reproductive organ in one reaction, and produces successful DNA methylation profiles in aged or mixed samples. Although it remains to be investigated whether this approach is more sensitive, more practical than RNA- or peptide-based assays and whether it can be successfully applied to forensic casework, the results of the present study will be useful for the forensic investigators dealing with body fluid samples.

  7. Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

    Science.gov (United States)

    Brant, Jason O; Riva, Alberto; Resnick, James L; Yang, Thomas P

    2014-01-01

    Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain. PMID:25482058

  8. Elevation of peripheral BDNF promoter methylation links to the risk of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Lan Chang

    Full Text Available Brain derived neurotrophic factor (BDNF has been known to play an important role in various mental disorders or diseases such as Alzheimer's disease (AD. The aim of our study was to assess whether BDNF promoter methylation in peripheral blood was able to predict the risk of AD. A total of 44 AD patients and 62 age- and gender-matched controls were recruited in the current case-control study. Using the bisulphite pyrosequencing technology, we evaluated four CpG sites in the promoter of the BDNF. Our results showed that BDNF methylation was significantly higher in AD cases than in the controls (CpG1: p = 10.021; CpG2: p = 0.002; CpG3: p = 0.007; CpG4: p = 0.005; average methylation: p = 0.004. In addition, BDNF promoter methylation was shown to be significantly correlated with the levels of alkaline phosphatase (ALP, glucose, Lp(a, ApoE and ApoA in males (ALP: r = -0.308, p = 0.042; glucose: r = -0.383, p = 0.010; Lp(a: r = 0.333, p = 0.027; ApoE: r = -0.345, p = 0.032;, ApoA levels in females (r = 0.362, p = 0.033, and C Reactive Protein (CRP levels in both genders (males: r = -0.373, p = 0.016; females: r = -0.399, p = 0.021. Our work suggested that peripheral BDNF promoter methylation might be a diagnostic marker of AD risk, although its underlying function remains to be elaborated in the future.

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

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    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.

  10. Methylation in hepatocellular carcinoma

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    Regina M. Santella

    2007-02-01

    Full Text Available

    The development of HCC is a complex, multistep, multistage process. The molecular pathogenesis of HCC appears to involve multiple genetic aberrations in the molecular control of hepatocyte proliferation, differentiation and death and the maintenance of genomic integrity. This process is influenced by the cumulative activation and inactivation of oncogenes, tumor suppressor genes and other genes. p53, a tumor suppressor gene, is the most frequently mutated gene in human cancers. There is also a striking sequence specific binding and induction of mutations by AFB1 at codon 249 of p53 in HCC.

    Epigenetic alterations are also involved in cancer development and progression. Methylation of promoter CpG islands is associated with inhibition of transcriptional initiation and permanent silencing of downstream genes.

    It is now known that most important tumor suppressor genes are inactivated, not only by mutations and deletions but also by promoter methylation. Several studies indicated that p16, p15, RASSF1A, MGMT, and GSTP1 promoter hypermethylation are prevalent in HCC. In addition, geographic variation in the methylation status of tumor DNA indicates that environmental factors may influence the frequent and concordant degree of hypermethylation in multiple genes in HCC and that epigeneticenvironmental interactions may be involved in hepatocarcinogenesis. We have found significant relationships between promoter methylation and AFB1-DNA adducts confirming the impact of environmental exposures on gene methylation.

    DNA isolated from serum or plasma of cancer patients frequently contains the same genetic and

  11. Age-associated DNA methylation changes in immune genes, histone modifiers and chromatin remodeling factors within 5 years after birth in human blood leukocytes

    DEFF Research Database (Denmark)

    Acevedo, Nathalie; Reinius, Lovisa E; Vitezic, Morana;

    2015-01-01

    the dynamics of DNA methylation. Serial blood samples were collected at 3, 6, 12, 24, 36, 48 and 60 months after birth in ten healthy girls born in Finland and participating in the Type 1 Diabetes Prediction and Prevention Study. DNA methylation was measured using the HumanMethylation450 BeadChip. RESULTS......: After filtering for the presence of polymorphisms and cell-lineage-specific signatures, 794 CpG sites showed significant DNA methylation differences as a function of age in all children (41.6% age-methylated and 58.4% age-demethylated, Bonferroni-corrected P value ... performing DNA methylation studies in children....

  12. DNA methylation profiles of the brain-derived neurotrophic factor (BDNF gene as a potent diagnostic biomarker in major depression.

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    Manabu Fuchikami

    Full Text Available Major depression, because of its recurring and life-threatening nature, is one of the top 10 diseases for global disease burden. Major depression is still diagnosed on the basis of clinical symptoms in patients. The search for specific biological markers is of great importance to advance the method of diagnosis for depression. We examined the methylation profile of 2 CpG islands (I and IV at the promoters of the brain-derived neurotrophic factor (BDNF gene, which is well known to be involved in the pathophysiology of depression. We analyzed genomic DNA from peripheral blood of 20 Japanese patients with major depression and 18 healthy controls to identify an appropriate epigenetic biomarker to aid in the establishment of an objective system for the diagnosis of depression. Methylation rates at each CpG unit was measured using a MassArray® system (SEQUENOM, and 2-dimensional hierarchical clustering analyses were undertaken to determine the validity of these methylation profiles as a diagnostic biomarker. Analyses of the dendrogram from methylation profiles of CpG I, but not IV, demonstrated that classification of healthy controls and patients at the first branch completely matched the clinical diagnosis. Despite the small number of subjects, our results indicate that classification based on the DNA methylation profiles of CpG I of the BDNF gene may be a valuable diagnostic biomarker for major depression.

  13. Methylation levels of P16 and TP53 that are involved in DNA strand breakage of 16HBE cells treated by hexavalent chromium.

    Science.gov (United States)

    Hu, Guiping; Li, Ping; Li, Yang; Wang, Tiancheng; Gao, Xin; Zhang, Wenxiao; Jia, Guang

    2016-05-13

    The correlations between methylation levels of p16 and TP53 with DNA strand breakage treated by hexavalent chromium [Cr(VI)] remain unknown. In this research, Human bronchial epithelial cells (16HBE cells) in vitro and bioinformatics analysis were used to analyze the epigenetic role in DNA damage and potential biomarkers. CCK-8 and single cell gel electrophoresis assay were chosen to detect the cellular biological damage. MALDI-TOF-MS was used to detect the methylation levels of p16 and TP53. qRT-PCR was used to measure their expression levels in different Cr(VI) treatment groups. The transcription factors with target sequences of p16 and TP53 were predicted using various bioinformatics software. The findings showed that the cellular toxicity and DNA strand damage were Cr(VI) concentration dependent. The hypermethylation of CpG1, CpG31 and CpG32 of p16 was observed in Cr(VI) treated groups. There was significant positive correlation between the CpG1 methylation level of p16 and cell damage. In Cr(VI) treated groups, the expression level of p16 was lower than that in control group. The expression level of TP53 increased when the Cr(VI)concentration above 5μM. About p16, there was significant negative correlation between the CpG1 methylation levels with its expression level. A lot of binding sites for transcription factors existed in our focused CpG islands of p16. All the results suggested that the CpG1 methylation level of p16 could be used as a biomarker of epigenetic effect caused by Cr(VI) treatment, which can enhance cell damage by regulating its expression or affecting some transcription factors to combine with their DNA strand sites.

  14. 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.

  15. The effects of cytosine methylation on general transcription factors

    Science.gov (United States)

    Jin, Jianshi; Lian, Tengfei; Gu, Chan; Yu, Kai; Gao, Yi Qin; Su, Xiao-Dong

    2016-07-01

    DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.

  16. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain

    OpenAIRE

    Nguyen, Anhthu; Rauch, Tibor A; Pfeifer, Gerd P.; Hu, Valerie W.

    2010-01-01

    Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well ...

  17. Leukocyte DNA methylation signature differentiates pancreatic cancer patients from healthy controls.

    Directory of Open Access Journals (Sweden)

    Katrina S Pedersen

    Full Text Available Pancreatic adenocarcinoma (PaC is one of most difficult tumors to treat. Much of this is attributed to the late diagnosis. To identify biomarkers for early detection, we examined DNA methylation differences in leukocyte DNA between PaC cases and controls in a two-phase study. In phase I, we measured methylation levels at 1,505 CpG sites in treatment-naïve leukocyte DNA from 132 never-smoker PaC patients and 60 never-smoker healthy controls. We found significant differences in 110 CpG sites (false discovery rate <0.05. In phase II, we tested and validated 88 of 96 phase I selected CpG sites in 240 PaC cases and 240 matched controls (p≤0.05. Using penalized logistic regression, we built a prediction model consisting of five CpG sites (IL10_P348, LCN2_P86, ZAP70_P220, AIM2_P624, TAL1_P817 that discriminated PaC patients from controls (C-statistic = 0.85 in phase I; 0.76 in phase II. Interestingly, one CpG site (LCN2_P86 alone could discriminate resectable patients from controls (C-statistic= 0.78 in phase I; 0.74 in phase II. We also performed methylation quantitative trait loci (methQTL analysis and identified three CpG sites (AGXT_P180_F, ALOX12_E85_R, JAK3_P1075_R where the methylation levels were significantly associated with single nucleotide polymorphisms (SNPs (false discovery rate <0.05. Our results demonstrate that epigenetic variation in easily obtainable leukocyte DNA, manifested by reproducible methylation differences, may be used to detect PaC patients. The methylation differences at certain CpG sites are partially attributable to genetic variation. This study strongly supports future epigenome-wide association study using leukocyte DNA for biomarker discovery in human diseases.

  18. Epigenetic regulation of HIV-1 latency by cytosine methylation.

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    Steven E Kauder

    2009-06-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 persists in a latent state within resting CD4+ T cells of infected persons treated with highly active antiretroviral therapy (HAART. This reservoir must be eliminated for the clearance of infection. Using a cDNA library screen, we have identified methyl-CpG binding domain protein 2 (MBD2 as a regulator of HIV-1 latency. Two CpG islands flank the HIV-1 transcription start site and are methylated in latently infected Jurkat cells and primary CD4+ T cells. MBD2 and histone deacetylase 2 (HDAC2 are found at one of these CpG islands during latency. Inhibition of cytosine methylation with 5-aza-2'deoxycytidine (aza-CdR abrogates recruitment of MBD2 and HDAC2. Furthermore, aza-CdR potently synergizes with the NF-kappaB activators prostratin or TNF-alpha to reactivate latent HIV-1. These observations confirm that cytosine methylation and MBD2 are epigenetic regulators of HIV-1 latency. Clearance of HIV-1 from infected persons may be enhanced by inclusion of DNA methylation inhibitors, such as aza-CdR, and NF-kappaB activators into current antiviral therapies.

  19. CpG oligodeoxynucleotide nanomedicines for the prophylaxis or treatment of cancers, infectious diseases, and allergies

    Science.gov (United States)

    Hanagata, Nobutaka

    2017-01-01

    Unmethylated cytosine-guanine dinucleotide-containing oligodeoxynucleotides (CpG ODNs), which are synthetic agonists of Toll-like receptor 9 (TLR9), activate humoral and cellular immunity and are being developed as vaccine adjuvants to prevent or treat cancers, infectious diseases, and allergies. Free CpG ODNs have been used in many clinical trials implemented to verify their effects. However, recent research has reported that self-assembled CpG ODNs, protein/peptide–CpG ODN conjugates, and nanomaterial–CpG ODN complexes demonstrate higher adjuvant effects than free CpG ODNs, owing to their improved uptake efficiency into cells expressing TLR9. Moreover, protein/peptide–CpG ODN conjugates and nanomaterial–CpG ODN complexes are able to deliver CpG ODNs and antigens (or allergens) to the same types of cells, which enables a higher degree of prophylaxis or therapeutic effect. In this review, the author describes recent trends in the research and development of CpG ODN nanomedicines containing self-assembled CpG ODNs, protein/peptide–CpG ODN conjugates, and nanomaterial–CpG ODN complexes, focusing mainly on the results of preclinical and clinical studies. PMID:28144136

  20. Multi-layered multi-pattern CPG for adaptive locomotion of humanoid robots.

    Science.gov (United States)

    Nassour, John; Hénaff, Patrick; Benouezdou, Fethi; Cheng, Gordon

    2014-06-01

    In this paper, we present an extended mathematical model of the central pattern generator (CPG) in the spinal cord. The proposed CPG model is used as the underlying low-level controller of a humanoid robot to generate various walking patterns. Such biological mechanisms have been demonstrated to be robust in locomotion of animal. Our model is supported by two neurophysiological studies. The first study identified a neural circuitry consisting of a two-layered CPG, in which pattern formation and rhythm generation are produced at different levels. The second study focused on a specific neural model that can generate different patterns, including oscillation. This neural model was employed in the pattern generation layer of our CPG, which enables it to produce different motion patterns-rhythmic as well as non-rhythmic motions. Due to the pattern-formation layer, the CPG is able to produce behaviors related to the dominating rhythm (extension/flexion) and rhythm deletion without rhythm resetting. The proposed multi-layered multi-pattern CPG model (MLMP-CPG) has been deployed in a 3D humanoid robot (NAO) while it performs locomotion tasks. The effectiveness of our model is demonstrated in simulations and through experimental results.

  1. All three classes of CpG ODNs up-regulate IP-10 gene in pigs.

    Science.gov (United States)

    Dar, Arshud; Nichani, Anil; Lai, Ken; Potter, Andy; Gerdts, Volker; Babiuk, Lorne A; Mutwiri, George

    2010-04-01

    The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-gamma, IL-12 (P40), IL-6, IL-4 and TNF-alpha mRNA, C-class CpG ODN induced significant level of IFN-gamma, IFN-alpha and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12h post stimulation. These in vitro and in vivo observations suggest that interferon-gamma inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.

  2. DNA-methyltransferase1 (DNMT1) binding to CpG rich GABAergic and BDNF promoters is increased in the brain of schizophrenia and bipolar disorder patients.

    Science.gov (United States)

    Dong, E; Ruzicka, W B; Grayson, D R; Guidotti, A

    2015-09-01

    The down regulation of glutamic acid decarboxylase67 (GAD1), reelin (RELN), and BDNF expression in brain of schizophrenia (SZ) and bipolar (BP) disorder patients is associated with overexpression of DNA methyltransferase1 (DNMT1) and ten-eleven translocase methylcytosine dioxygenase1 (TET1). DNMT1 and TET1 belong to families of enzymes that methylate and hydroxymethylate cytosines located proximal to and within cytosine phosphodiester guanine (CpG) islands of many gene promoters, respectively. Altered promoter methylation may be one mechanism underlying the down-regulation of GABAergic and glutamatergic gene expression. However, recent reports suggest that both DNMT1 and TET1 directly bind to unmethylated CpG rich promoters through their respective Zinc Finger (ZF-CXXC) domains. We report here, that the binding of DNMT1 to GABAergic (GAD1, RELN) and glutamatergic (BDNF-IX) promoters is increased in SZ and BP disorder patients and this increase does not necessarily correlate with enrichment in promoter methylation. The increased DNMT1 binding to these promoter regions is detected in the cortex but not in the cerebellum of SZ and BP disorder patients, suggesting a brain region and neuron specific dependent mechanism. Increased binding of DNMT1 positively correlates with increased expression of DNMT1 and with increased binding of MBD2. In contrast, the binding of TET1 to RELN, GAD1 and BDNF-IX promoters failed to change. These data are consistent with the hypothesis that the down-regulation of specific GABAergic and glutamatergic genes in SZ and BP disorder patients may be mediated, at least in part, by a brain region specific and neuronal-activity dependent DNMT1 action that is likely independent of its DNA methylation activity.

  3. A pyrosequencing assay for the quantitative methylation analysis of the PCDHB gene cluster, the major factor in neuroblastoma methylator phenotype.

    Science.gov (United States)

    Banelli, Barbara; Brigati, Claudio; Di Vinci, Angela; Casciano, Ida; Forlani, Alessandra; Borzì, Luana; Allemanni, Giorgio; Romani, Massimo

    2012-03-01

    Epigenetic alterations are hallmarks of cancer and powerful biomarkers, whose clinical utilization is made difficult by the absence of standardization and of common methods of data interpretation. The coordinate methylation of many loci in cancer is defined as 'CpG island methylator phenotype' (CIMP) and identifies clinically distinct groups of patients. In neuroblastoma (NB), CIMP is defined by a methylation signature, which includes different loci, but its predictive power on outcome is entirely recapitulated by the PCDHB cluster only. We have developed a robust and cost-effective pyrosequencing-based assay that could facilitate the clinical application of CIMP in NB. This assay permits the unbiased simultaneous amplification and sequencing of 17 out of 19 genes of the PCDHB cluster for quantitative methylation analysis, taking into account all the sequence variations. As some of these variations were at CpG doublets, we bypassed the data interpretation conducted by the methylation analysis software to assign the corrected methylation value at these sites. The final result of the assay is the mean methylation level of 17 gene fragments in the protocadherin B cluster (PCDHB) cluster. We have utilized this assay to compare the methylation levels of the PCDHB cluster between high-risk and very low-risk NB patients, confirming the predictive value of CIMP. Our results demonstrate that the pyrosequencing-based assay herein described is a powerful instrument for the analysis of this gene cluster that may simplify the data comparison between different laboratories and, in perspective, could facilitate its clinical application. Furthermore, our results demonstrate that, in principle, pyrosequencing can be efficiently utilized for the methylation analysis of gene clusters with high internal homologies.

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

    Science.gov (United States)

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

    2013-01-01

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

  5. Epigenetic DNA Methylation Profiling with MSRE: A Quantitative NGS Approach Using a Parkinson's Disease Test Case

    Directory of Open Access Journals (Sweden)

    Adam G. Marsh

    2016-11-01

    Full Text Available Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS data using methyl-sensitive restriction endonucleases (MSRE. An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1,008 statistically different CpG sites (p textless 0.0025, after false discovery rate correction. A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations. Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression.

  6. 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.

  7. 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.

  8. Use of DNA methylation for cancer detection and molecular classification.

    Science.gov (United States)

    Zhu, Jingde; Yao, Xuebiao

    2007-03-31

    Conjugation of the methyl group at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3' sequence (DNA methylation) is the best studied epigenetic mechanism, which acts together with other epigenetic entities: histone modification, chromatin remodeling and microRNAs to shape the chromatin structure of DNA according to its functional state. The cancer genome is frequently characterized by hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methyl cytosine, the pathological implication of which to the cancerous state has been well established. While the latest genome-wide technologies have been applied to classify and interpret the epigenetic layer of gene regulation in the physiological and disease states, the epigenetic testing has also been seriously explored in clinical practice for early detection, refining tumor staging and predicting disease recurrence. This critique reviews the latest research findings on the use of DNA methylation in cancer diagnosis, prognosis and staging/classification.

  9. DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood.

    Science.gov (United States)

    Lee, Hwan Young; Jung, Sang-Eun; Lee, Eun Hee; Yang, Woo Ick; Shin, Kyoung-Jin

    2016-09-01

    The ability to predict the type of tissues or cells from molecular profiles of crime scene samples has important practical implications in forensics. A previously reported multiplex assay using DNA methylation markers could only discriminate between 4 types of body fluids: blood, saliva, semen, and the body fluid which originates from female reproductive organ. In the present study, we selected 15 menstrual blood-specific CpG marker candidates based on analysis of 12 genome-wide DNA methylation profiles of vaginal fluid and menstrual blood. The menstrual blood-specificity of the candidate markers was confirmed by comparison with HumanMethylation450 BeadChip array data obtained for 58 samples including 12 blood, 12 saliva, 12 semen, 3 vaginal fluid, and 19 skin epidermis samples. Among 15CpG marker candidates, 3 were located in the promoter region of the SLC26A10 gene, and 2 of them (cg09696411 and cg18069290) showed high menstrual blood specificity. DNA methylation at the 2CpG markers was further tested by targeted bisulfite sequencing of 461 additional samples including 49 blood, 52 saliva, 34 semen, 125 vaginal fluid, and 201 menstrual blood. Because the 2 markers showed menstrual blood-specific methylation patterns, we modified our previous multiplex methylation SNaPshot reaction to include these 2 markers. In addition, a blood marker cg01543184 with cross reactivity to semen was replaced with cg08792630, and a semen-specific unmethylation marker cg17621389 was removed. The resultant multiplex methylation SNaPshot allowed positive identification of blood, saliva, semen, vaginal fluid and menstrual blood using the 9CpG markers which show a methylation signal only in the target body fluids. Because of the complexity in cell composition, menstrual bloods produced DNA methylation profiles that vary with menstrual cycle and sample collection methods, which are expected to provide more insight into forensic menstrual blood test. Moreover, because the developed

  10. Immunogenicity and safety of liposome-vaccine encapsulating hepatitis B surface antigen and phosphodiester CpG oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    CHUN YAN HE; QING LIANG LIU

    2006-01-01

    CpG oligodeoxynucleotides (CpG ODN) as adjuvant have been extensively studied in recent years. Phosphodiester CpG ODN (PO CpG ODN) can perfectly mimic bacterial DNA in enhancing immune response but are vulnerable to nucleases in vivo. This study aimed to evaluate the immunostimu latory potential and safety of phosphodiester CpG ODN encapsulated in nonphospholipid liposomes.BALB/c mice were immunized intramuscularly with different formulations of liposomes, CpG ODN and hepatitis B surface antigen (HBsAg). The results demonstrated that the encapsulated PO CpG ODN were protected against rapid degradation in vivo and retained their adjuvant activity. PO CpG ODN encapsulated with HBsAg in liposomes induced strong Th1-biased or Th1/Th2 mixed humoral immune response in mice with the magnitude similar to their phosphothioate equivalent in the same formulation.High IFN-gamma production induced by this formulation confirmed the generation of strong cellular immune response. Additionally, co-delivery of HBsAg and PO CpG ODN improved the immune response over that obtained with separate delivery. Safety experiment showed that liposome-encapsulaed PO CpG ODN and HBsAg caused mild systemic and moderate local adverse reaction. In conclusion, our data shows that PO CpG ODN encapsulated in liposomes fully exhibit their Th1-type adjuvant activity and act as a potential adjuvant for vaccines.

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

    Directory of Open Access Journals (Sweden)

    Deming Li

    2014-06-01

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

  12. DNA Methylation Patterns in the Hypothalamus of Female Pubertal Goats.

    Science.gov (United States)

    Yang, Chen; Ye, Jing; Li, Xiumei; Gao, Xiaoxiao; Zhang, Kaifa; Luo, Lei; Ding, Jianping; Zhang, Yunhai; Li, Yunsheng; Cao, Hongguo; Ling, Yinghui; Zhang, Xiaorong; Liu, Ya; Fang, Fugui

    2016-01-01

    Female pubertal development is tightly controlled by complex mechanisms, including neuroendocrine and epigenetic regulatory pathways. Specific gene expression patterns can be influenced by DNA methylation changes in the hypothalamus, which can in turn regulate timing of puberty onset. In order to understand the relationship between DNA methylation changes and gene expression patterns in the hypothalamus of pubertal goats, whole-genome bisulfite sequencing and RNA-sequencing analyses were carried out. There was a decline in DNA methylation levels in the hypothalamus during puberty and 268 differentially methylated regions (DMR) in the genome, with differential patterns in different gene regions. There were 1049 genes identified with distinct expression patterns. High levels of DNA methylation were detected in promoters, introns and 3'-untranslated regions (UTRs). Levels of methylation decreased gradually from promoters to 5'-UTRs and increased from 5'-UTRs to introns. Methylation density analysis demonstrated that methylation level variation was consistent with the density in the promoter, exon, intron, 5'-UTRs and 3'-UTRs. Analyses of CpG island (CGI) sites showed that the enriched gene contents were gene bodies, intergenic regions and introns, and these CGI sites were hypermethylated. Our study demonstrated that DNA methylation changes may influence gene expression profiles in the hypothalamus of goats during the onset of puberty, which may provide new insights into the mechanisms involved in pubertal onset.

  13. 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...

  14. Analysis of methylation microarray for tissue specific detection.

    Science.gov (United States)

    Muangsub, Tachapol; Samsuwan, Jarunya; Tongyoo, Pumipat; Kitkumthorn, Nakarin; Mutirangura, Apiwat

    2014-12-10

    The role of human DNA methylation has been extensively studied in genomic imprinting, X-inactivation, and disease. However, studies of tissue-specific methylation remain limited. In this study, we use bioinformatics methods to analyze methylation data and reveal loci that are exclusively methylated or unmethylated in individual tissues. We collect 39 previously published DNA methylation profiles using an Illumina® HumanMethylation 27 BeadChip Kit containing 22 common tissues and involving 27,578 CpG loci across the human genome. We found 86 positions of tissue specific methylation CpG (TSM) that encompass 34 hypermethylated TSMs (31 genes) and 52 hypomethylated TSMs (47 genes). Tissues were found to contain 1 to 25 TSM loci, with the majority in the liver (25), testis (18), and brain (16). Fewer TSM loci were found in the muscle (8), ovary (7), adrenal gland (3), pancreas (2-4), kidney, spleen, and stomach (1 each). TSMs are predominantly located 0-300 base pairs in the 3' direction after the transcription start site. Similar to known promoters of methylation, hypermethylated TSM genes suppress transcription, while hypomethylated TSMs allow gene transcription. The majority of hypermethylated TSM genes encode membrane proteins and receptors, while hypomethylated TSM genes primarily encode signal peptides and tissue-specific proteins. In summary, the database of TSM loci produced herein is useful for the selection of tissue-specific DNA markers as diagnostic tools, as well as for the further study of the mechanisms and roles of TSM. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. High-throughput engineering of a mammalian genome reveals building principles of methylation states at CG rich regions.

    Science.gov (United States)

    Krebs, Arnaud R; Dessus-Babus, Sophie; Burger, Lukas; Schübeler, Dirk

    2014-09-26

    The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.

  16. TET1 is a DNA-binding protein that modulates DNA methylation and gene transcription via hydroxylation of 5-methylcytosine

    Institute of Scientific and Technical Information of China (English)

    Haikuo Zhang; Xin Zhang; Erin Clark; Michelle Mulcahey; Stephen Huang; Yujiang Geno Shi

    2010-01-01

    @@ Dear Editor, DNA methylation, which often occurs at the 5-carbon position of cytosine (5mC) located in CpG dinucleotide, is a key epigenetic hallmark and serves as a major epigenetic mechanism for establishing X-inactivation, paren tal imprinting and silencing retrotransposable elements during early embryogenesis in mammals.

  17. PRKCZ methylation is associated with sunlight exposure in a North American but not a Mediterranean population.

    Science.gov (United States)

    Aslibekyan, Stella; Dashti, Hassan S; Tanaka, Toshiko; Sha, Jin; Ferrucci, Luigi; Zhi, Degui; Bandinelli, Stefania; Borecki, Ingrid B; Absher, Devin M; Arnett, Donna K; Ordovas, Jose M

    2014-11-01

    Sunlight exposure has been shown to alter DNA methylation patterns across several human cell-types, including T-lymphocytes. Since epigenetic changes establish gene expression profiles, changes in DNA methylation induced by sunlight exposure warrant investigation. The purpose of this study was to assess the effects of sunlight exposure on CD4+ T-cell methylation patterns on an epigenome-wide scale in a North American population of European origin (n=991). In addition, we investigated the genetic contribution to epigenetic variation (methylQTL). We used linear regression to test the associations between methylation scores at 461,281 cytosine-phosphate-guanine (CpG) sites and sunlight exposure, followed by a genome-wide association analysis (methylQTL) to test for associations between methylation at the top CpG locus and common genetic variants, assuming an additive genetic model. We observed an epigenome-wide significant association between sunlight exposure and methylation status at cg26930596 (p=9.2×10(-8)), a CpG site located in protein kinase C zeta (PRKCZ), a gene previously shown to be entrained by light. MethylQTL analysis resulted in significant associations between cg26930596 and two intergenic single nucleotide polymorphisms on chromosome 3, rs4574216 (p=1.5×10(-10)) and rs4405858 (p=1.9×10(-9)). These common genetic variants reside downstream of WWTR1, a transcriptional co-activator of PRKCZ. Associations observed in the North American population, however, did not replicate in an independent Mediterranean cohort. Our preliminary results support the role of sunlight exposure in epigenetic processes, and lay the groundwork for future studies of the molecular link between sunlight and physiologic processes such as tumorigenesis and metabolism.

  18. 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.

  19. Identification of 5 novel genes methylated in breast and other epithelial cancers

    Directory of Open Access Journals (Sweden)

    Ward Robyn L

    2010-03-01

    Full Text Available Abstract Background There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer. Results Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate. We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers. Conclusion The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.

  20. Gene-specific DNA methylation of DNMT3B and MTHFR and colorectal adenoma risk.

    Science.gov (United States)

    Ho, Vikki; Ashbury, Janet E; Taylor, Sherryl; Vanner, Stephen; King, Will D

    2015-12-01

    DNA methyltransferase 3B (DNMT3B) and methylenetetrahydrofolate reductase (MTHFR) are genes which encode enzymes critical to one-carbon metabolism. Polymorphisms in these genes have been implicated in colorectal cancer etiology; however, epigenetic modifications such as gene-specific DNA methylation also affect gene expression. DNA methylation of DNMT3B and MTHFR was quantified in blood leukocytes using Sequenom EpiTYPER® among 272 participants undergoing a screening colonoscopy. DNA methylation was quantified in 66 and 28CpG sites of DNMT3B and MTHFR respectively, and conceptualized using two approaches. First, measures representing average methylation across all CpG sites were created. Second, unsupervised principal component (PC) analysis was used to identify summary variables representing methylation around the transcription start site and in the gene-coding area for both DNMT3B and MTHFR. Logistic regression was used to compare methylation levels between participants diagnosed with colorectal adenoma(s) versus those with a normal colonoscopy via the estimation of odds ratios (ORs) and 95% confidence intervals (95% CIs) for the risk of colorectal adenomas. No association was observed between average DNA methylation of either DNMT3B or MTHFR and colorectal adenoma risk. For DNMT3B, increasing DNA methylation of CpG sites in the gene-coding area was associated with a higher risk of colorectal adenomas (OR=1.34; 95% CI: 1.01-1.79 per SD). This research provides preliminary evidence that methylation of DNMT3B may have functional significance with respect to colorectal adenomas, precursors to the vast majority of colorectal cancers.

  1. CpG ODN 免疫佐剂效应研究进展%Progress on Immunoadjuvant Effect of CpG ODN

    Institute of Scientific and Technical Information of China (English)

    董鹏; 程世鹏; 李真光; 杨洺扬; 胡桂学; 冷雪

    2015-01-01

    Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN)could combine with Toll-like receptor 9 (TLR-9)on endoplasmic reticulum from dendritic cells,B cells,monocytes and macrophages to mount an immune response characterized by the production of Th1 and proinflammatory cytokines,as an immune enhancer to resistance to pathogen invasion and antitumor in the hosts.In recent years,CpG ODN as a new immune adjuvant has received more attention.This article mainly introduced the research progress on the kinds of CpG ODN and its structure and function characteristics,mechanisms of action,immune en-hancement livestock and poultry in to strengthen and safety for CpG ODN as vaccine adjuvants to offer ref-erence to further research and its veterinary usage in clinic.%人工合成的含非甲基化 CpG 基序的寡聚脱氧核苷酸(CpG ODN)能够与树突状细胞、B 细胞、单核细胞及巨噬细胞等免疫细胞内质网膜上的 Toll 样受体9(TLR-9)结合,通过产生 Th1型免疫反应和分泌促炎因子来增强免疫应答的强度,在机体抵抗病原入侵和抗肿瘤等方面起到免疫增强剂的作用。近年来, CpG ODN 作为一种新型免疫佐剂受到了人们越来越多的关注。论文主要介绍了 CpG ODN 的种类及其结构和功能特征、作用机制、对畜禽的免疫增强作用、安全性等方面的研究进展,为 CpG ODN 作为疫苗佐剂的进一步研究和在兽医临床上的应用提供参考。

  2. 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.

  3. Real-time Walking Pattern Generation for a Biped Robot with Hybrid CPG-ZMP Algorithm

    Directory of Open Access Journals (Sweden)

    Bin He

    2014-10-01

    Full Text Available Biped robots have better mobility than conventional wheeled robots. The bio-inspired method based on a central pattern generator (CPG can be used to control biped robot walking in a manner like human beings. However, to achieve stable locomotion, it is difficult to modulate the parameters for the neural networks to coordinate every degree of freedom of the walking robot. The zero moment point (ZMP method is very popular for the stability control of biped robot walking. However, the reference trajectories have low energy efficiency, lack naturalness and need significant offline calculation. This paper presents a new method for biped real-time walking generation using a hybrid CPG-ZMP control algorithm. The method can realize a stable walking pattern by combining the ZMP criterion with rhythmic motion control. The CPG component is designed to generate the desired motion for each robot joint, which is modulated by phase resetting according to foot contact information. By introducing the ZMP location, the activity of the CPG output signal is adjusted to coordinate the limbs’ motion and allow the robot to maintain balance during the process of locomotion. The numerical simulation results show that, compared with the CPG method, the new hybrid CPG-ZMP algorithm can enhance the robustness of the CPG parameters and improve the stability of the robot. In addition, the proposed algorithm is more energy efficient than the ZMP method. The results also demonstrate that the control system can generate an adaptive walking pattern through interactions between the robot, the CPG and the environment.

  4. Design of a cyclic inhibitory CPG controller for the locomotion of a snakelike robot

    Institute of Scientific and Technical Information of China (English)

    LU Zhen-li; MA Shu-gen; LI Bin; WANG Yue-chao

    2006-01-01

    The rhythmic locomotion of a creature is a serf-excitation behavior of the CPG (central pattern generator),which makes it supremely adapted for environment.Based on this fact,firstly,a snake-like robot controller with cyclic inhibitory CPG model was designed,and then the stability of a single neuron,CPG model and the NON(neuron oscillator network) was analyzed.By implementing this control architecture to a simulator based on the mechanical dynamics of a real snake-like robot named Perambulator-I,we presented preliminary rules for parameter setting of the CPG controller to modulate the number of S shapes,the curve of the body shape,locomotion velocity,and the curve of the locomotion trajectory for serpentine locomotion.Moreover,we demonstrated that Perambulator-I can successfully exhibit serpentine locomotion by using the output of the proposed CPG controller.The results of this paper provide a realistic approach for designing an artificial CPG controller.

  5. CpG oligodeoxynucleotides with crude parasite antigens reduce worm recovery in Opisthorchis viverrini infected hamsters.

    Science.gov (United States)

    Kaewraemruaen, Chamraj; Sermswan, Rasana W; Wongratanacheewin, Surasakdi

    2016-12-01

    Opisthorchis viverrini, a human liver fluke, is still an endemic parasitic infection in Thailand and nearly all countries in Southeast Asia. O. viverrini induces a chronic stage of infection in hamsters. During the first 2 weeks of infection, Th1 inducing cytokine, IL-12, increased but was down regulated in chronic infection. In this study it was found that unmethylated-CpG ODN (oligodeoxynucleotides) 1826 increased hamster mononuclear cell proliferation and stimulated IFN-γ production in vitro. The IFN-γ levels in hamster sera were significantly increased in hamsters injected with CpG ODN 1826 alone or plus crude somatic antigens (CSAg). Further investigation using the flow cytometer found that CD4(+)T cells and IFN-γ(+) CD4(+)T cells (Th1-like cells) in the hamster blood were significantly increased. The role of these cells in the protective responses in hamsters was evaluated by challenging with 25 metacercaria and observation for 3 months. The number of worms recovered was significantly reduced in the hamsters injected with CpG ODN 1826 with CSAg, but not in CpG ODN 1826 alone groups when compared to PBS control. The percent of reduction in hamsters against this parasite were 32.95% and 21.49% in the CpG ODN 1826 with CSAg and CpG ODN 1826 alone. This study indicates that CpG ODN 1826 plus parasite antigens elicit a Th1-like response that leads to the enhancement of worm reduction.

  6. CpG ODN Enhances Immunization Effects of Hepatitis B Vaccine in Aged Mice

    Institute of Scientific and Technical Information of China (English)

    Weibing Qin; Jianwei Jiang; Qiaoer Chen; Ning Yang; Yifeng Wang; Xiangcai Wei; Ruqiang Ou

    2004-01-01

    Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides in contexts of unique sequence (CpG motifs) is active as adjuvant in induction of cellular and humoral immune responses in young mice. To date, there are only limited reports about effect of CpG ODN on immune responses against hepatitis B (HB) infection in aged mice. Our studies demonstrated there were significant increases in secreting of total anti-HB IgG, IgG1 and IgG2a, as well as of IL-12 and IFN-γ, when CpG ODNs were injected together with hepatitis B antigen in aged mice. Moreover, CpG ODN could stimulate proliferation of spleen lymphocytes in a dose-dependent manner. Taken together, the results we obtained indicate that the adding of CpG ODN into the vaccine antigen might be useful in development of more effe