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Sample records for repair gene methylation

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. Methylation Analysis of DNA Mismatch Repair Genes Using DNA Derived from the Peripheral Blood of Patients with Endometrial Cancer: Epimutation in Endometrial Carcinogenesis

    PubMed Central

    Takeda, Takashi; Banno, Kouji; Yanokura, Megumi; Adachi, Masataka; Iijima, Moito; Kunitomi, Haruko; Nakamura, Kanako; Iida, Miho; Nogami, Yuya; Umene, Kiyoko; Masuda, Kenta; Kobayashi, Yusuke; Yamagami, Wataru; Hirasawa, Akira; Tominaga, Eiichiro; Susumu, Nobuyuki; Aoki, Daisuke

    2016-01-01

    Germline mutation of DNA mismatch repair (MMR) genes is a cause of Lynch syndrome. Methylation of MutL homolog 1 (MLH1) and MutS homolog 2 (MSH2) has been detected in peripheral blood cells of patients with colorectal cancer. This methylation is referred to as epimutation. Methylation of these genes has not been studied in an unselected series of endometrial cancer cases. Therefore, we examined methylation of MLH1, MSH2, and MSH6 promoter regions of peripheral blood cells in 206 patients with endometrial cancer using a methylation-specific polymerase chain reaction (MSP). Germline mutation of MMR genes, microsatellite instability (MSI), and immunohistochemistry (IHC) were also analyzed in each case with epimutation. MLH1 epimutation was detected in a single patient out of a total of 206 (0.49%)—1 out of 58 (1.72%) with an onset age of less than 50 years. The patient with MLH1 epimutation showed high level MSI (MSI-H), loss of MLH1 expression and had developed endometrial cancer at 46 years old, complicated with colorectal cancer. No case had epimutation of MSH2 or MSH6. The MLH1 epimutation detected in a patient with endometrial cancer may be a cause of endometrial carcinogenesis. This result indicates that it is important to check epimutation in patients with endometrial cancer without a germline mutation of MMR genes. PMID:27754426

  4. Methylation Analysis of DNA Mismatch Repair Genes Using DNA Derived from the Peripheral Blood of Patients with Endometrial Cancer: Epimutation in Endometrial Carcinogenesis.

    PubMed

    Takeda, Takashi; Banno, Kouji; Yanokura, Megumi; Adachi, Masataka; Iijima, Moito; Kunitomi, Haruko; Nakamura, Kanako; Iida, Miho; Nogami, Yuya; Umene, Kiyoko; Masuda, Kenta; Kobayashi, Yusuke; Yamagami, Wataru; Hirasawa, Akira; Tominaga, Eiichiro; Susumu, Nobuyuki; Aoki, Daisuke

    2016-10-14

    Germline mutation of DNA mismatch repair (MMR) genes is a cause of Lynch syndrome. Methylation of MutL homolog 1 (MLH1) and MutS homolog 2 (MSH2) has been detected in peripheral blood cells of patients with colorectal cancer. This methylation is referred to as epimutation. Methylation of these genes has not been studied in an unselected series of endometrial cancer cases. Therefore, we examined methylation of MLH1, MSH2, and MSH6 promoter regions of peripheral blood cells in 206 patients with endometrial cancer using a methylation-specific polymerase chain reaction (MSP). Germline mutation of MMR genes, microsatellite instability (MSI), and immunohistochemistry (IHC) were also analyzed in each case with epimutation. MLH1 epimutation was detected in a single patient out of a total of 206 (0.49%)-1 out of 58 (1.72%) with an onset age of less than 50 years. The patient with MLH1 epimutation showed high level MSI (MSI-H), loss of MLH1 expression and had developed endometrial cancer at 46 years old, complicated with colorectal cancer. No case had epimutation of MSH2 or MSH6. The MLH1 epimutation detected in a patient with endometrial cancer may be a cause of endometrial carcinogenesis. This result indicates that it is important to check epimutation in patients with endometrial cancer without a germline mutation of MMR genes.

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

    PubMed

    Metcalf, Alexander M; Spurdle, Amanda B

    2014-03-01

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

  6. Programmed Genetic Instability: A Tumor-Permissive Mechanism for Maintaining the Evolvability of Higher Species through Methylation-Dependent Mutation of DNA Repair Genes in the Male Germ Line

    PubMed Central

    Zhao, Yongzhong

    2008-01-01

    Tumor suppressor genes are classified by their somatic behavior either as caretakers (CTs) that maintain DNA integrity or as gatekeepers (GKs) that regulate cell survival, but the germ line role of these disease-related gene subgroups may differ. To test this hypothesis, we have used genomic data mining to compare the features of human CTs (n = 38), GKs (n = 36), DNA repair genes (n = 165), apoptosis genes (n = 622), and their orthologs. This analysis reveals that repair genes are numerically less common than apoptosis genes in the genomes of multicellular organisms (P < 0.01), whereas CT orthologs are commoner than GK orthologs in unicellular organisms (P < 0.05). Gene targeting data show that CTs are less essential than GKs for survival of multicellular organisms (P < 0.0005) and that CT knockouts often permit offspring viability at the cost of male sterility. Patterns of human familial oncogenic mutations confirm that isolated CT loss is commoner than is isolated GK loss (P < 0.00001). In sexually reproducing species, CTs appear subject to less efficient purifying selection (i.e., higher Ka/Ks) than GKs (P = 0.000003); the faster evolution of CTs seems likely to be mediated by gene methylation and reduced transcription-coupled repair, based on differences in dinucleotide patterns (P = 0.001). These data suggest that germ line CT/repair gene function is relatively dispensable for survival, and imply that milder (e.g., epimutational) male prezygotic repair defects could enhance sperm variation—and hence environmental adaptation and speciation—while sparing fertility. We submit that CTs and repair genes are general targets for epigenetically initiated adaptive evolution, and propose a model in which human cancers arise in part as an evolutionarily programmed side effect of age- and damage-inducible genetic instability affecting both somatic and germ line lineages. PMID:18535014

  7. Protein expression and methylation of MGMT, a DNA repair gene and their correlation with clinicopathological parameters in invasive ductal carcinoma of the breast.

    PubMed

    Asiaf, Asia; Ahmad, Shiekh Tanveer; Malik, Ajaz Ahmad; Aziz, Shiekh Aejaz; Rasool, Zubaida; Masood, Akbar; Zargar, Mohammad Afzal

    2015-08-01

    Epigenetic mechanisms such as DNA methylation are being increasingly recognized to play an important role in cancer and may serve as a cancer biomarker. The aim of this study was to evaluate the promoter methylation status of MGMT (O6-methylguanine-DNA methyltransferase) and a possible correlation with the expression of MGMT and standard clinicopathological parameters in invasive ductal breast carcinoma patients (IDC) of Kashmir. Methylation-specific PCR was carried out to investigate the promoter methylation status of MGMT in breast tumors paired with the corresponding normal tissue samples from 128 breast cancer patients. The effect of promoter methylation on protein expression in the primary breast cancer and adjacent normal tissues was evaluated by immunohistochemistry (n = 128) and western blotting (n = 30). The frequency of tumor hypermethylation was 39.8 % and a significant difference in methylation frequency among breast tumors were found (p < 0.001) when compared with the corresponding normal tissue. Immunohistochemical analysis showed no detectable expression of MGMT in 68/128 (53.1 %) tumors. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression (rs = -0.285, p = 0.001, OR = 3.38, 95 % CI = 1.59-7.17). A significant correlation was seen between loss of MGMT and lymph node involvement (p = 0.030), tumor grade (p < 0.0001), loss of estrogen receptors (ER; p = 0.021) and progesterone receptors (PR) (p = 0.016). Also, MGMT methylation was found to be associated with tumor grade (p = 0.011), tumor stage (p = 0.009), and loss of ER (p = 0.003) and PR receptors (p = 0.009). To our knowledge, our findings, for the first time, in Kashmiri population, indicate that MGMT is aberrantly methylated in breast cancer and promoter hypermethylation could be attributed to silencing of MGMT gene expression in breast cancer. Our data suggests that MGMT promoter

  8. The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice

    PubMed Central

    Langie, Sabine A. S.; Cameron, Kerry M.; Ficz, Gabriella; Oxley, David; Tomaszewski, Bartłomiej; Gorniak, Joanna P.; Maas, Lou M.; Godschalk, Roger W. L.; van Schooten, Frederik J.; Reik, Wolf; von Zglinicki, Thomas; Mathers, John C.

    2017-01-01

    Base excision repair (BER) may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases. The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events (e.g., DNA methylation) contribute to the ageing process and may be functionally important through the regulation of the expression of DNA repair genes. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain. Brains from male mice were isolated at 3–32 months of age. Pyrosequencing analyses revealed significantly increased Ogg1 methylation with ageing, which correlated inversely with Ogg1 expression. The reduced Ogg1 expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2. A significant inverse correlation between Neil1 methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2′-deoxyguanosine levels. These data indicate that Ogg1 and Neil1 expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain. PMID:28218666

  9. Methylating agents and DNA repair responses: methylated bases and sources of strand breaks

    PubMed Central

    Wyatt, Michael D.; Pittman, Douglas L.

    2008-01-01

    The chemical methylating agents methylmethane sulfonate (MMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) have been used for decades as classical DNA damaging agents. These agents have been utilized to uncover and explore pathways of DNA repair, DNA damage response, and mutagenesis. MMS and MNNG modify DNA by adding methyl groups to a number of nucleophilic sites on the DNA bases, although MNNG produces a greater percentage of O-methyl adducts. There has been substantial progress elucidating direct reversal proteins that remove methyl groups and base excision repair (BER), which removes and replaces methylated bases. Direct reversal proteins and BER thus counteract the toxic, mutagenic and clastogenic effects of methylating agents. Despite recent progress, the complexity of DNA damage responses to methylating agents is still being discovered. In particular, there is growing understanding of pathways such as homologous recombination, lesion bypass, and mismatch repair that react when the response of direct reversal proteins and BER is insufficient. Furthermore, the importance of proper balance within the steps in BER has been uncovered with the knowledge that DNA structural intermediates during BER are deleterious. A number of issues complicate elucidating the downstream responses when direct reversal is insufficient or BER is imbalanced. These include inter-species differences, cell-type specific differences within mammals and between cancer cell lines, and the type of methyl damage or BER intermediate encountered. MMS also carries a misleading reputation of being a ‘radiomimetic,’ i.e., capable of directly producing strand breaks. This review focuses on the DNA methyl damage caused by MMS and MNNG for each site of potential methylation to summarize what is known about the repair of such damage and the downstream responses and consequences if not repaired. PMID:17173371

  10. Correlation of tumour BRAF mutations and MLH1 methylation with germline mismatch repair (MMR) gene mutation status: a literature review assessing utility of tumour features for MMR variant classification.

    PubMed

    Parsons, Michael T; Buchanan, Daniel D; Thompson, Bryony; Young, Joanne P; Spurdle, Amanda B

    2012-03-01

    Colorectal cancer (CRC) that demonstrates microsatellite instability (MSI) is caused by either germline mismatch repair (MMR) gene mutations, or 'sporadic' somatic tumour MLH1 promoter methylation. MLH1 promoter methylation is reportedly correlated with tumour BRAF V600E mutation status. No systematic review has been undertaken to assess the value of BRAF V600E mutation and MLH1 promoter methylation tumour markers as negative predictors of germline MMR mutation status. A literature review of CRC cohorts tested for MMR mutations, and tumour BRAF V600E mutation and/or MLH1 promoter methylation was conducted using PubMed. Studies were assessed for tumour features, stratified by tumour MMR status based on immunohistochemistry or MSI where possible. Pooled frequencies and 95% CIs were calculated using a random effects model. BRAF V600E results for 4562 tumours from 35 studies, and MLH1 promoter methylation results for 2975 tumours from 43 studies, were assessed. In 550 MMR mutation carriers, the BRAF V600E mutation frequency was 1.40% (95% CI 0.06% to 3%). In MMR mutation-negative cases, the BRAF V600E mutation frequency was 5.00% (95% CI 4% to 7%) in 1623 microsatellite stable (MSS) cases and 63.50% (95% CI 47% to 79%) in 332 cases demonstrating MLH1 methylation or MLH1 expression loss. MLH1 promoter methylation of the 'A region' was reported more frequently than the 'C region' in MSS CRCs (17% vs 0.06%, p<0.0001) and in MLH1 mutation carriers (42% vs 6%, p<0.0001), but not in MMR mutation-negative MSI-H CRCs (40% vs 47%, p=0.12). Methylation of the 'C region' was a predictor of MMR mutation-negative status in MSI-H CRC cases (47% vs 6% in MLH1 mutation carriers, p<0.0001). This review demonstrates that tumour BRAF V600E mutation, and MLH1 promoter 'C region' methylation specifically, are strong predictors of negative MMR mutation status. It is important to incorporate these features in multifactorial models aimed at predicting MMR mutation status.

  11. Gene methylation in gastric cancer.

    PubMed

    Qu, Yiping; Dang, Siwen; Hou, Peng

    2013-09-23

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

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

    PubMed

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

    2005-01-26

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

  13. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  14. Repair of Alkylation Damage: Stability of Methyl Groups in Bacillus subtilis Treated with Methyl Methanesulfonate

    PubMed Central

    Prakash, Louise; Strauss, Bernard

    1970-01-01

    Bacillus subtilis was not inactivated and was able to replicate even though approximately 3 × 104 methyl groups added by methyl methanesulfonate (MMS) were bound to the deoxyribonucleic acid (DNA) of each organism. No significant loss of methyl groups from the DNA occurred for several generations upon incubation of methylated wild-type or MMS-sensitive cells. Single-strand breaks were not observed in the DNA from cells treated at this low MMS dose. Higher doses of MMS resulted in significant killing of both wild-type and MMS-sensitive strains, and the DNA extracted from such treated cells sedimented more slowly than control DNA through alkaline sucrose gradients, indicating the presence of breaks or apurinic sites (or both). These breaks were repaired upon incubation of wild-type but not of MMS-sensitive strains. Repair of damage induced by alkylating agents is probably the repair of breaks which occur as a consequence of high levels of alkylation. PMID:4988041

  15. DNA Methylation of BDNF Gene in Schizophrenia.

    PubMed

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

    2016-02-06

    BACKGROUND Although genetic factors are risk factors for schizophrenia, some environmental factors are thought to be required for the manifestation of disease. Epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of DNA. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. It is established that methylation status of the BDNF gene is associated with fear learning, memory, and stressful social interactions. In this study, we aimed to investigate the DNA methylation status of BDNF gene in patients with schizophrenia. MATERIAL AND METHODS The study included 49 patients (33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (46 male and 19 female). Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA results in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA. RESULTS There was no significant difference in methylated or un-methylated status for BDNF promoters between schizophrenia patients and controls. The mean duration of illness was significantly lower in the hemi-methylated group compared to the non-methylated group for BDNF gene CpG island-1 in schizophrenia patients. CONCLUSIONS Although there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls, there was an association between duration of illness and DNA methylation.

  16. DNA Methylation of BDNF Gene in Schizophrenia

    PubMed Central

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

    2016-01-01

    Background Although genetic factors are risk factors for schizophrenia, some environmental factors are thought to be required for the manifestation of disease. Epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of DNA. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. It is established that methylation status of the BDNF gene is associated with fear learning, memory, and stressful social interactions. In this study, we aimed to investigate the DNA methylation status of BDNF gene in patients with schizophrenia. Material/Methods The study included 49 patients (33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (46 male and 19 female). Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA results in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA. Results There was no significant difference in methylated or un-methylated status for BDNF promoters between schizophrenia patients and controls. The mean duration of illness was significantly lower in the hemi-methylated group compared to the non-methylated group for BDNF gene CpG island-1 in schizophrenia patients. Conclusions Although there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls, there was an association between duration of illness and DNA methylation. PMID:26851233

  17. DNA repair genes in the Megavirales pangenome.

    PubMed

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  18. The clinical value of aberrant epigenetic changes of DNA damage repair genes in human cancer

    PubMed Central

    Gao, Dan; Herman, James G.; Guo, Mingzhou

    2016-01-01

    The stability and integrity of the human genome are maintained by the DNA damage repair (DDR) system. Unrepaired DNA damage is a major source of potentially mutagenic lesions that drive carcinogenesis. In addition to gene mutation, DNA methylation occurs more frequently in DDR genes in human cancer. Thus, DNA methylation may play more important roles in DNA damage repair genes to drive carcinogenesis. Aberrant methylation patterns in DNA damage repair genes may serve as predictive, diagnostic, prognostic and chemosensitive markers of human cancer. MGMT methylation is a marker for poor prognosis in human glioma, while, MGMT methylation is a sensitive marker of glioma cells to alkylating agents. Aberrant epigenetic changes in DNA damage repair genes may serve as therapeutic targets. Treatment of MLH1-methylated colon cancer cell lines with the demethylating agent 5′-aza-2′-deoxycytidine induces the expression of MLH1 and sensitizes cancer cells to 5-fluorouracil. Synthetic lethality is a more exciting approach in patients with DDR defects. PARP inhibitors are the most effective anticancer reagents in BRCA-deficient cancer cells. PMID:26967246

  19. Transactivation of repair genes by BRCA1.

    PubMed

    El-Deiry, Wafik S

    2002-01-01

    Recent studies have identified a link between the BRCA1 tumor suppressor and transcriptional regulation of a group of genes involved in nucleotide excision repair. There is some controversy regarding the precise mechanism of upregulation of XPE DDB2 or XPC by BRCA1, with some evidence suggesting that p53 is involved in their regulation. Some evidence suggests BRCA1 may stabilize p53 and direct regulation of DNA repair genes, although how BRCA1 stabilizes p53 remains unclear and whether BRCA1 can upregulate DNA repair genes in a p53-independent manner remains a possibility. A transcriptional component to the action of BRCA1 and involvement of XP genes brings up new and interesting questions about breast cancer development and therapy.

  20. Exercise effects on methylation of ASC gene.

    PubMed

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

    2010-09-01

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

  1. Gene Therapy for Fracture Repair

    DTIC Science & Technology

    2005-12-01

    chemotactic factor for human mast cells. J. Immunol. 153: 3717-3723. 36 41. Ono I, Yamashita T, Hida T, Jin HY, Ito Y, Hamada H, Akasaka Y, Ishii T...1994;153:3717–23. [37] Ono I, Yamashita T, Hida T, Jin HY, Ito Y, Hamada H, et al. Local administration of hepatocyte growth factor gene enhances the

  2. A mediator methylation mystery: JMJD1C demethylates MDC1 to regulate DNA repair.

    PubMed

    Lu, Jian; Matunis, Michael J

    2013-12-01

    Mediator of DNA-damage checkpoint 1 (MDMDC1) has a central role in repair of DNA double-strand breaks (DSBs) by both homologous recombination and nonhomologous end joining, and its function is regulated by post-translational phosphorylation, ubiquitylation and sumoylation. In this issue, a new study by Watanabe et al. reveals that methylation of MDMDC1 is also critical for its function in DSB repair and specifically affects repair through BRCA1-dependent homologous recombination.

  3. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome.

    PubMed

    Sneppen, Kim; Semsey, Szabolcs

    2014-12-05

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome.

  4. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome

    NASA Astrophysics Data System (ADS)

    Sneppen, Kim; Semsey, Szabolcs

    2014-12-01

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome.

  5. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome

    PubMed Central

    Sneppen, Kim; Semsey, Szabolcs

    2014-01-01

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome. PMID:25475788

  6. Gene Therapy for Fracture Repair

    DTIC Science & Technology

    2003-12-01

    relative transgene expression efficiencies for the MLV-based and lentiviral-based vectors, the Enhanced Green Fluorescent Protein (EGFP) was used as...for both Cy3 and Cy5 2,-15i Hybridized to to Aigilent Rat -s 2-- Gene Chip - iGnTrr. . tea 2 ug universal RNAw silx sl59 (?es) Cy310-0 (control) 1...fractures were also examined at sacrifice for evidence of fibrosis due to irritation or migration of the stabilizing pin. None was observed and the fracture

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

    SciTech Connect

    Hagensee, M.E.; Bryan, S.K.; Moses, R.E.

    1987-10-01

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

  8. GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

    PubMed Central

    Lee, Bongyong; Morano, Annalisa; Porcellini, Antonio; Muller, Mark T.

    2012-01-01

    In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair. PMID:22135303

  9. Both base excision repair and O6-methylguanine-DNA methyltransferase protect against methylation-induced colon carcinogenesis

    PubMed Central

    Wirtz, Stefan; Nagel, Georg; Eshkind, Leonid; Neurath, Markus F.; Samson, Leona D.; Kaina, Bernd

    2010-01-01

    Methylating agents are widely distributed environmental carcinogens. Moreover, they are being used in cancer chemotherapy. The primary target of methylating agents is DNA, and therefore, DNA repair is the first-line barrier in defense against their toxic and carcinogenic effects. Methylating agents induce in the DNA O6-methylguanine (O6MeG) and methylations of the ring nitrogens of purines. The lesions are repaired by O6-methylguanine-DNA methyltransferase (Mgmt) and by enzymes of the base excision repair (BER) pathway, respectively. Whereas O6MeG is well established as a pre-carcinogenic lesion, little is known about the carcinogenic potency of base N-alkylation products such as N3-methyladenine and N3-methylguanine. To determine their role in cancer formation and the role of BER in cancer protection, we checked the response of mice with a targeted gene disruption of Mgmt or N-alkylpurine-DNA glycosylase (Aag) or both Mgmt and Aag, to azoxymethane (AOM)-induced colon carcinogenesis, using non-invasive mini-colonoscopy. We demonstrate that both Mgmt- and Aag-null mice show a higher colon cancer frequency than the wild-type. With a single low dose of AOM (3 mg/kg) Aag-null mice showed an even stronger tumor response than Mgmt-null mice. The data provide evidence that both BER initiated by Aag and O6MeG reversal by Mgmt are required for protection against alkylation-induced colon carcinogenesis. Further, the data indicate that non-repaired N-methylpurines are not only pre-toxic but also pre-carcinogenic DNA lesions. PMID:20732909

  10. DNA repair genes of mammalian cells

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Salazar, E.P.; Fuscoe, J.C.; Weber, C.A.

    1985-09-27

    In the CHO cell line various mutations affecting DNA repair have been obtained. Mutants that belong to five genetic complementation groups for UV sensitivity and resemble the cells from individuals having the cancer-prone genetic disorder xeroderma pigmentosum were previously identified. Each mutant is defective in the incision step of nucleotide excision repair and hypersensitive to bulky DNA lesions. A sixth genetic complementation group for UV sensitivity has now been identified with UV27-1. These UV mutants can be divided into two subgroups; only Groups 2 and 4 are extremely sensitive to mitomycin C and other DNA cross-linking agents. The clear-cut phenotypes of the CHO mutants have allowed us to construct hybrid cells by fusion with human lymphocytes and thereby identify which human chromosomes carry genes that correct the CHO mutations. The first two mutants analyzed, UV20 (excision-repair deficient; UV Group 2) and EM9, which has very high SCE, are both corrected by chromosome 19. 46 refs., 3 figs.

  11. Gene repair and transposon-mediated gene therapy.

    PubMed

    Richardson, Paul D; Augustin, Lance B; Kren, Betsy T; Steer, Clifford J

    2002-01-01

    The main strategy of gene therapy has traditionally been focused on gene augmentation. This approach typically involves the introduction of an expression system designed to express a specific protein in the transfected cell. Both the basic and clinical sciences have generated enough information to suggest that gene therapy would eventually alter the fundamental practice of modern medicine. However, despite progress in the field, widespread clinical applications and success have not been achieved. The myriad deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. One, derived primarily from the pioneering work of homologous recombination, is gene repair. Simply stated, the process involves targeting the mutation in situ for gene correction and a return to normal gene function. Site-specific genetic repair has many advantages over augmentation although it too is associated with significant limitations. This review outlines the advantages and disadvantages of gene correction. In particular, we discuss technologies based on chimeric RNA/DNA oligonucleotides, single-stranded and triplex-forming oligonucleotides, and small fragment homologous replacement. While each of these approaches is different, they all share a number of common characteristics, including the need for efficient delivery of nucleic acids to the nucleus. In addition, we review the potential application of a novel and exciting nonviral gene augmentation strategy--the Sleeping Beauty transposon system.

  12. Inactivation of ultraviolet repair in normal and xeroderma pigmentosum cells by methyl methanesulfonate

    SciTech Connect

    Cleaver, J.E.

    1982-03-01

    Excision repair of ultraviolet damage in the DNA of normal and xeroderma pigmentosum (Groups C, D, and variant) cells was inactivated by exposure of cells to methyl methanesulfonate immediately before irradiation independent of the presence of 0 to 10% fetal calf serum. The inactivation could be represented by a semilog relationship between the amount of repair and methyl methanesulfonate concentration up to approximately 5 mM. The inactivation can be considered to occur as the result of alkylation of a large (about 10(6) daltons) repair enzyme complex, and the dose required to reduce repair to 37% for most cells types was between 4 and 7 mM. No consistent, large difference in sensitivity to methyl methanesulfonate was found in any xeroderma pigmentosum complementation group compared to normal cells, implying that reduced repair in these groups may be caused by small inherited changes in the amino acid composition (i.e., point mutations or small deletions) rather than by losses of major components of the repair enzyme complex.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  15. Mutation and repair induced by the carcinogen 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in the dihydrofolate reductase gene of Chinese hamster ovary cells and conformational modeling of the dG-C8-PhIP adduct in DNA.

    PubMed

    Carothers, A M; Yuan, W; Hingerty, B E; Broyde, S; Grunberger, D; Snyderwine, E G

    1994-01-01

    Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. Body Mass Index is Associated with Gene Methylation in Estrogen Receptor-Positive Breast Tumors

    PubMed Central

    Hair, Brionna Y.; Troester, Melissa A.; Edmiston, Sharon N.; Parrish, Eloise A.; Robinson, Whitney R.; Wu, Michael C.; Olshan, Andrew F.; Swift-Scanlan, Theresa; Conway, Kathleen

    2015-01-01

    Background Although obesity is associated with breast cancer incidence and prognosis, the underlying mechanisms are poorly understood. Identification of obesity-associated epigenetic changes in breast tissue may advance mechanistic understanding of breast cancer initiation and progression. The goal of this study, therefore, was to investigate associations between obesity and gene methylation in breast tumors. Methods Using the Illumina GoldenGate Cancer I Panel, we estimated the association between body mass index (BMI) and gene methylation in 345 breast tumor samples from Phase I of the Carolina Breast Cancer Study, a population based case-control study. Multivariable linear regression was used to identify sites that were differentially methylated by BMI. Stratification by tumor estrogen receptor status was also conducted. Results In the majority of the 935 probes analyzed (87%), the average beta value increased with obesity (BMI ≥ 30). Obesity was significantly associated with differential methylation (false discovery rate q-value < 0.05) in just 2 gene loci in breast tumor tissue overall and in 21 loci among estrogen receptor (ER)-positive tumors. Obesity was associated with methylation of genes that function in immune response, cell growth, and DNA repair. Conclusions Obesity is associated with altered methylation overall, and with hypermethylation among ER-positive tumors in particular, suggesting that obesity may influence the methylation of genes with known relevance to cancer. Some of these differences in methylation by obese status may influences levels of gene expression within breast cells. Impact If our results are validated, obesity-associated methylation sites could serve as targets for prevention and treatment research. PMID:25583948

  19. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  20. Incomplete methylation of the FMR gene in amniotic cells

    SciTech Connect

    Skare, J.C.; Townes, P.L.

    1994-09-01

    Fragile X mental retardation is usually caused by expansion of triplet repeats near the 5{prime} end of the FMR gene. It has been reported that expansions over 600 bp (full mutations) result in mental retardation of males. Furthermore, FMR genes with full mutations have methylation of certain CpG dinucleotides upstream of the gene, one of which is in an Eag I recognition site. Methylation of the Eag I site correlates with transcriptional inactivation. We report a pregnancy with twin males which were shown to be dizygotic by RFLP analysis. The mother possessed an expansion of 150 bp in one of her FMR genes. Amniocentesis was performed. One fetus had an FMR gene with a 600 bp expansion and the other had a heterogeneous expansion with an average of 1100 bp. The gene with a 600 bp expansion had no methylation of its Eag I site, while about half of the FMR genes with the 1100 bp expansion had methylated Eag I sites. At birth, peripheral blood DNA was examined. The extent of methylation in the newborn with the 600 bp expansion had increased to about 50%. The newborn with the 1000 bp expansion was almost completely methylated. Therefore, methylation of FMR genes progressed prenatally in both, fetuses, and the larger expansion was methylated earliest. Furthermore, it would appear that methylation analysis is of limited value in prenatal diagnosis of fragile X mental retardation.

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

    PubMed

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

    2016-05-01

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

  2. Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage.

    PubMed

    Li, Qi; Huang, Yue; Liu, Xichun; Gan, Jianhua; Chen, Hao; Yang, Cai-Guang

    2016-05-20

    The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.

  3. DNA Methylation is Developmentally Regulated for Genes Essential for Cardiogenesis

    PubMed Central

    Chamberlain, Alyssa A.; Lin, Mingyan; Lister, Rolanda L.; Maslov, Alex A.; Wang, Yidong; Suzuki, Masako; Wu, Bingruo; Greally, John M.; Zheng, Deyou; Zhou, Bin

    2014-01-01

    Background DNA methylation is a major epigenetic mechanism altering gene expression in development and disease. However, its role in the regulation of gene expression during heart development is incompletely understood. The aim of this study is to reveal DNA methylation in mouse embryonic hearts and its role in regulating gene expression during heart development. Methods and Results We performed the genome‐wide DNA methylation profiling of mouse embryonic hearts using methyl‐sensitive, tiny fragment enrichment/massively parallel sequencing to determine methylation levels at ACGT sites. The results showed that while global methylation of 1.64 million ACGT sites in developing hearts remains stable between embryonic day (E) 11.5 and E14.5, a small fraction (2901) of them exhibit differential methylation. Gene Ontology analysis revealed that these sites are enriched at genes involved in heart development. Quantitative real‐time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2). Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer. Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co‐expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function. Conclusions DNA methylation is developmentally regulated for genes essential to heart development, and abnormal DNA methylation may contribute to congenital heart disease. PMID:24947998

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

    PubMed

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

    2007-11-19

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2016-01-01

    Neuroblastoma is a very heterogeneous tumor of childhood. The clinical spectra range from very aggressive metastatic disease to spontaneous regression, even without therapy. Aberrant DNA methylation pattern is a common feature of most cancers. For neuroblastoma, it has been demonstrated both for single genes as well as genome-wide, where a so-called methylator phenotype has been described. Here, we present a study using Illumina 450K methylation arrays on 60 neuroblastoma tumors. We show that aggressive tumors, characterized by International Neuroblastoma Risk Group (INRG) as stage M, are hypermethylated compared to low-grade tumors. On the contrary, INRG stage L tumors display more non-CpG methylation. The genes with the highest number of hypermethylated CpG sites in INRG M tumors are TERT, PCDHGA4, DLX5, and DLX6-AS1. Gene ontology analysis showed a representation of neuronal tumor relevant gene functions among the differentially methylated genes. For validation, we used a set of independent tumors previously analyzed with the Illumina 27K methylation arrays, which confirmed the differentially methylated sites. Top candidate genes with aberrant methylation were analyzed for altered gene expression through the R2 platform ( http://r2.amc.nl), and for correlations between methylation and gene expression in a public dataset. Altered expression in nonsurvivors was found for the genes B3GALT4 and KIAA1949, CLIC5, DLX6-AS, TERT, and PIRT, and strongest correlations were found for TRIM36, KIAA0513, and PIRT. Our data indicate that methylation profiling can be used for patient stratification and informs on epigenetically deregulated genes with the potential of increasing our knowledge about the underlying mechanisms of tumor development.

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

    PubMed Central

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

    2016-01-01

    ABSTRACT Neuroblastoma is a very heterogeneous tumor of childhood. The clinical spectra range from very aggressive metastatic disease to spontaneous regression, even without therapy. Aberrant DNA methylation pattern is a common feature of most cancers. For neuroblastoma, it has been demonstrated both for single genes as well as genome-wide, where a so-called methylator phenotype has been described. Here, we present a study using Illumina 450K methylation arrays on 60 neuroblastoma tumors. We show that aggressive tumors, characterized by International Neuroblastoma Risk Group (INRG) as stage M, are hypermethylated compared to low-grade tumors. On the contrary, INRG stage L tumors display more non-CpG methylation. The genes with the highest number of hypermethylated CpG sites in INRG M tumors are TERT, PCDHGA4, DLX5, and DLX6-AS1. Gene ontology analysis showed a representation of neuronal tumor relevant gene functions among the differentially methylated genes. For validation, we used a set of independent tumors previously analyzed with the Illumina 27K methylation arrays, which confirmed the differentially methylated sites. Top candidate genes with aberrant methylation were analyzed for altered gene expression through the R2 platform (http://r2.amc.nl), and for correlations between methylation and gene expression in a public dataset. Altered expression in nonsurvivors was found for the genes B3GALT4 and KIAA1949, CLIC5, DLX6-AS, TERT, and PIRT, and strongest correlations were found for TRIM36, KIAA0513, and PIRT. Our data indicate that methylation profiling can be used for patient stratification and informs on epigenetically deregulated genes with the potential of increasing our knowledge about the underlying mechanisms of tumor development. PMID:26786290

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

    PubMed Central

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

    2014-01-01

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

  10. Mismatch repair genes in renal cortical neoplasms.

    PubMed

    Baiyee, Daniel; Banner, Barbara

    2006-02-01

    Mutation of human mutL homolog 1 (MLH-1) and human mutS homolog 2 (MSH-2) has been linked with the pathogenesis of colorectal carcinoma in hereditary nonpolyposis colorectal cancer syndrome and other carcinomas. Mutations of these genes in renal cell carcinomas were recently described. The aim of this study was to examine the expression of MLH-1 and MSH-2 in renal cortical neoplasms of various histological types by immunohistochemistry. Thirty-eight (n = 38) resected renal tumors were obtained from the surgical pathology files of the UMass Memorial Healthcare, including clear cell carcinomas (CLEARs, n = 20), papillary carcinomas (PAPs, n = 8), chromophobe carcinomas (CHRs, n = 4), and oncocytomas (ONCs, n = 6). Positive immunostaining for MLH-1 and MSH-2 was graded by the number of positive tumor cell nuclei, as follows: 0, negative; 1, up to one third of positive nuclei; 2, one to two thirds positive; and 3, greater than two thirds positive. Loss of MLH-1 or MSH-2 was defined as a tumor with grade 0 or 1, compared with the normal tubules. Normal tubules and intercalated ducts contained cells positive for MLH-1 and MSH-2 in all cases. For both antibodies, positive staining in tumors ranged from grade 1 to 3 in the CLEAR and PAP but was only grade 2 to 3 in the CHR and ONC. Loss of MLH-1 and/or MSH-2 occurred in malignant tumors but not in ONC. Loss of MLH-1 was present in 8 (40%) of 20 CLEARs and 4 (50%) of 8 PAPs, compared with loss of MSH-2 in 4 (20%) of 20 CLEARs and 1 (25%) of 4 CHRs. Our results suggest that loss of mismatch repair genes is involved in the malignant transformation in some renal carcinomas, particularly those derived from the proximal tubules.

  11. Bi-allelic alterations in DNA repair genes underpin homologous recombination DNA repair defects in breast cancer.

    PubMed

    Mutter, Robert W; Riaz, Nadeem; Ng, Charlotte K Y; Delsite, Rob; Piscuoglio, Salvatore; Edelweiss, Marcia; Martelotto, Luciano G; Sakr, Rita A; King, Tari A; Giri, Dilip D; Drobnjak, Maria; Brogi, Edi; Bindra, Ranjit; Bernheim, Giana; Lim, Raymond S; Blecua, Pedro; Desrichard, Alexis; Higginson, Dan; Towers, Russell; Jiang, Ruomu; Lee, William; Weigelt, Britta; Reis-Filho, Jorge S; Powell, Simon N

    2017-03-15

    Homologous recombination (HR) DNA repair deficient (HRD) breast cancers have been shown to be sensitive to DNA repair targeted therapies. Burgeoning evidence suggests that sporadic breast cancers, lacking germline BRCA1/BRCA2 mutations, may also be HRD. We developed a functional ex vivo RAD51-based test to identify HRD primary breast cancers. An integrated approach examining methylation, gene expression and whole-exome sequencing was employed to ascertain the etiology of HRD. Functional HRD breast cancers displayed genomic features of lack of competent HR, including large-scale state transitions and specific mutational signatures. Somatic and/or germline genetic alterations resulting in bi-allelic loss-of-function of HR genes underpinned functional HRD in 89% of cases, and were observed in only one of the 15 HR-proficient samples tested. These findings indicate the importance of a comprehensive genetic assessment of bi-allelic alterations in the HR pathway to deliver a precision medicine-based approach to select patients for therapies targeting tumor-specific DNA repair defects.

  12. Methylated MicroRNA Genes of the Developing Murine Palate

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-04-01

    Previous studies showed sex-specific DNA methylation and expression of candidate genes in bisexual flowers of andromonoecious poplar, but the regulatory relationship between methylation and microRNAs (miRNAs) remains unclear. To investigate whether the methylation of miRNA genes regulates gene expression in bisexual flower development, the methylome, microRNA, and transcriptome were examined in female and male flowers of andromonoecious poplar. 27 636 methylated coding genes and 113 methylated miRNA genes were identified. In the coding genes, 64.5% of the methylated reads mapped to the gene body region; by contrast, 60.7% of methylated reads in miRNA genes mainly mapped in the 5' and 3' flanking regions. CHH methylation showed the highest methylation levels and CHG showed the lowest methylation levels. Correlation analysis showed a significant, negative, strand-specific correlation of methylation and miRNA gene expression (r=0.79, P <0.05). The methylated miRNA genes included eight long miRNAs (lmiRNAs) of 24 nucleotides and 11 miRNAs related to flower development. miRNA172b might play an important role in the regulation of bisexual flower development-related gene expression in andromonoecious poplar, via modification of methylation. Gynomonoecious, female, and male poplars were used to validate the methylation patterns of the miRNA172b gene, implying that hyper-methylation in andromonoecious and gynomonoecious poplar might function as an important regulator in bisexual flower development. Our data provide a useful resource for the study of flower development in poplar and improve our understanding of the effect of epigenetic regulation on genes other than protein-coding genes.

  14. Influence of methyl mercaptan on the repair bond strength of composites fabricated using self-etch adhesives.

    PubMed

    Yokokawa, Miho; Rikuta, Akitomo; Tsujimoto, Akimasa; Tsuchiya, Kenji; Shibasaki, Syo; Matsuyoshi, Saki; Miyazaki, Masashi

    2015-02-01

    The influence of methyl mercaptan on the repair bond strength of composites fabricated using self-etch adhesives was investigated. The surface free-energies were determined by measuring the contact angles of test liquids placed on composites that had been immersed in different concentrations of methyl mercaptan (0.01, 0.1, and 1.0 M). To determine the repair bond strength, self-etch adhesives were applied to the aged composite, and then newly added composites were condensed. Ten samples of each specimen were subjected to shear testing at a crosshead speed of 1.0 mm min(-1). Samples were analyzed using two-way ANOVA followed by Tukey's honestly significant difference (HSD) test. Although the dispersion force of the composites remained relatively constant, their polar force increased slightly as the concentration of methyl mercaptan increased. The hydrogen-bonding forces were significantly higher after immersion in 1.0 M methyl mercaptan, leading to higher surface-free energies. However, the repair bond strengths for the repair restorations prepared from composites immersed in 1.0 M methyl mercaptan were significantly lower than for those immersed in 0.01 and 0.10 M methyl mercaptan. Considering the results of this study, it can be concluded that the repair bond strengths of both the aged and newly added composites were affected by immersion in methyl mercaptan solutions.

  15. Cigarette smoke induces methylation of the tumor suppressor gene NISCH

    PubMed Central

    Ostrow, Kimberly Laskie; Michalidi, Christina; Guerrero-Preston, Rafael; Hoque, Mohammad O.; Greenberg, Alissa; Rom, William; Sidransky, David

    2013-01-01

    We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (< 20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients’ plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer. PMID:23503203

  16. Base excision repair of tandem modifications in a methylated CpG dinucleotide.

    PubMed

    Sassa, Akira; Çağlayan, Melike; Dyrkheeva, Nadezhda S; Beard, William A; Wilson, Samuel H

    2014-05-16

    Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3'-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5'-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide.

  17. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    PubMed

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  18. Control of gene editing by manipulation of DNA repair mechanisms.

    PubMed

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  19. DNA damage, repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans.

    PubMed

    Georgieva, Mariyana; Rashydov, Namik M; Hajduch, Martin

    2017-02-01

    This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident. Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products.

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

    PubMed

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

    2013-11-01

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

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

    PubMed

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

    2003-02-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2006-09-28

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

  4. Genome-Wide Methylation and Gene Expression Changes in Newborn Rats following Maternal Protein Restriction and Reversal by Folic Acid

    PubMed Central

    Stupka, Elia; Clark, Adrian J. L.; Langley-Evans, Simon

    2013-01-01

    A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures. PMID:24391732

  5. Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest.

    PubMed

    Zhang, Ye; Rohde, Larry H; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish K; Jeevarajan, Antony S; Pierson, Duane L; Wu, Honglu

    2008-11-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that

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

    PubMed

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

    2015-11-03

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2005-10-01

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

  9. Non-functional genes repaired at the RNA level.

    PubMed

    Burger, Gertraud

    2016-01-01

    Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years.

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

    PubMed

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

    2015-11-24

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

  11. Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix

    PubMed Central

    Rieke, Damian T.; Ochsenreither, Sebastian; Klinghammer, Konrad; Seiwert, Tanguy Y.; Klauschen, Frederick; Tinhofer, Inge; Keilholz, Ulrich

    2016-01-01

    Immune checkpoints are emerging treatment targets, but mechanisms underlying checkpoint expression are poorly understood. Since alterations in DNA repair genes have been connected to the efficacy of checkpoint inhibitors, we investigated associations between methylation of DNA repair genes and CTLA4 and CD274 (PD-L1) expression. A list of DNA repair genes (179 genes) was selected from the literature, methylation status and expression of inflammation-associated genes (The Cancer Genome Atlas data) was correlated in head and neck squamous cell carcinoma (HNSCC), cervical and lung squamous cell carcinoma. A significant positive correlation of the methylation status of 15, 3 and 2 genes with checkpoint expression was identified, respectively. RAD51B methylation was identified in all cancer subtypes. In HNSCC and cervical cancer, there was significant enrichment for homologous recombination genes. Methylation of the candidate genes was also associated with expression of other checkpoints, ligands, MHC- and T-cell associated genes as well as an interferon-inflammatory immune gene signature, predictive for the efficacy of PD-1 inhibition in HNSCC. Homologous recombination deficiency might therefore be mediated by DNA repair gene hypermethylation and linked to an immune-evasive phenotype in SCC. The methylation status of these genes could represent a new predictive biomarker for immune checkpoint inhibition. PMID:27683114

  12. Targeted gene repair: the ups and downs of a promising gene therapy approach.

    PubMed

    de Semir, David; Aran, Josep M

    2006-08-01

    As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cell's endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.

  13. Methylation of miRNA genes and oncogenesis.

    PubMed

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

    2015-02-01

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

  14. Methylization analysis of the FMR1 gene in carrier females

    SciTech Connect

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

    1994-09-01

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

  15. Polymorphisms in DNA repair genes and associations with cancer risk.

    PubMed

    Goode, Ellen L; Ulrich, Cornelia M; Potter, John D

    2002-12-01

    Common polymorphisms in DNA repair genes may alter protein function and an individual's capacity to repair damaged DNA; deficits in repair capacity may lead to genetic instability and carcinogenesis. To establish our overall understanding of possible in vivo relationships between DNA repair polymorphisms and the development of cancer, we performed a literature review of epidemiological studies that assessed associations between such polymorphisms and risk of cancer. Thirty studies of polymorphisms in OGG1, XRCC1, ERCC1, XPC, XPD, XPF, BRCA2, and XRCC3 were identified in the April 30, 2002 MEDLINE database (National Center for Biotechnology Information. PubMed Database: http://www.ncbi.nlm.nih.gov/entrez). These studies focused on adult glioma, bladder cancer, breast cancer, esophageal cancer, lung cancer, prostate cancer, skin cancer (melanoma and nonmelanoma), squamous cell carcinoma of the head and neck, and stomach cancer. We found that a small proportion of the published studies were large and population-based. Nonetheless, published data were consistent with associations between: (a) the OGG1 S326C variant and increased risk of various types of cancer; (b) the XRCC1 R194W variant and reduced risk of various types of cancer; and (c) the BRCA2 N372H variant and increased risk of breast cancer. Suggestive results were seen for polymorphisms in other genes; however, small sample sizes may have contributed to false-positive or false-negative findings. We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of cancer in the presence of reduced DNA repair capacity.

  16. uv excision-repair gene transfer in Chinese hamster ovary (CHO) cells

    SciTech Connect

    MacInnes, M.A.; Bingham, J.M.; Strniste, G.F.; Thompson, L.H.

    1983-01-01

    uvc-sensitive mutants of CHO cells provide a model system for molecular studies of DNA repair. We present our recent results which show that these mutants are competent recipients for plasmid marker gene transfer and incorporation of a putative CHO repair gene. The applicability and advantages of this system for interspecies human repair gene identification are discussed.

  17. Bone tissue engineering and repair by gene therapy.

    PubMed

    Betz, Volker M; Betz, Oliver B; Harris, Mitchel B; Vrahas, Mark S; Evans, Christopher H

    2008-01-01

    Many clinical conditions require the stimulation of bone growth. The use of recombinant bone morphogenetic proteins does not provide a satisfying solution to these conditions due to delivery problems and high cost. Gene therapy has emerged as a very promising approach for bone repair that overcomes limitations of protein-based therapy. Several preclinical studies have shown that gene transfer technology has the ability to deliver osteogenic molecules to precise anatomical locations at therapeutic levels for sustained periods of time. Both in-vivo and ex-vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Genetic engineering of adult stem cells from various sources with osteogenic genes has led to enhanced fracture repair, spinal fusion and rapid healing of bone defects in animal models. This review describes current viral and non-viral gene therapy strategies for bone tissue engineering and repair including recent work from the author's laboratory. In addition, the article discusses the potential of gene-enhanced tissue engineering to enter widespread clinical use.

  18. Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

    PubMed

    Major, G N; Gardner, E J; Carne, A F; Lawley, P D

    1990-03-25

    DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).

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

    PubMed Central

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

    2017-01-01

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

  20. Feedback regulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 via ATM/Chk2 pathway contributes to the resistance of MCF-7 breast cancer cells to cisplatin.

    PubMed

    Lv, Juan; Qian, Ying; Ni, Xiaoyan; Xu, Xiuping; Dong, Xuejun

    2017-03-01

    The methyl methanesulfonate and ultraviolet-sensitive gene clone 81 protein is a structure-specific nuclease that plays important roles in DNA replication and repair. Knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 has been found to sensitize cancer cells to chemotherapy. However, the underlying molecular mechanism is not well understood. We found that methyl methanesulfonate and ultraviolet-sensitive gene clone 81 was upregulated and the ATM/Chk2 pathway was activated at the same time when MCF-7 cells were treated with cisplatin. By using lentivirus targeting methyl methanesulfonate and ultraviolet-sensitive gene clone 81 gene, we showed that knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 enhanced cell apoptosis and inhibited cell proliferation in MCF-7 cells under cisplatin treatment. Abrogation of ATM/Chk2 pathway inhibited cell viability in MCF-7 cells in response to cisplatin. Importantly, we revealed that ATM/Chk2 was required for the upregulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81, and knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 resulted in inactivation of ATM/Chk2 pathway in response to cisplatin. Meanwhile, knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 activated the p53/Bcl-2 pathway in response to cisplatin. These data suggest that the ATM/Chk2 may promote the repair of DNA damage caused by cisplatin by sustaining methyl methanesulfonate and ultraviolet-sensitive gene clone 81, and the double-strand breaks generated by methyl methanesulfonate and ultraviolet-sensitive gene clone 81 may activate the ATM/Chk2 pathway in turn, which provide a novel mechanism of how methyl methanesulfonate and ultraviolet-sensitive gene clone 81 modulates DNA damage response and repair.

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

    PubMed

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

    2014-04-15

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

  2. Gene therapy and peripheral nerve repair: a perspective

    PubMed Central

    Hoyng, Stefan A.; de Winter, Fred; Tannemaat, Martijn R.; Blits, Bas; Malessy, Martijn J. A.; Verhaagen, Joost

    2015-01-01

    Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan’s, Parkinson’s (PD) and Alzheimer’s disease (AD), retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral vectors (AAV) and the first AAV-based therapeutic, a vector encoding lipoprotein lipase, is now marketed in Europe under the name Glybera. These remarkable advances may become relevant to translational research on gene therapy to promote peripheral nervous system (PNS) repair. This short review first summarizes the results of gene therapy in animal models for peripheral nerve repair. Secondly, we identify key areas of future research in the domain of PNS-gene therapy. Finally, a perspective is provided on the path to clinical translation of PNS-gene therapy for traumatic nerve injuries. In the latter section we discuss the route and mode of delivery of the vector to human patients, the efficacy and safety of the vector, and the choice of the patient population for a first possible proof-of-concept clinical study. PMID:26236188

  3. Gene therapy and peripheral nerve repair: a perspective.

    PubMed

    Hoyng, Stefan A; de Winter, Fred; Tannemaat, Martijn R; Blits, Bas; Malessy, Martijn J A; Verhaagen, Joost

    2015-01-01

    Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan's, Parkinson's (PD) and Alzheimer's disease (AD), retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral vectors (AAV) and the first AAV-based therapeutic, a vector encoding lipoprotein lipase, is now marketed in Europe under the name Glybera. These remarkable advances may become relevant to translational research on gene therapy to promote peripheral nervous system (PNS) repair. This short review first summarizes the results of gene therapy in animal models for peripheral nerve repair. Secondly, we identify key areas of future research in the domain of PNS-gene therapy. Finally, a perspective is provided on the path to clinical translation of PNS-gene therapy for traumatic nerve injuries. In the latter section we discuss the route and mode of delivery of the vector to human patients, the efficacy and safety of the vector, and the choice of the patient population for a first possible proof-of-concept clinical study.

  4. Gene- and strand-specific repair in vitro: Partial purification of a transcription-repair coupling factor

    SciTech Connect

    Selby, C.P.; Sancar, A. )

    1991-09-15

    In eukaryotic and prokaryotic cells, actively transcribed genes and, in some instances, the template strand of these genes have been found to be repaired 2-10 times more rapidly than nontranscribed genes or the coding strand of transcribed genes. The authors demonstrate here gene- and template strand-specific repair synthesis in vitro by using an Escherichia coli cell-free extract and a plasmid carrying a gene with the strong tac promoter. Strand-specific repair of UV, 4{prime}-hydroxymethyl-4,5{prime}, 8-trimethylpsoralen, and cis-dicholorodiammine platinum(II) damage was dependent upon transcription and a functional nucleotide excision repair system and was stimulated by 6% (wt/vol) polyethylene glycol. A defined system consisting of the transcription and repair proteins in highly purified form did not perform strand-specific repair; however, active fractions of extract conferred strand specificity to the defined system. Transcription-repair coupling activity was partially purified from extract by successive DEAE-agarose and gel filtration chromatography. The coupling factor is heat-labile, with an estimated M{sub r} of 100,000.

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

    PubMed Central

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

    2011-01-01

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

  6. Methyl-CpG binding domain protein acts to regulate the repair of cyclobutane pyrimidine dimers on rice DNA

    PubMed Central

    Fang, Changxun; Chen, Weisi; Li, Chengxun; Jian, Xin; Li, Yingzhe; Lin, Hongmei; Lin, Wenxiong

    2016-01-01

    UVB radiation causes cyclobutane pyrimidine dimers (CPDs) to form on the DNA of living organisms. This study found that overexpression of the silicon absorbance gene Lsi1 reduced the accumulation of CPDs in rice, which profited from the reactivation by photolyase. The transcript abundance of deoxyribodipyrimidine photolyase (Os10g0167600) was generally correlated with the silicon content of the rice, and the up-regulation of Os10g0167600 was found to be highest in the UVB-treated Lsi1-overexpressed (Lsi1-OX) rice. A trans-acting factor, methyl-CpG binding domain protein (OsMeCP), was found to interact with the cis-element of Os10g0167600. The nucleic location of OsMeCP effectively enabled the transcriptional regulation. Compared with the WT, the level of OsMeCP was lower in the Lsi1-OX rice but higher in the Lsi1-RNAi line. Rice cultured in a high silicate-concentration solution also exhibited less OsMeCP abundance. Overexpression of OsMeCP led to lower Os10g0167600 transcript levels and a higher CPD content than in the WT, but the reverse was true in the OsMeCP-RNAi line. These findings indicate that OsMeCP acts as a negative regulator of silicon, and can mediate the repression of the transcription from Os10g0167600, which inhibits the photoreactivation of the photolyase involved in the repair of CPDs. PMID:27694845

  7. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

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

    PubMed Central

    Olson, Claire E.; Roberts, Steven B.

    2014-01-01

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

  9. Genetic variants involved in oxidative stress, base excision repair, DNA methylation, and folate metabolism pathways influence myeloid neoplasias susceptibility and prognosis.

    PubMed

    Gonçalves, Ana Cristina; Alves, Raquel; Baldeiras, Inês; Cortesão, Emília; Carda, José Pedro; Branco, Claudia C; Oliveiros, Bárbara; Loureiro, Luísa; Pereira, Amélia; Nascimento Costa, José Manuel; Sarmento-Ribeiro, Ana Bela; Mota-Vieira, Luisa

    2017-01-01

    Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) share common features: elevated oxidative stress, DNA repair deficiency, and aberrant DNA methylation. We performed a hospital-based case-control study to evaluate the association in variants of genes involved in oxidative stress, folate metabolism, DNA repair, and DNA methylation with susceptibility and prognosis of these malignancies. To that end, 16 SNPs (one per gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2, and XRCC1) were genotyped in 191 patients (101 MDS and 90 AML) and 261 controls. We also measured oxidative stress (reactive oxygen species/total antioxidant status ratio), DNA damage (8-hydroxy-2'-deoxyguanosine), and DNA methylation (5-methylcytosine) in 50 subjects (40 MDS and 10 controls). Results showed that five genes (GPX1, NEIL1, NFE2L2, OGG1, and SOD2) were associated with MDS, two (DNMT3B and SLC19A1) with AML, and two (CYBA and DNMT1) with both diseases. We observed a correlation of CYBA TT, GPX1 TT, and SOD2 CC genotypes with increased oxidative stress levels, as well as NEIL1 TT and OGG1 GG genotypes with higher DNA damage. The 5-methylcytosine levels were negatively associated with DNMT1 CC, DNMT3A CC, and MTRR AA genotypes, and positively with DNMT3B CC genotype. Furthermore, DNMT3A, MTRR, NEIL1, and OGG1 variants modulated AML transformation in MDS patients. Additionally, DNMT3A, OGG1, GPX1, and KEAP1 variants influenced survival of MDS and AML patients. Altogether, data suggest that genetic variability influence predisposition and prognosis of MDS and AML patients, as well AML transformation rate in MDS patients. © 2016 Wiley Periodicals, Inc.

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

    PubMed

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

    2016-04-01

    Genomes of invertebrates and vertebrates exhibit highly divergent patterns of DNA methylation. Invertebrate genomes tend to be sparsely methylated, and DNA methylation is mostly targeted to a subset of transcription units (gene bodies). In a drastic contrast, vertebrate genomes are generally globally and heavily methylated, punctuated by the limited local hypo-methylation of putative regulatory regions such as promoters. These genomic differences also translate into functional differences in DNA methylation and gene regulation. Although promoter DNA methylation is an important regulatory component of vertebrate gene expression, its role in invertebrate gene regulation has been little explored. Instead, gene body DNA methylation is associated with expression of invertebrate genes. However, the evolutionary steps leading to the differentiation of invertebrate and vertebrate genomic DNA methylation remain unresolved. Here we analyzed experimentally determined DNA methylation maps of several species across the invertebrate-vertebrate boundary, to elucidate how vertebrate gene methylation has evolved. We show that, in contrast to the prevailing idea, a substantial number of promoters in an invertebrate basal chordate Ciona intestinalis are methylated. Moreover, gene expression data indicate significant, epigenomic context-dependent associations between promoter methylation and expression in C. intestinalis. However, there is no evidence that promoter methylation in invertebrate chordate has been evolutionarily maintained across the invertebrate-vertebrate boundary. Rather, body-methylated invertebrate genes preferentially obtain hypo-methylated promoters among vertebrates. Conversely, promoter methylation is preferentially found in lineage- and tissue-specific vertebrate genes. These results provide important insights into the evolutionary origin of epigenetic regulation of vertebrate gene expression.

  11. An RNA polymerase II-coupled function for histone H3K36 methylation in checkpoint activation and DSB repair.

    PubMed

    Jha, Deepak Kumar; Strahl, Brian D

    2014-06-09

    Histone modifications are major determinants of DNA double-strand break (DSB) response and repair. Here we elucidate a DSB repair function for transcription-coupled Set2 methylation at H3 lysine 36 (H3K36me). Cells devoid of Set2/H3K36me are hypersensitive to DNA-damaging agents and site-specific DSBs, fail to properly activate the DNA-damage checkpoint, and show genetic interactions with DSB-sensing and repair machinery. Set2/H3K36me3 is enriched at DSBs, and loss of Set2 results in altered chromatin architecture and inappropriate resection during G1 near break sites. Surprisingly, Set2 and RNA polymerase II are programmed for destruction after DSBs in a temporal manner--resulting in H3K36me3 to H3K36me2 transition that may be linked to DSB repair. Finally, we show a requirement of Set2 in DSB repair in transcription units--thus underscoring the importance of transcription-dependent H3K36me in DSB repair.

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    1996-01-01

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

  14. Reversible histone methylation regulates brain gene expression and behavior

    PubMed Central

    Xu, Jun; Andreassi, Megan

    2011-01-01

    Epigenetic chromatin remodeling, including reversible histone methylation, regulates gene transcription in brain development and synaptic plasticity. Aberrant chromatin modifications due to mutant chromatin enzymes or chemical exposures have been associated with neurological or psychiatric disorders such as mental retardation, schizophrenia, depression, and drug addiction. Some chromatin enzymes, such as histone demethylases JARID1C and UTX, are coded by X-linked genes which are not X-inactivated in females. The higher expression of JARID1C and UTX in females could contribute to sex differences in brain development and behavior. PMID:20816965

  15. Structural Rearrangements in DNA Repair Genes in Breast Cancer

    DTIC Science & Technology

    2012-10-01

    bind RAD51B and XRCC3 , components of the homologous recombination pathway. RAD51C was found to bind both RAD51B and XRCC3 , however, the RAD51C...repair due to lack of binding partners in its C-terminus was not correct. Figure 11: RAD51C:ATXN7 fusion gene binds RAD51B and XRCC3 similar to wild...terminus. In vitro expressed GST fusion proteins were incubated with in vitro expressed RAD51B or XRCC3 and interaction tested via GST pull down assay

  16. Simulated microgravity influenced the expression of DNA damage repair genes

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Sun, Yeqing; Jiawei, Liu; Wang, Ting

    2016-07-01

    Ionizing radiation and microgravity were considered to be the most important stress factors of space environmental the respective study of the biological effects of the radiation and microgravity carried out earlier, but the interaction of the effects of radiation with microgravity started later, and due to difference of the materials and methods the result of this experiment were not consistent. To further investigate the influence of microgravity on the expression of the radiation damage repair genes, the seed of Arabidopsis (Col) and its gravity-insensitive mutant (PIN2) were exposed to 0.1Gy of the dose of energetic carbon-ion beam radiation (LET = 30KeV / μm), and the germinated seed were than fixed in the 3D random positioning apparatus immediately for a 10-day simulated microgravity. By measuring the deflection angle of root tip and the changes of the expression of Ku70 and RAD51 protein, we investigated the impact of microgravity effect on radiation damage repair systems. The results shown that radiation, microgravity and microgravity with radiation could increase the angle of the root of the Col significantly, but no obvious effect on PIN2 type. The radiation could increase the expression of Ku70 significantly in both Col and PIN2, microgravity does not affect the expression, but the microgravity with radiation could decrease the expression of Ku70. This result shown that the microgravity could influence the radiation damage repair systems in molecular level. Moreover, our findings were important to understand the molecular mechanism of the impact of microgravity effect on radiation damage repair systems in vivo.

  17. Differential mutagenicity of N-methyl-N-nitrosocarbamate insectides in Escherichia coli strains having different DNA repair capacities.

    PubMed

    Yoshikawa, K; Uchino, H; Kurata, H

    1978-12-01

    Four isogenic strains of Escherichia coli with the same auxotrophic marker (arg Fam--namely wild-type, uvrA-, polA- and recA-) were used for testing the lethalities and mutagenicities of 1-naphthyl N-methyl-N-nitrosocarbamate (nitroso-NAC), 3-methylphenyl N-methyl-N-nitrosocarbamate (nitroso-MTMC), and 3,4-dimethylphenyl N-methyl-N-nitrosocarbamate (nitroso-MPMC). The strains recA- and polA- showed a similarly higher sensitivity to killing than wild-type and uvrA- after treatments with each of the three chemicals, whereas the strains wild-type, uvrA-, and polA- were equally mutable by these compounds at equal doses. The strain recA- was hardly mutable by nitroso-NAC, but significant levels of Arg+ mutations were observed after treatments with nitroso-MTMC and nitroso-MPMC. These and previous results suggest that both nitroso-MTMC and nitroso-MPMC are similar in their mutagenicity pattern to N-methyl-N'-nitro-N-nitrosoguanidine whereas nitroso-NAC is similar to methyl methanesulfonate or X-rays, and that the major damage to DNA of the three agents is not excisable by the uvrA+-dependent excision repair, probably methylation in DNA.

  18. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    PubMed Central

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

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

    PubMed

    Zaker, Farhad; Amirizadeh, Naser; Nasiri, Nahid; Razavi, Seyed Mohsen; Teimoori-Toolabi, Ladan; Yaghmaie, Marjan; Mehrasa, Roya

    2016-01-01

    Myelodysplastic syndromes (MDSs) are a clonal bone marrow (BM) disease characterized by ineffective hematopoiesis, dysplastic maturation and progression to acute myeloid leukemia (AML). Methylation silencing of HRK has been found in several human malignancies. In this study, we explored the association of HRK methylation status with its expression, clinical parameters and MDS subtypes in MDS patients. To study the methylation status of HRK gene, we applied Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) in MDS patients, as well as healthy controls and EpiTect®PCR Control DNA. Real time RT-PCR was used for gene expression analysis. Methylation frequency in promoter region of HRK in patient samples was 20.37%. Methylation of HRK was significantly related to transcriptional downregulation (P=0.023). The difference in frequency of hypermethylated HRK gene was significant between good (10%) and poor (71.42%) cytogenetic risk groups (P= 0.001), advanced stage MDS patients (66.66%) in comparison with early stage MDS patients (2.56%) (P= 0.00), higher- risk MDS group (61.53%) and lower- risk MDS group (7.31%) (P= 0.00). HRK hypermethylation was associated with advanced- stage MDS and downregulation of HRK gene may play a role in the progression of MDS.

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

    PubMed Central

    Zaker, Farhad; Amirizadeh, Naser; Nasiri, Nahid; Razavi, Seyed Mohsen; Teimoori-Toolabi, Ladan; Yaghmaie, Marjan; Mehrasa, Roya

    2016-01-01

    Myelodysplastic syndromes (MDSs) are a clonal bone marrow (BM) disease characterized by ineffective hematopoiesis, dysplastic maturation and progression to acute myeloid leukemia (AML). Methylation silencing of HRK has been found in several human malignancies. In this study, we explored the association of HRK methylation status with its expression, clinical parameters and MDS subtypes in MDS patients. To study the methylation status of HRK gene, we applied Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) in MDS patients, as well as healthy controls and EpiTect®PCR Control DNA. Real time RT-PCR was used for gene expression analysis. Methylation frequency in promoter region of HRK in patient samples was 20.37%. Methylation of HRK was significantly related to transcriptional downregulation (P=0.023). The difference in frequency of hypermethylated HRK gene was significant between good (10%) and poor (71.42%) cytogenetic risk groups (P= 0.001), advanced stage MDS patients (66.66%) in comparison with early stage MDS patients (2.56%) (P= 0.00), higher- risk MDS group (61.53%) and lower- risk MDS group (7.31%) (P= 0.00). HRK hypermethylation was associated with advanced- stage MDS and downregulation of HRK gene may play a role in the progression of MDS. PMID:27478805

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

    PubMed

    Wedd, Laura; Maleszka, Ryszard

    2016-01-01

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

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

    PubMed

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

    2006-11-15

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

  3. groE genes affect SOS repair in Escherichia coli

    SciTech Connect

    Liu, S.K.; Tessman, I. )

    1990-10-01

    Repair of UV-irradiated bacteriophage in Escherichia coli by Weigle reactivation requires functional recA+ and umuD+C+ genes. When the cells were UV irradiated, the groE heat shock gene products, GroES and GroEL, were needed for at least 50% of the Weigle reactivation of the single-stranded DNA phage S13. Because of repression of the umuDC and recA genes, Weigle reactivation is normally blocked by the lexA3(Ind-) mutation (which creates a noncleavable LexA protein), but it was restored by a combination of a high-copy-number umuD+C+ plasmid and a UV dose that increases groE expression. Maximal reactivation was achieved by elevated amounts of the Umu proteins, which was accomplished in part by UV-induced expression of the groE genes. By increasing the number of copies of the umuD+C+ genes, up to 50% of the normal amount of reactivation of S13 was achieved in an unirradiated recA+ host.

  4. Synergism of Dam, MutH, and MutS in methylation-directed mismatch repair in Escherichia coli.

    PubMed

    Hu, Changkun; Zhao, Yunqi; Sun, Huiyun; Yang, Yixin

    2017-01-01

    DNA mismatch repair (MMR) is a critical mutation surveillance system for recognizing and repairing erroneous insertion, deletion, and disincorporation of base. Major components of mismatch repair system consist of MutH, MutL, and MutS. Dam methylates adenine to distinguish newly synthesized daughter strands from the parent strands. Employing a tyrosine-auxotrophic E. coli FX-11 strain, the mutation frequency can be determined by the number of tyrosine revertants and the cell viability of FX-11 with deficiencies in dam and mismatch repair proteins. This study showed that mutS defect produced a higher mutation frequency than mutH did. Interestingly, double defects in dam and mutS synergistically produced a dramatically higher spontaneous mutation frequency than the summation of mutation frequencies of FX-11 strains with individual deficiency of dam or mutS, suggesting that Dam may work with MutHL to partially accomplish the task of recognizing the mismatch sites to retain partial mismatch repair capacity.

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

    PubMed Central

    Zhongli, Cui; Shunpeng, Li; Guoping, Fu

    2001-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    Takuno, Shohei; Gaut, Brandon S.

    2013-01-01

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

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

    PubMed Central

    2014-01-01

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

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

    PubMed

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

    2013-04-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Alterations in Gene Expression and DNA Methylation during Murine and Human Lung Alveolar Septation

    PubMed Central

    Cuna, Alain; Halloran, Brian; Faye-Petersen, Ona; Kelly, David; Crossman, David K.; Cui, Xiangqin; Pandit, Kusum; Kaminski, Naftali; Bhattacharya, Soumyaroop; Ahmad, Ausaf; Mariani, Thomas J.

    2015-01-01

    DNA methylation, a major epigenetic mechanism, may regulate coordinated expression of multiple genes at specific time points during alveolar septation in lung development. The objective of this study was to identify genes regulated by methylation during normal septation in mice and during disordered septation in bronchopulmonary dysplasia. In mice, newborn lungs (preseptation) and adult lungs (postseptation) were evaluated by microarray analysis of gene expression and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-Seq). In humans, microarray gene expression data were integrated with genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm and term lung. Genes with reciprocal changes in expression and methylation, suggesting regulation by DNA methylation, were identified. In mice, 95 genes with inverse correlation between expression and methylation during normal septation were identified. In addition to genes known to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its extracellular matrix (Tnc, Eln, etc.), genes involved with immune and antioxidant defense (Stat4, Sod3, Prdx6, etc.) were also observed. In humans, 23 genes were differentially methylated with reciprocal changes in expression in bronchopulmonary dysplasia compared with preterm or term lung. Genes of interest included those involved with detoxifying enzymes (Gstm3) and transforming growth factor-β signaling (bone morphogenetic protein 7 [Bmp7]). In terms of overlap, 20 genes and three pathways methylated during mouse lung development also demonstrated changes in methylation between preterm and term human lung. Changes in methylation correspond to altered expression of a number of genes associated with lung development, suggesting that DNA methylation of these genes may regulate normal and abnormal alveolar septation. PMID:25387348

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

    PubMed Central

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

    2016-01-01

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

  15. Sister chromatid exchange, DNA repair, and single-gene mutation

    SciTech Connect

    Carrano, A.V.; Thompson, L.H.

    1982-01-01

    Sister chromatid exchange (SCE) has been studied in cultured mammalian cells with regard to the nature of the inducing lesion, mutation induction, and factors that modify the observed frequency following mutagen exposure, SCEs can be induced by a wide spectrum of DNA lesions and, for nine agents examined, the frequency of induced SCE is linearly related to induced single-gene mutation. Further, a deficiency in DNA repair may alter the expression of both SCE and mutation in a qualitatively similar manner. The frequency of SCE induced by mitomycin-C is suppressed in heterochromatic relative to euchromatin and, in nondividing lymphocytes, the lesions leading to the formation of SCEs may persist for several months.

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

    PubMed

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

    2016-12-05

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

  17. Oligonucleotide-mediated gene repair at DNA level: the potential applications for gene therapy.

    PubMed

    Liu, Chang-Mei; Liu, De-Pei; Liang, Chih-Chuan

    2002-10-01

    Mutations in gene sequence can cause many genetic disorders, and researchers have attempted to develop treatments or cures at the DNA level for these diseases. Several strategies including triple-helix-forming oligonucleotides (TFOs), chimeric RNA/DNA oligonucleotide (RDO), and short single-stranded oligodeoxynucleotide (ODN) have been used to correct the dysfunctional genes in situ in the chromosome. Experimental data from cells and animal models suggest that all these strategies can repair the mutations in situ at DNA level. More effective structures of oligonucleotide, efficient delivery systems, and gene correction efficiency should be improved. Development of these strategies holds great potentials for treatments of genetic defects and other disorders.

  18. CHST11 gene expression and DNA methylation in breast cancer

    PubMed Central

    HERMAN, DAMIR; LEAKEY, TATIANA I.; BEHRENS, ALICE; YAO-BORENGASSER, AIWEI; COONEY, CRAIG A.; JOUSHEGHANY, FARIBA; PHANAVANH, BOUNLEUT; SIEGEL, ERIC R.; SAFAR, A. MAZIN; KOROURIAN, SOHEILA; KIEBER-EMMONS, THOMAS; MONZAVI-KARBASSI, BEHJATOLAH

    2015-01-01

    methylation status of this gene also has potential as a prognostic biomarker. PMID:25586191

  19. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

    PubMed Central

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  20. Molecular epidemiology of DNA repair gene polymorphisms and head and neck cancer

    PubMed Central

    Wang, Meilin; Chu, Haiyan; Zhang, Zhengdong; Wei, Qingyi

    2013-01-01

    Although tobacco and alcohol consumption are two common risk factors of head and neck cancer (HNC), other specific etiologic causes, such as viral infection and genetic susceptibility factors, remain to be understood. Human DNA is often damaged by numerous endogenous and exogenous mutagens or carcinogens, and genetic variants in interaction with environmental exposure to these agents may explain interindividual differences in HNC risk. Single nucleotide polymorphisms (SNPs) in genes involved in the DNA damage-repair response are reported to be risk factors for various cancer types, including HNC. Here, we reviewed epidemiological studies that have assessed the associations between HNC risk and SNPs in DNA repair genes involved in base-excision repair, nucleotide-excision repair, mismatch repair, double-strand break repair and direct reversion repair pathways. We found, however, that only a few SNPs in DNA repair genes were found to be associated with significantly increased or decreased risk of HNC, and, in most cases, the effects were moderate, depending upon locus-locus interactions among the risk SNPs in the pathways. We believe that, in the presence of exposure, additional pathway-based analyses of DNA repair genes derived from genome-wide association studies (GWASs) in HNC are needed. PMID:23720673

  1. DNA Adenine Methylation Regulates Virulence Gene Expression in Salmonella enterica Serovar Typhimurium▿

    PubMed Central

    Balbontín, Roberto; Rowley, Gary; Pucciarelli, M. Graciela; López-Garrido, Javier; Wormstone, Yvette; Lucchini, Sacha; García-del Portillo, Francisco; Hinton, Jay C. D.; Casadesús, Josep

    2006-01-01

    Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam− mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam− mutants in the mouse model may be caused by altered patterns of gene expression. PMID:16997949

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

    PubMed Central

    Li, Xinxin; Miclaus, Mihai; Messing, Joachim

    2016-01-01

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

  3. Genetic analysis of DNA methylation and gene expression levels in whole blood of healthy human subjects

    PubMed Central

    2012-01-01

    Background The predominant model for regulation of gene expression through DNA methylation is an inverse association in which increased methylation results in decreased gene expression levels. However, recent studies suggest that the relationship between genetic variation, DNA methylation and expression is more complex. Results Systems genetic approaches for examining relationships between gene expression and methylation array data were used to find both negative and positive associations between these levels. A weighted correlation network analysis revealed that i) both transcriptome and methylome are organized in modules, ii) co-expression modules are generally not preserved in the methylation data and vice-versa, and iii) highly significant correlations exist between co-expression and co-methylation modules, suggesting the existence of factors that affect expression and methylation of different modules (i.e., trans effects at the level of modules). We observed that methylation probes associated with expression in cis were more likely to be located outside CpG islands, whereas specificity for CpG island shores was present when methylation, associated with expression, was under local genetic control. A structural equation model based analysis found strong support in particular for a traditional causal model in which gene expression is regulated by genetic variation via DNA methylation instead of gene expression affecting DNA methylation levels. Conclusions Our results provide new insights into the complex mechanisms between genetic markers, epigenetic mechanisms and gene expression. We find strong support for the classical model of genetic variants regulating methylation, which in turn regulates gene expression. Moreover we show that, although the methylation and expression modules differ, they are highly correlated. PMID:23157493

  4. Germline mutations in DNA repair genes may predict neoadjuvant therapy response in triple negative breast patients.

    PubMed

    Spugnesi, Laura; Gabriele, Michele; Scarpitta, Rosa; Tancredi, Mariella; Maresca, Luisa; Gambino, Gaetana; Collavoli, Anita; Aretini, Paolo; Bertolini, Ilaria; Salvadori, Barbara; Landucci, Elisabetta; Fontana, Andrea; Rossetti, Elena; Roncella, Manuela; Naccarato, Giuseppe Antonio; Caligo, Maria Adelaide

    2016-12-01

    Triple negative breast cancers (TNBCs) represent about 15-20% of all breast cancer cases and are characterized by a complex molecular heterogeneity. Some TNBCs exhibit clinical and pathological properties similar to BRCA-mutated tumors, without actually bearing a mutation in BRCA genes. This "BRCAness" phenotype may be explained by germline mutations in other genes involved in DNA repair. Although respond to chemotherapy with alkylating agents, they have a high risk of recurrence and progression. Some studies have shown the efficacy of neoadjuvant therapy in TNBC patients with DNA repair defects, but proper biomarkers of DNA repair deficiency are still needed. Here, we investigated if mutations in DNA repair genes may be correlated with anthracyclines/taxanes neoadjuvant therapy response. DNA from 19 TNBC patients undergoing neoadjuvant therapy were subjected to next generation sequencing of a panel of 24 genes in DNA repair and breast cancer predisposition. In this study, 5 of 19 patients (26%) carried a pathogenic mutation in BRCA1, PALB2, RAD51C and two patients carried a probable pathogenic missense variant. Moreover, VUS (Variants of Unknown Significance) in other genes, predicted to be deleterious by in silico tools, were detected in five patients. Germline mutations in DNA repair genes were found to be associated with the group of TNBC patients who responded to therapy. We conclude that a subgroup of TNBC patients have defects in DNA repair genes, other than BRCA1, and such patients respond favourably to neoadjuvant anthracyclines/taxanes therapy. © 2016 Wiley Periodicals, Inc.

  5. Dystrophin gene replacement and gene repair therapy for Duchenne muscular dystrophy in 2016.

    PubMed

    Duan, Dongsheng

    2016-03-04

    After years of relentless efforts, gene therapy has now begun to deliver its therapeutic promise in several diseases. A number of gene therapy products have received regulatory approval in Europe and Asia. Duchenne muscular dystrophy (DMD) is an X-linked inherited lethal muscle disease. It is caused by mutations in the dystrophin gene. Replacing and/or repair the mutated dystrophin gene holds great promises to treated DMD at the genetic level. Last several years have evidenced significant developments in preclinical experimentations in murine and canine models of DMD. There has been a strong interest in moving these promising findings to clinical trials. In light of rapid progress in this field, the Parent Project Muscular Dystrophy (PPMD) recently interviewed me on the current status of DMD gene therapy. Here I summarized the interview with PPMD.

  6. Dystrophin Gene Replacement and Gene Repair Therapy for Duchenne Muscular Dystrophy in 2016: An Interview.

    PubMed

    Duan, Dongsheng

    2016-03-01

    After years of relentless efforts, gene therapy has now begun to deliver its therapeutic promise in several diseases. A number of gene therapy products have received regulatory approval in Europe and Asia. Duchenne muscular dystrophy (DMD) is an X-linked inherited lethal muscle disease. It is caused by mutations in the dystrophin gene. Replacing and/or repairing the mutated dystrophin gene holds great promises to treated DMD at the genetic level. Last several years have evidenced significant developments in preclinical experimentations in murine and canine models of DMD. There has been a strong interest in moving these promising findings to clinical trials. In light of rapid progress in this field, the Parent Project Muscular Dystrophy (PPMD) recently interviewed me on the current status of DMD gene therapy and readiness for clinical trials. Here I summarized the interview with PPMD.

  7. Dystrophin Gene Replacement and Gene Repair Therapy for Duchenne Muscular Dystrophy in 2016: An Interview

    PubMed Central

    Duan, Dongsheng

    2016-01-01

    After years of relentless efforts, gene therapy has now begun to deliver its therapeutic promise in several diseases. A number of gene therapy products have received regulatory approval in Europe and Asia. Duchenne muscular dystrophy (DMD) is an X-linked inherited lethal muscle disease. It is caused by mutations in the dystrophin gene. Replacing and/or repairing the mutated dystrophin gene holds great promises to treated DMD at the genetic level. Last several years have evidenced significant developments in preclinical experimentations in murine and canine models of DMD. There has been a strong interest in moving these promising findings to clinical trials. In light of rapid progress in this field, the Parent Project Muscular Dystrophy (PPMD) recently interviewed me on the current status of DMD gene therapy and readiness for clinical trials. Here I summarized the interview with PPMD. PMID:27003751

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

    PubMed

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

    2016-11-01

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

  9. A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy

    SciTech Connect

    Stefanini, M.; Giliani, S. ); Vermuelen, W.; Weeda, G.; Hoeijmakers, H.J.; Mezzina, M.; Sarasin, A.; Harper, J.I.; Arlett, C.F.; Lehmann, A.R.

    1993-10-01

    The sun-sensitive, cancer-prone genetic disorder xeroderma pigmentosum (XP) is associated in most cases with a defect in the ability to carry out excision repair of UV damage. Seven genetically distinct complementation groups (i.e., A-G) have been identified. A large proportion of patients with the unrelated disorder trichothiodystrophy (TTD), which is characterized by hair-shaft abnormalities, as well as by physical and mental retardation, are also deficient in excision repair of UV damage. In most of these cases the repair deficiency is in the same complementation group as is XP group D. The authors report here on cells from a patient, TTD1BR, in which the repair defect complements all known XP groups (including XP-D). Furthermore, microinjection of various cloned human repair genes fails to correct the repair defect in this cell strain. The defect in TTD1BR cells is therefore in a new gene involved in excision repair in human cells. The finding of a second DNA repair gene that is associated with the clinical features of TTD argues strongly for an involvement of repair proteins in hair-shaft development. 20 refs., 2 figs., 1 tab.

  10. Gene structure, DNA methylation, and imprinted expression of the human SNRPN gene

    SciTech Connect

    Glenn, C.C.; Jong, T.C.; Filbrandt, M.M.

    1996-02-01

    The human SNRPN (small nuclear ribonucleoprotein polypeptide N) gene is one of a gene family that encode proteins involved in pre-mRNA splicing and maps to the smallest deletion region involved in the Prader-Willi syndrome (PWS) within chromosome 15q11-q13. Paternal only expression of SNRPN has previously been demonstrated by use of cell lines from PWS patients (maternal allele only) and Angelman syndrome (AS) patients (paternal allele only). We have characterized two previously unidentified 5{prime} exons of the SNRPN gene and demonstrate that exons -1 and 0 are included in the full-length transcript. This gene is expressed in a wide range of somatic tissues and at high, approximately equal levels in all regions of the brain. Both the first exon of SNRPN (exon -1) and the putative transcription start site are embedded within a CpG island. This CpG island is extensively methylated on the repressed maternal allele and is unmethylated on the expressed paternal allele, in a wide range of fetal and adult somatic cells. This provides a quick and highly reliable diagnostic assay for PWS and AS, which is based on DNA-methylation analysis that has been tested on >100 patients in a variety of tissues. Conversely, several CpG sites {approximately}22 kb downstream of the transcription start site in intron 5 are preferentially methylated on the expressed paternal allele in somatic tissues and male germ cells, whereas these same sites are unmethylated in fetal oocytes. These findings are consistent with a key role for DNA methylation in the imprinted inheritance and subsequent gene expression of the human SNRPN gene. 59 refs., 9 figs., 1 tab.

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

    EPA Science Inventory

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

  12. A study of molecular signals deregulating mismatch repair genes in prostate cancer compared to benign prostatic hyperplasia.

    PubMed

    Basu, Sanmitra; Majumder, Subhadipa; Bhowal, Ankur; Ghosh, Alip; Naskar, Sukla; Nandy, Sumit; Mukherjee, Subhabrata; Sinha, Rajan Kumar; Basu, Keya; Karmakar, Dilip; Banerjee, Soma; Sengupta, Sanghamitra

    2015-01-01

    Prostate cancer is one of the leading causes of mortality among aging males. There is an unmet requirement of clinically useful biomarkers for early detection of prostate cancer to reduce the liabilities of overtreatment and accompanying morbidity. The present population-based study investigates the factors disrupting expression of multiple functionally related genes of DNA mismatch repair pathway in prostate cancer patients to identify molecular attributes distinguishing adenocarcinoma from benign hyperplasia of prostate. Gene expression was compared between tissue samples from prostate cancer and benign prostatic hyperplasia using real-time-PCR, western blot and immunohistochemistry. Assessment of genotypes of seven single-nucleotide-polymorphisms of three MMR genes was conducted using PCR-coupled RFLP and sequencing. Promoter methylation was interrogated by methylation-specific-PCR and bisulfite-sequencing. Interaction between microRNAs and MMR genes was verified by 3'UTR-based dual luciferase assays. Concurrent reduction of three MMR genes namely hMLH1, hMSH6 and hMSH2 (34-85%, P<0.05) was observed in prostate cancer tissues. hMSH6 polymorphism rs1800932(Pro92Pro) conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75). Relative transcript level of hMLH1 was inversely related (r = -0.59, P<0.05) with methylation quotient of its promoter which showed a significantly higher methylation density (P = 0.008, Z = -2.649) in cancer patients. hsa-miR-155, hsa-miR-141 and hsa-miR-21 gene expressions were significantly elevated (66-85%, P<0.05) in tumor specimens and negatively correlated (r = -0.602 to -0.527, P<0.05) with that of MMR genes. hsa-miR-155 & hsa-miR-141 and hsa-miR-155 & hsa-miR-21 were demonstrated to bind to their putative seed sequences in hMLH1 and hMSH6 3'UTRs respectively. Relatively higher expression of DNA methyl-transferases (DNMT1 and DNMT3b) and HIF-1α genes (34-50%, P<0.05) were also detected in tumor tissues. This

  13. A Study of Molecular Signals Deregulating Mismatch Repair Genes in Prostate Cancer Compared to Benign Prostatic Hyperplasia

    PubMed Central

    Basu, Sanmitra; Majumder, Subhadipa; Bhowal, Ankur; Ghosh, Alip; Naskar, Sukla; Nandy, Sumit; Mukherjee, Subhabrata; Sinha, Rajan Kumar; Basu, Keya; Karmakar, Dilip; Banerjee, Soma; Sengupta, Sanghamitra

    2015-01-01

    Prostate cancer is one of the leading causes of mortality among aging males. There is an unmet requirement of clinically useful biomarkers for early detection of prostate cancer to reduce the liabilities of overtreatment and accompanying morbidity. The present population-based study investigates the factors disrupting expression of multiple functionally related genes of DNA mismatch repair pathway in prostate cancer patients to identify molecular attributes distinguishing adenocarcinoma from benign hyperplasia of prostate. Gene expression was compared between tissue samples from prostate cancer and benign prostatic hyperplasia using real-time-PCR, western blot and immunohistochemistry. Assessment of genotypes of seven single-nucleotide-polymorphisms of three MMR genes was conducted using PCR-coupled RFLP and sequencing. Promoter methylation was interrogated by methylation-specific-PCR and bisulfite-sequencing. Interaction between microRNAs and MMR genes was verified by 3'UTR-based dual luciferase assays. Concurrent reduction of three MMR genes namely hMLH1, hMSH6 and hMSH2 (34-85%, P<0.05) was observed in prostate cancer tissues. hMSH6 polymorphism rs1800932(Pro92Pro) conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75). Relative transcript level of hMLH1 was inversely related (r = -0.59, P<0.05) with methylation quotient of its promoter which showed a significantly higher methylation density (P = 0.008, Z = -2.649) in cancer patients. hsa-miR-155, hsa-miR-141 and hsa-miR-21 gene expressions were significantly elevated (66-85%, P<0.05) in tumor specimens and negatively correlated (r = -0.602 to -0.527, P<0.05) with that of MMR genes. hsa-miR-155 & hsa-miR-141 and hsa-miR-155 & hsa-miR-21 were demonstrated to bind to their putative seed sequences in hMLH1 and hMSH6 3’UTRs respectively. Relatively higher expression of DNA methyl-transferases (DNMT1 and DNMT3b) and HIF-1α genes (34-50%, P<0.05) were also detected in tumor tissues

  14. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    PubMed Central

    Andreis, Daniele; Bertoni, Ramona; Giardini, Roberto; Fox, Stephen B.; Broggini, Massimo; Bottini, Alberto; Zanoni, Vanessa; Bazzola, Letizia; Foroni, Chiara; Generali, Daniele; Damia, Giovanna

    2013-01-01

    DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia) in paraffin embedded samples of triple negative breast cancer (TNBC) compared to luminal A breast cancer (LABC). Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects. PMID:23825533

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

    PubMed

    Schroeder, Diane I; Jayashankar, Kartika; Douglas, Kory C; Thirkill, Twanda L; York, Daniel; Dickinson, Pete J; Williams, Lawrence E; Samollow, Paul B; Ross, Pablo J; Bannasch, Danika L; Douglas, Gordon C; LaSalle, Janine M

    2015-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair

    PubMed Central

    Mikhed, Yuliya; Görlach, Agnes; Knaus, Ulla G.; Daiber, Andreas

    2015-01-01

    Reactive oxygen and nitrogen species (e.g. H2O2, nitric oxide) confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. In addition, classical regulation of gene expression or activity, including gene transcription to RNA followed by translation to the protein level, by transcription factors (e.g. NF-κB, HIF-1α) and mRNA binding proteins (e.g. GAPDH, HuR) is subject to redox regulation. This review will give an update of recent discoveries in this field, and specifically highlight the impact of reactive oxygen and nitrogen species on DNA repair systems that contribute to genomic stability. Emphasis will be placed on the emerging role of redox mechanisms regulating epigenetic pathways (e.g. miRNA, DNA methylation and histone modifications). By providing clinical correlations we discuss how oxidative stress can impact on gene regulation/activity and vise versa, how epigenetic processes, other gene regulatory mechanisms and DNA repair can influence the cellular redox state and contribute or prevent development or progression of disease. PMID:26079210

  18. The Polycomb Group Protein EZH2 Impairs DNA Damage Repair Gene Expression in Human Uterine Fibroids1

    PubMed Central

    Yang, Qiwei; Nair, Sangeeta; Laknaur, Archana; Ismail, Nahed; Diamond, Michael P.; Al-Hendy, Ayman

    2016-01-01

    Uterine fibroids are benign, smooth muscle tumors that occur in approximately 70%–80% of women by age 50 yr. The cellular and molecular mechanism(s) by which uterine fibroids (UFs) develop are not fully understood. Accumulating evidence demonstrates that several genetic abnormalities, including deletions, rearrangements, translocations, as well as mutations, have been found in UFs. These genetic anomalies suggest that low DNA damage repair capacity may be involved in UF formation. The objective of this study was to determine whether expression levels of DNA damage repair-related genes were altered, and how they were regulated in the pathogenesis of UFs. Expression levels of DNA repair-related genes RAD51 and BRCA1 were deregulated in fibroid tissues as compared to adjacent myometrial tissues. Expression levels of chromatin protein enhancer of zeste homolog 2 (EZH2) were higher in a subset of fibroids as compared to adjacent myometrial tissues by both immunohistochemistry and Western blot analysis. Treatment with an inhibitor of EZH2 markedly increased expression levels of RAD51 and BRCA1 in fibroid cells and inhibited cell proliferation paired with cell cycle arrest. Restoring the expression of RAD51 and BRCA1 by treatment with EZH2 inhibitor was dependent on reducing the enrichment of trimethylation of histone 3 lysine 27 epigenetic mark in their promoter regions. This study reveals the important role of EZH2-regulated DNA damage-repair genes via histone methylation in fibroid biology, and may provide novel therapeutic targets for the medical treatment of women with symptomatic UFs. PMID:26888970

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

    PubMed

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

    2013-09-26

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-03-03

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

  3. Methylation of miRNA genes in the response to temperature stress in Populus simonii

    PubMed Central

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

    2015-01-01

    DNA methylation and miRNAs provide crucial regulation of the transcriptional and post-transcriptional responses to abiotic stress. In this study, we used methylation-sensitive amplification polymorphisms to identify 1066 sites that were differentially methylated in response to temperature stress in Populus simonii. Among these loci, BLAST searches of miRBase identified seven miRNA genes. Expression analysis by quantitative real-time PCR suggested that the methylation pattern of these miRNA genes probably influences their expression. Annotation of these miRNA genes in the sequenced genome of Populus trichocarpa found three target genes (Potri.007G090400, Potri.014G042200, and Potri.010G176000) for the miRNAs produced from five genes (Ptc-MIR396e and g, Ptc-MIR156i and j, and Ptc-MIR390c) respectively. The products of these target genes function in lipid metabolism to deplete lipid peroxide. We also constructed a network based on the interactions between DNA methylation and miRNAs, miRNAs and target genes, and the products of target genes and the metabolic factors that they affect, including H2O2, malondialdehyde, catalase (CAT), and superoxide dismutase. Our results suggested that DNA methylation probably regulates the expression of miRNA genes, thus affecting expression of their target genes, likely through the gene-silencing function of miRNAs, to maintain cell survival under abiotic stress conditions. PMID:26579167

  4. Network-based characterization and prediction of human DNA repair genes and pathways

    PubMed Central

    Li, Yan-Hui; Zhang, Gai-Gai

    2017-01-01

    Network biology is a useful strategy to understand cell’s functional organization. In this study, for the first time, we successfully introduced network approaches to study properties of human DNA repair genes. Compared with non-DNA repair genes, we found distinguishing features for DNA repair genes: (i) they tend to have higher degrees; (ii) they tend to be located at global network center; (iii) they tend to interact directly with each other. Based on these features, we developed the first algorithm to predict new DNA repair genes. We tested several machine-learning models and found that support vector machine with kernel function of radial basis function (RBF) achieve the best performance, with precision = 0.74 and area under curve (AUC) = 0.96. In the end, we applied the algorithm to predict new DNA repair genes and got 32 new candidates. Literature supporting four of the predictions was found. We believe the network approaches introduced here might open a new avenue to understand DNA repair genes and pathways. The suggested algorithm and the predicted genes might be helpful for scientists in the field. PMID:28368026

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

    PubMed

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

    2007-01-01

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

  6. An integrative study on the impact of highly differentially methylated genes on expression and cancer etiology

    PubMed Central

    2017-01-01

    DNA methylation is an important epigenetic phenomenon that plays a key role in the regulation of expression. Most of the studies on the topic of methylation’s role in cancer mechanisms include analyses based on differential methylation, with the integration of expression information as supporting evidence. In the present study, we sought to identify methylation-driven patterns by also integrating protein-protein interaction information. We performed integrative analyses of DNA methylation, expression, SNP and copy number data on paired samples from six different cancer types. As a result, we found that genes that show a methylation change larger than 32.2% may influence cancer-related genes via fewer interaction steps and with much higher percentages compared with genes showing a methylation change less than 32.2%. Additionally, we investigated whether there were shared cancer mechanisms among different cancer types. Specifically, five cancer types shared a change in AGTR1 and IGF1 genes, which implies that there may be similar underlying disease mechanisms among these cancers. Additionally, when the focus was placed on distinctly altered genes within each cancer type, we identified various cancer-specific genes that are also supported in the literature and may play crucial roles as therapeutic targets. Overall, our novel graph-based approach for identifying methylation-driven patterns will improve our understanding of the effects of methylation on cancer progression and lead to improved knowledge of cancer etiology. PMID:28178311

  7. Aberrant DNA methylation impacts gene expression and prognosis in breast cancer subtypes.

    PubMed

    Győrffy, Balázs; Bottai, Giulia; Fleischer, Thomas; Munkácsy, Gyöngyi; Budczies, Jan; Paladini, Laura; Børresen-Dale, Anne-Lise; Kristensen, Vessela N; Santarpia, Libero

    2016-01-01

    DNA methylation has a substantial impact on gene expression, affecting the prognosis of breast cancer (BC) patients dependent on molecular subtypes. In this study, we investigated the prognostic relevance of the expression of genes reported as aberrantly methylated, and the link between gene expression and DNA methylation in BC subtypes. The prognostic value of the expression of 144 aberrantly methylated genes was evaluated in ER+/HER2-, HER2+, and ER-/HER2- molecular BC subtypes, in a meta-analysis of two large transcriptomic cohorts of BC patients (n = 1,938 and n = 1,640). The correlation between gene expression and DNA methylation in distinct gene regions was also investigated in an independent dataset of 104 BCs. Survival and Pearson correlation analyses were computed for each gene separately. The expression of 48 genes was significantly associated with BC prognosis (p < 0.05), and 32 of these prognostic genes exhibited a direct expression-methylation correlation. The expression of several immune-related genes, including CD3D and HLA-A, was associated with both relapse-free survival (HR = 0.42, p = 3.5E-06; HR = 0.35, p = 1.7E-08) and overall survival (HR = 0.50, p = 5.5E-04; HR = 0.68, p = 4.5E-02) in ER-/HER2- BCs. On the overall, the distribution of both positive and negative expression-methylation correlation in distinct gene regions have different effects on gene expression and prognosis in BC subtypes. This large-scale meta-analysis allowed the identification of several genes consistently associated with prognosis, whose DNA methylation could represent a promising biomarker for prognostication and clinical stratification of patients with distinct BC subtypes.

  8. DNA methylation of genes of the main components of the telomerase complex in Danio rerio.

    PubMed

    Belova, E V; Kozlov, A E; Shubernetskaya, O S; Zvereva, M I; Shpanchenko, O V; Dontsova, O A

    2015-01-01

    The methylation status of the genes of telomerase reverse transcriptase (tert) and telomerase RNA (terc) was determined in brain tissues of Danio rerio of different age. It is found that, regardless of the age of fish, the regulatory region of the tert gene was completely methylated, whereas the coding region remained unmethylated in all cases. The level of methylation of the region located downstream of the coding region of the terc gene changes with age. This region was analyzed in the samples of other tissues, and its methylation status was also nonuniform. The alteration of the methylation status in the 3'-untranslated region of the terc gene suggests the possibility of transcription of the antisense strand in this region.

  9. Genes and junk in plant mitochondria-repair mechanisms and selection.

    PubMed

    Christensen, Alan C

    2014-06-05

    Plant mitochondrial genomes have very low mutation rates. In contrast, they also rearrange and expand frequently. This is easily understood if DNA repair in genes is accomplished by accurate mechanisms, whereas less accurate mechanisms including nonhomologous end joining or break-induced replication are used in nongenes. An important question is how different mechanisms of repair predominate in coding and noncoding DNA, although one possible mechanism is transcription-coupled repair (TCR). This work tests the predictions of TCR and finds no support for it. Examination of the mutation spectra and rates in genes and junk reveals what DNA repair mechanisms are available to plant mitochondria, and what selective forces act on the repair products. A model is proposed that mismatches and other DNA damages are repaired by converting them into double-strand breaks (DSBs). These can then be repaired by any of the DSB repair mechanisms, both accurate and inaccurate. Natural selection will eliminate coding regions repaired by inaccurate mechanisms, accounting for the low mutation rates in genes, whereas mutations, rearrangements, and expansions generated by inaccurate repair in noncoding regions will persist. Support for this model includes the structure of the mitochondrial mutS homolog in plants, which is fused to a double-strand endonuclease. The model proposes that plant mitochondria do not distinguish a damaged or mismatched DNA strand from the undamaged strand, they simply cut both strands and perform homology-based DSB repair. This plant-specific strategy for protecting future generations from mitochondrial DNA damage has the side effect of genome expansions and rearrangements.

  10. New differentially expressed genes and differential DNA methylation underlying refractory epilepsy

    PubMed Central

    Xu, Tao; Liu, Shiyong; Yuan, Jinxian; Huang, Hao; Qin, Lu; Yang, Hui; Chen, Lifen; Tan, Xinjie; Chen, Yangmei

    2016-01-01

    Epigenetics underlying refractory epilepsy is poorly understood, especially in patients without distinctive genetic alterations. DNA methylation may affect gene expression in epilepsy without affecting DNA sequences. Herein, we analyzed genome-wide DNA methylation and gene expression in brain tissues of 10 patients with refractory epilepsy using methylated DNA immunoprecipitation linked with sequencing and mRNA Sequencing. Diverse distribution of differentially methylated genes was found in X chromosome, while differentially methylated genes appeared rarely in Y chromosome. 62 differentially expressed genes, such as MMP19, AZGP1, DES, and LGR6 were correlated with refractory epilepsy for the first time. Although general trends of differentially enriched gene ontology terms and Kyoto Encyclopedia of Genes and Genome pathways in this study are consistent with previous researches, differences also exist in many specific gene ontology terms and Kyoto Encyclopedia of Genes and Genome pathways. These findings provide a new genome-wide profiling of DNA methylation and gene expression in brain tissues of patients with refractory epilepsy, which may provide a basis for further study on the etiology and mechanisms of refractory epilepsy. PMID:27903967

  11. The Genomic Impact of DNA CpG Methylation on Gene Expression; Relationships in Prostate Cancer.

    PubMed

    Long, Mark D; Smiraglia, Dominic J; Campbell, Moray J

    2017-02-14

    The process of DNA CpG methylation has been extensively investigated for over 50 years and revealed associations between changing methylation status of CpG islands and gene expression. As a result, DNA CpG methylation is implicated in the control of gene expression in developmental and homeostasis processes, as well as being a cancer-driver mechanism. The development of genome-wide technologies and sophisticated statistical analytical approaches has ushered in an era of widespread analyses, for example in the cancer arena, of the relationships between altered DNA CpG methylation, gene expression, and tumor status. The remarkable increase in the volume of such genomic data, for example, through investigators from the Cancer Genome Atlas (TCGA), has allowed dissection of the relationships between DNA CpG methylation density and distribution, gene expression, and tumor outcome. In this manner, it is now possible to test that the genome-wide correlations are measurable between changes in DNA CpG methylation and gene expression. Perhaps surprisingly is that these associations can only be detected for hundreds, but not thousands, of genes, and the direction of the correlations are both positive and negative. This, perhaps, suggests that CpG methylation events in cancer systems can act as disease drivers but the effects are possibly more restricted than suspected. Additionally, the positive and negative correlations suggest direct and indirect events and an incomplete understanding. Within the prostate cancer TCGA cohort, we examined the relationships between expression of genes that control DNA methylation, known targets of DNA methylation and tumor status. This revealed that genes that control the synthesis of S-adenosyl-l-methionine (SAM) associate with altered expression of DNA methylation targets in a subset of aggressive tumors.

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

    PubMed Central

    Pilon, Catia; Rebellato, Andrea; Urbanet, Riccardo; Guzzardo, Vincenza; Cappellesso, Rocco; Sasano, Hironobu; Fassina, Ambrogio

    2015-01-01

    We previously showed a decreased expression of vitamin D receptor (VDR) mRNA/protein in a small group of adrenocortical carcinoma (ACC) tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5′ regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas) were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesis. PMID:26843863

  13. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    PubMed

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  14. Promoter CpG methylation of multiple genes in pituitary adenomas: frequent involvement of caspase-8.

    PubMed

    Bello, M Josefa; De Campos, Jose M; Isla, Alberto; Casartelli, Cacilda; Rey, Juan A

    2006-02-01

    The epigenetic changes in pituitary adenomas were identified by evaluating the methylation status of nine genes (RB1, p14(ARF), p16(INK4a), p73, TIMP-3, MGMT, DAPK, THBS1 and caspase-8) in a series of 35 tumours using methylation-specific PCR analysis plus sequencing. The series included non-functional adenomas (n=23), prolactinomas (n=6), prolactinoma plus thyroid-stimulating hormone adenoma (n=1), growth hormone adenomas (n=4), and adrenocorticotropic adenoma (n=1). All of the tumours had methylation of at least one of these genes and 40% of samples (14 of 35) displayed concurrent methylation of at least three genes. The frequencies of aberrant methylation were: 20% for RB1, 17% for p14(ARF), 34% for p16(INK4a), 29% for p73, 11% for TIMP-3, 23% for MGMT, 6% for DAPK, 43% for THBS1 and 54% for caspase-8. No aberrant methylation was observed in two non-malignant pituitary samples from healthy controls. Although some differences in the frequency of gene methylation between functional and non-functional adenomas were detected, these differences did not reach statistical significance. Our results suggest that promoter methylation is a frequent event in pituitary adenoma tumourigenesis, a process in which inactivation of apoptosis-related genes (DAPK, caspase-8) might play a key role.

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

    PubMed

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

    2014-12-01

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

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

    PubMed

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

    2016-06-01

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

  17. Transcript RNA supports precise repair of its own DNA gene.

    PubMed

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

    The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

  18. Aberrant DNA methylation of some tumor suppressor genes in lung cancers from workers with chromate exposure.

    PubMed

    Ali, Abdellah H K; Kondo, Kazuya; Namura, Toshiaki; Senba, Yoshitaka; Takizawa, Hiromitsu; Nakagawa, Yasushi; Toba, Hiroaki; Kenzaki, Koichiro; Sakiyama, Shoji; Tangoku, Akira

    2011-02-01

    Our previous studies revealed a variety of genetic changes in lung cancers from chromate-exposed workers (chromate lung cancer). In the present study, we examined epigenetic changes in chromate lung cancers. Nested-methylation-specific PCR was employed in studying the methylation of CpG islands in the APC, MGMT, hMLH1 genes in 36 chromate lung cancers and 25 nonchromate lung cancers. Methylation in chromate lung cancers was detected at 86% for APC, 20% for MGMT, and 28% for hMLH1. Whereas, it occurred at lower frequencies in nonchromate lung cancers, particularly in APC (44%) and hMLH1 (0%) genes. Our previous study showed that methylation of p16 gene in chromate lung cancer and nonchromate lung cancer was 33% and 26%, respectively. The mean methylation index (MI), a reflection of the overall methylation status, was significantly higher in chromate lung cancers than nonchromate lung cancers (0.41 vs. 0.21, P=0.001). Methylation of multiple genes (particularly hMLH1, p16, and APC genes) had experienced more than 15 yr of chromate exposure in chromate lung cancer (MI: <15 yr; 0.19, ≥ 15 yr, 0.42). There is a significant correlation of p16 and hMLH1 methylation with the expressional decrease or loss of the corresponding gene products (P=0.037 and 0.024) respectively, and an inverse correlation between APC and MGMT methylation (P = 0.014). This study provides a novel evidence for the chromium carcinogenesis that chromate lung cancer is linked to the progressive methylation of some tumor suppressor genes, which may be related to genomic instability.

  19. Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

    PubMed Central

    Nelson, William; Luo, Meizhong; Ma, Jianxin; Estep, Matt; Estill, James; He, Ruifeng; Talag, Jayson; Sisneros, Nicholas; Kudrna, David; Kim, HyeRan; Ammiraju, Jetty SS; Collura, Kristi; Bharti, Arvind K; Messing, Joachim; Wing, Rod A; SanMiguel, Phillip; Bennetzen, Jeffrey L; Soderlund, Carol

    2008-01-01

    Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely

  20. Pancreatic Cancer Patient Survival Correlates with DNA Methylation of Pancreas Development Genes

    PubMed Central

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

    2015-01-01

    DNA methylation is an epigenetic mark associated with regulation of transcription and genome structure. These markers have been investigated in a variety of cancer settings for their utility in differentiating normal tissue from tumor tissue. Here, we examine the direct correlation between DNA methylation and patient survival. We find that changes in the DNA methylation of key pancreatic developmental genes are strongly associated with patient survival. PMID:26039411

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-01-01

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

  3. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

    PubMed Central

    Singh, K K; Samson, L

    1995-01-01

    Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761422

  4. The effect of acute dose charge particle radiation on expression of DNA repair genes in mice.

    PubMed

    Tariq, Muhammad Akram; Soedipe, Ayodotun; Ramesh, Govindarajan; Wu, Honglu; Zhang, Ye; Shishodia, Shishir; Gridley, Daila S; Pourmand, Nader; Jejelowo, Olufisayo

    2011-03-01

    The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the

  5. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene

    PubMed Central

    Hollywood, Jennifer A.; Lee, Ciaran M.; Scallan, Martina F.; Harrison, Patrick T.

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair. PMID:27557525

  6. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene.

    PubMed

    Hollywood, Jennifer A; Lee, Ciaran M; Scallan, Martina F; Harrison, Patrick T

    2016-08-25

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair.

  7. Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene

    SciTech Connect

    Buschhausen, G.; Wittig, B.; Graessmann, M.; Graessmann, A.

    1987-03-01

    Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of approx. 8 hr microinjection of the DNA into TK/sup -/ rat 2 and mouse LTK/sup -/ cells. The authors have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with (/sup 3/H)thymidine and by RNA dot blot analysis. However, in transformed cell lines, reactivation of the methylated chromatic occurred after treatment with 5-azacytidine. Furthermore, integration of the TK chromatin into the host genome is not required to block expression of the methylated TK gene. Mouse cells that contained the pML-BPV-TK4 chromatin permanently in an episomal state also did not support TK gene expression as long as the TK DNA remained methylated.

  8. Methylation of a panel of genes in peripheral blood leukocytes is associated with colorectal cancer

    PubMed Central

    Luo, Xiang; Huang, Rong; Sun, Hongru; Liu, Yupeng; Bi, Haoran; Li, Jing; Yu, Hongyuan; Sun, Jiamei; Lin, Shangqun; Cui, Binbin; Zhao, Yashuang

    2016-01-01

    The relationship between the DNA methylation status of the CpG islands of multiple genes in blood leukocytes in CRC susceptibility and prognosis, as well as possible interactions with dietary factors on CRC risk are unclear. We carried out a case-control study including 421 CRC patients and 506 controls to examine the associations between six genes (AOX-1, RARB2, RERG, ADAMTS9, IRF4, and FOXE-1), multiple CpG site methylation (MCSM) and susceptibility to CRC. High-level MCSM (MCSM-H) was defined as methylation of greater than or equal to 2 of 5 candidate genes (except for RARB2); low-level MCSM (MCSM-L) was when 1 candidate gene was methylated; non-MCSM was when none of the candidate genes were methylated. Blood cell-derived DNA methylation status was detected using methylation-sensitive high-resolution melting analysis. The hypermethylation status of each individual gene was statistically significantly associated with CRC. MCSM status was also associated with CRC (OR = 1.54, 95% CI: 1.15–2.05, P = 0.004). We observed interactions between a high level of dietary intake of cereals, pungent food, and stewed fish with brown sauce, age (older than 60 yrs), smoking and hypermethylation on risk of CRC. MCSM in peripheral blood DNA may be an important biomarker for susceptibility to CRC. PMID:27453436

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2017-03-08

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

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

    PubMed Central

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

    2017-01-01

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

  12. Protein Methylation and Interaction with the Antiproliferative Gene, BTG2/TIS21/Pc3

    PubMed Central

    Kim, Sangduk

    2014-01-01

    The last one and half a decade witnessed an outstanding re-emergence of attention and remarkable progress in the field of protein methylation. In the present article, we describe the early discoveries in research and review the role protein methylation played in the biological function of the antiproliferative gene, BTG2/TIS21/PC3. PMID:24532495

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

    ERIC Educational Resources Information Center

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

    2016-01-01

    Accumulating evidence suggests that early adversity is linked to methylation of the glucocorticoid receptor (GR) gene, "NR3C1," which is a key regulator of the hypothalamic-pituitary-adrenal axis. Yet no prior work has considered the contribution of methylation of "NR3C1" to emerging behavior problems and psychopathology in…

  14. Differentially methylated obligatory epialleles modulate context-dependent LAM gene expression in the honeybee Apis mellifera

    PubMed Central

    Wedd, Laura; Kucharski, Robert; Maleszka, Ryszard

    2016-01-01

    ABSTRACT Differential intragenic methylation in social insects has been hailed as a prime mover of environmentally driven organismal plasticity and even as evidence for genomic imprinting. However, very little experimental work has been done to test these ideas and to prove the validity of such claims. Here we analyze in detail differentially methylated obligatory epialleles of a conserved gene encoding lysosomal α-mannosidase (AmLAM) in the honeybee. We combined genotyping of progenies derived from colonies founded by single drone inseminated queens, ultra-deep allele-specific bisulfite DNA sequencing, and gene expression to reveal how sequence variants, DNA methylation, and transcription interrelate. We show that both methylated and non-methylated states of AmLAM follow Mendelian inheritance patterns and are strongly influenced by polymorphic changes in DNA. Increased methylation of a given allele correlates with higher levels of context-dependent AmLAM expression and appears to affect the transcription of an antisense long noncoding RNA. No evidence of allelic imbalance or imprinting involved in this process has been found. Our data suggest that by generating alternate methylation states that affect gene expression, sequence variants provide organisms with a high level of epigenetic flexibility that can be used to select appropriate responses in various contexts. This study represents the first effort to integrate DNA sequence variants, gene expression, and methylation in a social insect to advance our understanding of their relationships in the context of causality. PMID:26507253

  15. Differentially methylated obligatory epialleles modulate context-dependent LAM gene expression in the honeybee Apis mellifera.

    PubMed

    Wedd, Laura; Kucharski, Robert; Maleszka, Ryszard

    2016-01-01

    Differential intragenic methylation in social insects has been hailed as a prime mover of environmentally driven organismal plasticity and even as evidence for genomic imprinting. However, very little experimental work has been done to test these ideas and to prove the validity of such claims. Here we analyze in detail differentially methylated obligatory epialleles of a conserved gene encoding lysosomal α-mannosidase (AmLAM) in the honeybee. We combined genotyping of progenies derived from colonies founded by single drone inseminated queens, ultra-deep allele-specific bisulfite DNA sequencing, and gene expression to reveal how sequence variants, DNA methylation, and transcription interrelate. We show that both methylated and non-methylated states of AmLAM follow Mendelian inheritance patterns and are strongly influenced by polymorphic changes in DNA. Increased methylation of a given allele correlates with higher levels of context-dependent AmLAM expression and appears to affect the transcription of an antisense long noncoding RNA. No evidence of allelic imbalance or imprinting involved in this process has been found. Our data suggest that by generating alternate methylation states that affect gene expression, sequence variants provide organisms with a high level of epigenetic flexibility that can be used to select appropriate responses in various contexts. This study represents the first effort to integrate DNA sequence variants, gene expression, and methylation in a social insect to advance our understanding of their relationships in the context of causality.

  16. A novel approach identifies new differentially methylated regions (DMRs) associated with imprinted genes

    PubMed Central

    Choufani, Sanaa; Shapiro, Jonathan S.; Susiarjo, Martha; Butcher, Darci T.; Grafodatskaya, Daria; Lou, Youliang; Ferreira, Jose C.; Pinto, Dalila; Scherer, Stephen W.; Shaffer, Lisa G.; Coullin, Philippe; Caniggia, Isabella; Beyene, Joseph; Slim, Rima; Bartolomei, Marisa S.; Weksberg, Rosanna

    2011-01-01

    Imprinted genes are critical for normal human growth and neurodevelopment. They are characterized by differentially methylated regions (DMRs) of DNA that confer parent of origin-specific transcription. We developed a new strategy to identify imprinted gene-associated DMRs. Using genome-wide methylation profiling of sodium bisulfite modified DNA from normal human tissues of biparental origin, candidate DMRs were identified by selecting CpGs with methylation levels consistent with putative allelic differential methylation. In parallel, the methylation profiles of tissues of uniparental origin, i.e., paternally-derived androgenetic complete hydatidiform moles (AnCHMs), and maternally-derived mature cystic ovarian teratoma (MCT), were examined and then used to identify CpGs with parent of origin-specific DNA methylation. With this approach, we found known DMRs associated with imprinted genomic regions as well as new DMRs for known imprinted genes, NAP1L5 and ZNF597, and novel candidate imprinted genes. The paternally methylated DMR for one candidate, AXL, a receptor tyrosine kinase, was also validated in experiments with mouse embryos that demonstrated Axl was expressed preferentially from the maternal allele in a DNA methylation-dependent manner. PMID:21324877

  17. Integration and bioinformatics analysis of DNA-methylated genes associated with drug resistance in ovarian cancer

    PubMed Central

    YAN, BINGBING; YIN, FUQIANG; WANG, QI; ZHANG, WEI; LI, LI

    2016-01-01

    The main obstacle to the successful treatment of ovarian cancer is the development of drug resistance to combined chemotherapy. Among all the factors associated with drug resistance, DNA methylation apparently plays a critical role. In this study, we performed an integrative analysis of the 26 DNA-methylated genes associated with drug resistance in ovarian cancer, and the genes were further evaluated by comprehensive bioinformatics analysis including gene/protein interaction, biological process enrichment and annotation. The results from the protein interaction analyses revealed that at least 20 of these 26 methylated genes are present in the protein interaction network, indicating that they interact with each other, have a correlation in function, and may participate as a whole in the regulation of ovarian cancer drug resistance. There is a direct interaction between the phosphatase and tensin homolog (PTEN) gene and at least half of the other genes, indicating that PTEN may possess core regulatory functions among these genes. Biological process enrichment and annotation demonstrated that most of these methylated genes were significantly associated with apoptosis, which is possibly an essential way for these genes to be involved in the regulation of multidrug resistance in ovarian cancer. In addition, a comprehensive analysis of clinical factors revealed that the methylation level of genes that are associated with the regulation of drug resistance in ovarian cancer was significantly correlated with the prognosis of ovarian cancer. Overall, this study preliminarily explains the potential correlation between the genes with DNA methylation and drug resistance in ovarian cancer. This finding has significance for our understanding of the regulation of resistant ovarian cancer by methylated genes, the treatment of ovarian cancer, and improvement of the prognosis of ovarian cancer. PMID:27347118

  18. Relationship between DNA mismatch repair genes expression, Ku-genes expression and ploidy-related parameters in the progression of pigmented lesions of the skin.

    PubMed

    Korabiowska, Monika; Tscherny, Michael; Stachura, Jerzy; Ruschenburg, Ilka; Cordon-Cardo, Carlos; Brinck, Ulrich

    2002-01-01

    Defects of DNA repair systems in cutaneous tumours are related to DNA mismatch repair genes (MLH1, MSH2, PMS1, PMS2) and Ku70/80 genes involved in double- strand repair. In this study we investigated the statistical relationship between these systems and DNA-ploidy-related parameters in 19 naevus cell naevi, 23 lentigos maligna, 76 primary melanomas and 31 melanoma metastases, applying the correlation coefficient according to Spearman. In naevi significant correlations were found between Ku70/80 gene expression and some ploidy-related parameters. In lentigos, additionally, some significant correlations between the expression of DNA mismatch repair genes were found. Similar results were demonstrated for primary melanomas. In metastases no one significant correlation between DNA mismatch repair genes and Ku-genes was present. We postulate that DNA mismatch repair genes and Ku70/80 genes are functionally independent and that some of them are able to influence ploidy-related parameters.

  19. Preferential repair of DNA double-strand break at the active gene in vivo.

    PubMed

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K; Bhaumik, Sukesh R

    2012-10-19

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

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

    PubMed

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

    2015-05-15

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

  1. DDMGD: the database of text-mined associations between genes methylated in diseases from different species.

    PubMed

    Bin Raies, Arwa; Mansour, Hicham; Incitti, Roberto; Bajic, Vladimir B

    2015-01-01

    Gathering information about associations between methylated genes and diseases is important for diseases diagnosis and treatment decisions. Recent advancements in epigenetics research allow for large-scale discoveries of associations of genes methylated in diseases in different species. Searching manually for such information is not easy, as it is scattered across a large number of electronic publications and repositories. Therefore, we developed DDMGD database (http://www.cbrc.kaust.edu.sa/ddmgd/) to provide a comprehensive repository of information related to genes methylated in diseases that can be found through text mining. DDMGD's scope is not limited to a particular group of genes, diseases or species. Using the text mining system DEMGD we developed earlier and additional post-processing, we extracted associations of genes methylated in different diseases from PubMed Central articles and PubMed abstracts. The accuracy of extracted associations is 82% as estimated on 2500 hand-curated entries. DDMGD provides a user-friendly interface facilitating retrieval of these associations ranked according to confidence scores. Submission of new associations to DDMGD is provided. A comparison analysis of DDMGD with several other databases focused on genes methylated in diseases shows that DDMGD is comprehensive and includes most of the recent information on genes methylated in diseases.

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2017-01-15

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

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

    PubMed Central

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

    2017-01-01

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

  5. Gene-specific DNA methylation of DNMT3B and MTHFR and colorectal adenoma risk.

    PubMed

    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.

  6. Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma.

    PubMed

    Gonzalez-Gomez, Pilar; Bello, M Josefa; Inda, M Mar; Alonso, M Eva; Arjona, Dolores; Amiñoso, Cinthia; Lopez-Marin, Isabel; de Campos, Jose M; Sarasa, Jose L; Castresana, Javier S; Rey, Juan A

    2004-09-01

    Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  9. Survival of UV-irradiated mammalian cells correlates with efficient DNA repair in an essential gene

    SciTech Connect

    Bohr, V.A.; Okumoto, D.S.; Hanawalt, P.C.

    1986-06-01

    The survival of UV-irradiated mammalian cells is not necessarily correlated with their overall capacity to carry out DNA repair. Human cells typically remove 80% of the pyrimidine dimers produced by a UV dose of 5 J/m2 within 24 hr. In contrast, a Chinese hamster ovary (CHO) cell line survives UV irradiation equally well while removing only 15% of the dimers. Using a newly developed technique to measure dimer frequencies in single-copy specific sequences, we find that the CHO cells remove 70% of the dimers from the essential dihydrofolate reductase (DHFR) gene but only 20% from sequences located 30 kilobases or more upstream from the 5' end of the gene in a 24-hr period. Repair-deficient human cells from xeroderma pigmentosum complementation group C (XPC) are similar to the CHO cells in overall repair levels, but they are extremely sensitive to killing by UV irradiation. In the XPC cells, we find little or no repair in the DHFR gene; in contrast, in normal human fibroblasts and epidermal keratinocytes, greater than 80% of the dimers induced in the gene by 20 J/m2 are removed in 24 hr. Since the CHO and normal human cells exhibit similar UV resistance, much higher than that of XPC cells, our findings suggest a correlation between efficient repair of essential genes and resistance to DNA-damaging agents such as UV light.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Chromatin immunoprecipitation microarrays for identification of genes silenced by histone H3 lysine 9 methylation.

    PubMed

    Kondo, Yutaka; Shen, Lanlan; Yan, Pearlly S; Huang, Tim Hui-Ming; Issa, Jean-Pierre J

    2004-05-11

    Switching from acetylation to methylation at histone H3 lysine 9 (K9) has recently been shown to contribute to euchromatin gene silencing. To identify genes silenced by K9 modifications, we probed a human CpG island microarray with DNA obtained by chromatin immunoprecipitation (ChIP) in a cancer cell line using an anti-H3-K9 methylated antibody or an anti-H3-K9 acetylated antibody. Of the 27 clones with the highest signal ratio of K9 methylation over acetylation (Me/Ac), 13 contained repetitive sequences. Among 14 nonrepetitive clones, we identified 11 genes (seven known and four previously undescribed), one EST, and two unknown fragments. Using ChIP-PCR, all 18 examined clones showed higher ratios of H3-K9 Me/Ac than the active gene control, P21, thus confirming the microarray data. In addition, we found a strong correlation between the K9 Me/Ac ratio and CpG island DNA methylation (R = 0.92, P < 0.01), and five of seven genes examined (megalin, thrombospondin-4, KR18, latrophilin-3, and phosphatidylinositol-3-OH kinase P101 subunit) showed lack of expression by RT-PCR and reactivation by DNA methylation and/or histone deacetylase inhibition, suggesting that these genes are true targets of silencing through histone modifications. All five genes also showed significant DNA methylation in a cell line panel and in primary colon cancers. Our data suggest that CpG island microarray coupled with ChIP can identify novel targets of gene silencing in cancer. This unbiased approach confirms the tight coupling between DNA methylation and histone modifications in cancer and could be used to probe gene silencing in nonneoplastic conditions as well.

  14. Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

    PubMed Central

    Bokranz, M; Bäumner, G; Allmansberger, R; Ankel-Fuchs, D; Klein, A

    1988-01-01

    The genes coding for methyl coenzyme M reductase were cloned from a genomic library of Methanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, alpha, beta, and gamma. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes. Images PMID:2448287

  15. Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

    PubMed

    Das, Raima; Ghosh, Sankar Kumar

    2017-04-01

    DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future.

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

    PubMed

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

    2013-01-01

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

  17. Effects of arsenic exposure on DNA methylation and epigenetic gene regulation.

    PubMed

    Reichard, John F; Puga, Alvaro

    2010-02-01

    Arsenic is a nonmutagenic human carcinogen that induces tumors through unknown mechanisms. A growing body of evidence suggests that its carcinogenicity results from epigenetic changes, particularly in DNA methylation. Changes in gene methylation status, mediated by arsenic, have been proposed to activate oncogene expression or silence tumor suppressor genes, leading to long-term changes in the activity of genes controlling cell transformation. Mostly descriptive, and often contradictory, studies have demonstrated that arsenic exposure is associated with both hypo- and hyper-methylation at various genetic loci in vivo or in vitro. This ambiguity has made it difficult to assess whether the changes induced by arsenic are causally involved in the transformation process or are simply a reflection of the altered physiology of rapidly dividing cancer cells. Here, we discuss the evidence supporting changes in DNA methylation as a cause of arsenic carcinogenesis and highlight the strengths and limitations of these studies, as well as areas where consistencies and inconsistencies exist.

  18. Polymorphisms in DNA Repair Genes, Recreational Physical Activity and Breast Cancer Risk

    PubMed Central

    McCullough, Lauren E.; Santella, Regina M.; Cleveland, Rebecca J.; Millikan, Robert C.; Olshan, Andrew F.; North, Kari E.; Bradshaw, Patrick T.; Eng, Sybil M.; Terry, Mary Beth; Shen, Jing; Crew, Katherine D.; Rossner, Pavel; Teitelbaum, Susan L.; Neugut, Alfred I.; Gammon, Marilie D.

    2013-01-01

    The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single-strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Polymorphisms in MMR or other DNA repair gene variants may modify the association between RPA and breast cancer incidence. We investigated the individual and joint effects of variants in three MMR pathway genes (MSH3, MLH1 and MSH2) on breast cancer occurrence using resources from the Long Island Breast Cancer Study Project. We additionally characterized interactions between RPA and genetic polymorphisms in MMR, BER and NER pathways. We found statistically significant multiplicative interactions (p<0.05) between MSH2 and MLH1, as well as between postmenopausal RPA and four variants in DNA repair (XPC-Ala499Val, XPF-Arg415Gln, XPG-Asp1104His and MLH1-lle219Val). Significant risk reductions were observed among highly active women with the common genotype for XPC (OR=0.54; 95% CI, 0.36–0.81) and XPF (OR=0.62; 95% CI, 0.44–0.87), as well as among active women who carried at least one variant allele in XPG (OR=0.46; 95% CI, 0.29–0.77) and MLH1 (OR=0.46; 95% CI, 0.30–0.71). Our data show that women with minor alleles in both MSH2 and MLH1 could be at increased breast cancer risk. RPA may be modified by genes in the DNA repair pathway, and merit further investigation. PMID:23852586

  19. Tobacco smoking and methylation of genes related to lung cancer development

    PubMed Central

    Gao, Xu; Zhang, Yan; Breitling, Lutz Philipp; Brenner, Hermann

    2016-01-01

    Lung cancer is a leading cause of cancer-related mortality worldwide, and cigarette smoking is the major environmental hazard for its development. This study intended to examine whether smoking could alter methylation of genes at lung cancer risk loci identified by genome-wide association studies (GWASs). By systematic literature review, we selected 75 genomic candidate regions based on 120 single-nucleotide polymorphisms (SNPs). DNA methylation levels of 2854 corresponding cytosine-phosphate-guanine (CpG) candidates in whole blood samples were measured by the Illumina Infinium Human Methylation450 Beadchip array in two independent subsamples of the ESTHER study. After correction for multiple testing, we successfully confirmed associations with smoking for one previously identified CpG site within the KLF6 gene and identified 12 novel sites located in 7 genes: STK32A, TERT, MSH5, ACTA2, GATA3, VTI1A and CHRNA5 (FDR <0.05). Current smoking was linked to a 0.74% to 2.4% decrease of DNA methylation compared to never smoking in 11 loci, and all but one showed significant associations (FDR <0.05) with life-time cumulative smoking (pack-years). In conclusion, our study demonstrates the impact of tobacco smoking on DNA methylation of lung cancer related genes, which may indicate that lung cancer susceptibility genes might be regulated by methylation changes in response to smoking. Nevertheless, this mechanism warrants further exploration in future epigenetic and biomarker studies. PMID:27323854

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

    PubMed

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

    2015-09-01

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

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

    PubMed Central

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

    2016-01-01

    Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5′-cytosine–phosphate–guanine-3′) sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental

  2. Identification of uterine leiomyoma-specific marker genes based on DNA methylation and their clinical application

    PubMed Central

    Sato, Shun; Maekawa, Ryo; Yamagata, Yoshiaki; Tamura, Isao; Lee, Lifa; Okada, Maki; Jozaki, Kosuke; Asada, Hiromi; Tamura, Hiroshi; Sugino, Norihiro

    2016-01-01

    Differential diagnosis of uterine leiomyomas and leiomyosarcomas is needed to determine whether the uterus can be retained. Therefore, biomarkers for uterine leiomyomas, and reliable and objective diagnostic methods have been desired besides the pathological diagnosis. In the present study, we identified 12 genes specific to uterine leiomyomas based on DNA methylation. Using these marker genes specific to uterine leiomyomas, we established a hierarchical clustering system based on the DNA methylation level of the marker genes, which could completely differentiate between uterine leiomyomas and normal myometrium. Furthermore, our hierarchical clustering system completely discriminated uterine cancers and differentiated between uterine leiomyosarcomas and leiomyomas with more than 70% accuracy. In conclusion, this study identified DNA methylation-based marker genes specific to uterine leiomyomas, and our hierarchical clustering system using these marker genes was useful for differential diagnosis of uterine leiomyomas and leiomyosarcomas. PMID:27498619

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Liu, Jinghe; Liang, Xingwei; Zhu, Jiaqiao; Wei, Liang; Hou, Yi; Chen, Da-Yuan; Sun, Qing-Yuan

    2008-09-01

    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases (DNMTs), therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmt1 and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones (AF1, AF2 and AF3) showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF) fetuses. Besides, we found that the 5' regions of Dnmt1 and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  5. Methylation analysis of multiple genes in blood DNA of Alzheimer's disease and healthy individuals.

    PubMed

    Tannorella, Pierpaola; Stoccoro, Andrea; Tognoni, Gloria; Petrozzi, Lucia; Salluzzo, Maria Grazia; Ragalmuto, Alda; Siciliano, Gabriele; Haslberger, Alexander; Bosco, Paolo; Bonuccelli, Ubaldo; Migliore, Lucia; Coppedè, Fabio

    2015-07-23

    We collected blood DNA from 120 late-onset Alzheimer's disease (AD) patients and 115 healthy matched controls and analysed the methylation levels of genes involved in amyloid-beta peptide production (PSEN1 and BACE1), in DNA methylation (DNMT1, DNMT3A and DNMT3B), and in one-carbon metabolism (MTHFR), searching for correlation with age and gender, with biomarkers of one-carbon metabolism (plasma homocysteine, and serum folate and vitamin B12 levels), and with disease status (being healthy or having AD). We also evaluated the contribution of the APOE ϵ4 allele, the major late-onset AD genetic risk factor, to the studied gene methylation levels. All the genes showed low mean methylation levels (<5%) in both AD and control DNA, no difference between groups, and no correlation with the studied biomarkers, except for MTHFR that showed methylation levels ranging from 5% to 75%, and correlation with circulating biomarkers of one-carbon metabolism. However, mean MTHFR methylation levels were similar between groups (31.1% in AD and 30.7% in controls, P=0.58). Overall, present data suggest that none of the studied regions is differently methylated in blood DNA between AD and control subjects.

  6. Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

    PubMed Central

    Lock, L F; Melton, D W; Caskey, C T; Martin, G R

    1986-01-01

    It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt

  7. Gene Expression in Experimental Aortic Coarctation and Repair: Candidate Genes for Therapeutic Intervention?

    PubMed

    LaDisa, John F; Bozdag, Serdar; Olson, Jessica; Ramchandran, Ramani; Kersten, Judy R; Eddinger, Thomas J

    2015-01-01

    Coarctation of the aorta (CoA) is a constriction of the proximal descending thoracic aorta and is one of the most common congenital cardiovascular defects. Treatments for CoA improve life expectancy, but morbidity persists, particularly due to the development of chronic hypertension (HTN). Identifying the mechanisms of morbidity is difficult in humans due to confounding variables such as age at repair, follow-up duration, coarctation severity and concurrent anomalies. We previously developed an experimental model that replicates aortic pathology in humans with CoA without these confounding variables, and mimics correction at various times using dissolvable suture. Here we present the most comprehensive description of differentially expressed genes (DEGs) to date from the pathology of CoA, which were obtained using this model. Aortic samples (n=4/group) from the ascending aorta that experiences elevated blood pressure (BP) from induction of CoA, and restoration of normal BP after its correction, were analyzed by gene expression microarray, and enriched genes were converted to human orthologues. 51 DEGs with >6 fold-change (FC) were used to determine enriched Gene Ontology terms, altered pathways, and association with National Library of Medicine Medical Subject Headers (MeSH) IDs for HTN, cardiovascular disease (CVD) and CoA. The results generated 18 pathways, 4 of which (cell cycle, immune system, hemostasis and metabolism) were shared with MeSH ID's for HTN and CVD, and individual genes were associated with the CoA MeSH ID. A thorough literature search further uncovered association with contractile, cytoskeletal and regulatory proteins related to excitation-contraction coupling and metabolism that may explain the structural and functional changes observed in our experimental model, and ultimately help to unravel the mechanisms responsible for persistent morbidity after treatment for CoA.

  8. Gene Expression in Experimental Aortic Coarctation and Repair: Candidate Genes for Therapeutic Intervention?

    PubMed Central

    LaDisa, John F.; Bozdag, Serdar; Olson, Jessica; Ramchandran, Ramani; Kersten, Judy R.; Eddinger, Thomas J.

    2015-01-01

    Coarctation of the aorta (CoA) is a constriction of the proximal descending thoracic aorta and is one of the most common congenital cardiovascular defects. Treatments for CoA improve life expectancy, but morbidity persists, particularly due to the development of chronic hypertension (HTN). Identifying the mechanisms of morbidity is difficult in humans due to confounding variables such as age at repair, follow-up duration, coarctation severity and concurrent anomalies. We previously developed an experimental model that replicates aortic pathology in humans with CoA without these confounding variables, and mimics correction at various times using dissolvable suture. Here we present the most comprehensive description of differentially expressed genes (DEGs) to date from the pathology of CoA, which were obtained using this model. Aortic samples (n=4/group) from the ascending aorta that experiences elevated blood pressure (BP) from induction of CoA, and restoration of normal BP after its correction, were analyzed by gene expression microarray, and enriched genes were converted to human orthologues. 51 DEGs with >6 fold-change (FC) were used to determine enriched Gene Ontology terms, altered pathways, and association with National Library of Medicine Medical Subject Headers (MeSH) IDs for HTN, cardiovascular disease (CVD) and CoA. The results generated 18 pathways, 4 of which (cell cycle, immune system, hemostasis and metabolism) were shared with MeSH ID’s for HTN and CVD, and individual genes were associated with the CoA MeSH ID. A thorough literature search further uncovered association with contractile, cytoskeletal and regulatory proteins related to excitation-contraction coupling and metabolism that may explain the structural and functional changes observed in our experimental model, and ultimately help to unravel the mechanisms responsible for persistent morbidity after treatment for CoA. PMID:26207811

  9. Candidate Luminal B Breast Cancer Genes Identified by Genome, Gene Expression and DNA Methylation Profiling

    PubMed Central

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

    2014-01-01

    Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype. PMID:24416132

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

    PubMed

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

    2014-01-01

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

  11. Aberrant promoter methylation of multiple genes in sputum from individuals exposed to smoky coal emissions

    PubMed Central

    Liu, Yang; Lan, Qing; Shen, Min; Mumford, Judy; Keohavong, Phouthone

    2010-01-01

    Summary Aberrant methylation in the promoter region of cancer-related genes leads to gene transcriptional inactivation and plays an integral role in lung tumorigenesis. Recent studies demonstrated that promoter methylation was detected not only in lung tumors from patients with lung cancer but also in sputum of smokers without the disease, suggesting the potential for aberrant gene promoter methylation in sputum as a predictive marker for lung cancer. In the present study, we investigated promoter methylation of 4 genes frequently detected in lung tumors, including p16, MGMT, RASSF1A and DAPK genes, in sputum samples obtained from 107 individuals, including 34 never-smoking females and 73 mostly smoking males, who had no evidence of lung cancer but who were exposed to smoky coal emission in Xuan Wei County, China, where lung cancer rate is more than 6 times the Chinese national average rate. Forty nine of the individuals showed evidence of chronic bronchitis while the remaining 58 individuals showed no such a symptom. Promoter methylation of p16, MGMT, RASSF1A and DAPK was detected in 51.4% (55/107), 17.8% (19/107), 29.9% (32/107), and 15.9% (17/107) of the sputum samples from these individuals, respectively. There were no differences in promoter methylation frequencies of any of these genes according to smoking status or gender of the subjects or between individuals with chronic bronchitis and those without evidence of such a symptom. Therefore, individuals exposed to smoky coal emissions in this region harbored in their sputum frequent promoter methylation of these genes that have been previously found in lung tumors and implicated in lung cancer development. PMID:18751376

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

    PubMed Central

    2013-01-01

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

  13. Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

    PubMed Central

    Ngô, V; Gourdji, D; Laverrière, J N

    1996-01-01

    The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo. PMID:8668139

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

    PubMed

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

    2009-04-01

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

  15. Genome-wide gene expression and DNA methylation differences in abnormally cloned and normally natural mating piglets.

    PubMed

    Zou, C; Fu, Y; Li, C; Liu, H; Li, G; Li, J; Zhang, H; Wu, Y; Li, C

    2016-08-01

    Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways (e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer.

  16. Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates.

    PubMed

    Guo, Xiaoge; Jinks-Robertson, Sue

    2013-12-01

    Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral

  17. Functional characterization of polymorphisms in DNA repair genes using cytogenetic challenge assays.

    PubMed Central

    Au, William W; Salama, Salama A; Sierra-Torres, Carlos H

    2003-01-01

    A major barrier to understanding the role of polymorphic DNA repair genes for environmental cancer is that the functions of variant genotypes are largely unknown. Using our cytogenetic challenge assays, we conducted an investigation to address the deficiency. Using X-rays or ultraviolet (UV) light, we irradiated blood lymphocytes from 80 nonsmoking donors to challenge the cells to repair the induced DNA damage, and we analyzed expression of chromosome aberrations (CA) specific to the inducing agents. We have genotyped polymorphic DNA repair genes preferentially involved with base excision repair (BER) and nucleotide excision repair (NER) activities (XRCC1, XRCC3, APE1, XPD) corresponding to the repair of X-ray- and UV light-induced DNA damage, respectively. We expected that defects in specific DNA repair pathways due to polymorphisms would cause corresponding increases of specific CA. From our data, XRCC1 399Gln and XRCC3 241Met were associated with significant increases in chromosome deletions compared with the corresponding homozygous wild types (18.27 1.1 vs 14.79 1.2 and 18.22 0.99 vs 14.20 1.39, respectively); XPD 312Asn and XPD 751Gln were associated with significant increases in chromatid breaks compared with wild types (16.09 1.36 vs 11.41 0.98 and 16.87 1.27 vs 10.54 0.87, respectively), p < 0.05. The data indicate that XRCC1 399Gln and XRCC3 241Met are significantly defective in BER, and the XPD 312Asn and XPD 751Gln are significantly defective in NER. In addition, the variant genotypes interact significantly, with limited overlap of the two different repair pathways. PMID:14630517

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

    PubMed

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

    2009-03-01

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

  19. Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

    SciTech Connect

    Mudgett, J.S.; MacInnes, M.A. )

    1990-12-01

    The complete human nucleotide exicision repair gene ERCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-09-01

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

  2. Genome-Wide Screening of Genes Regulated by DNA Methylation in Colon Cancer Development

    PubMed Central

    Galamb, Orsolya; Wichmann, Barna; Sipos, Ferenc; Péterfia, Bálint; Csabai, István; Kovalszky, Ilona; Semsey, Szabolcs; Tulassay, Zsolt; Molnár, Béla

    2012-01-01

    Tumorigenesis is accompanied by changes in the DNA methylation pattern. Our aim was to test a novel approach for identification of transcripts at whole transcript level which are regulated by DNA methylation. Our approach is based on comparison of data obtained from transcriptome profiling of primary human samples and in vitro cell culture models. Epithelial cells were collected by LCM from normal, adenoma, and tumorous colonic samples. Using gene expression analysis, we identified downregulated genes in the tumors compared to normal tissues. In parallel 3000 upregulated genes were determined in HT-29 colon adenocarcinoma cell culture model after DNA demethylation treatment. Of the 2533 transcripts showing reduced expression in the tumorous samples, 154 had increased expression as a result of DNA demethylation treatment. Approximately 2/3 of these genes had decreased expression already in the adenoma samples. Expression of five genes (GCG, NMES-1, LRMP, FAM161B and PTGDR), was validated using RT-PCR. PTGDR showed ambiguous results, therefore it was further studied to verify the extent of DNA methylation and its effect on the protein level. Results confirmed that our approach is suitable for genome-wide screening of genes which are regulated or inactivated by DNA methylation. Activity of these genes possibly interferes with tumor progression, therefore genes identified can be key factors in the formation and in the progression of the disease. PMID:23049694

  3. [DNA methylation and epigenetics].

    PubMed

    Vaniushin, B F

    2006-09-01

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

  4. Epigenetic Studies Point to DNA Replication/Repair Genes as a Basis for the Heritable Nature of Long Term Complications in Diabetes

    PubMed Central

    Leontovich, Alexey A.; Intine, Robert V.; Sarras, Michael P.

    2016-01-01

    Metabolic memory (MM) is defined as the persistence of diabetic (DM) complications even after glycemic control is pharmacologically achieved. Using a zebrafish diabetic model that induces a MM state, we previously reported that, in this model, tissue dysfunction was of a heritable nature based on cell proliferation studies in limb tissue and this correlated with epigenetic DNA methylation changes that paralleled alterations in gene expression. In the current study, control, DM, and MM excised fin tissues were further analyzed by MeDIP sequencing and microarray techniques. Bioinformatics analysis of the data found that genes of the DNA replication/DNA metabolism process group (with upregulation of the apex1, mcm2, mcm4, orc3, lig1, and dnmt1 genes) were altered in the DM state and these molecular changes continued into MM. Interestingly, DNA methylation changes could be found as far as 6–13 kb upstream of the transcription start site for these genes suggesting potential higher levels of epigenetic control. In conclusion, DNA methylation changes in members of the DNA replication/repair process group best explain the heritable nature of cell proliferation impairment found in the zebrafish DM/MM model. These results are consistent with human diabetic epigenetic studies and provide one explanation for the persistence of long term tissue complications as seen in diabetes. PMID:26981540

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

    PubMed

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

    2015-06-03

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

  6. Gene-Specific DNA Methylation may Mediate Atypical Antipsychotic-Induced Insulin Resistance

    PubMed Central

    Burghardt, Kyle J.; Goodrich, Jacyln M.; Dolinoy, Dana C.; Ellingrod, Vicki L.

    2017-01-01

    Objectives Atypical Antipsychotics (AAPs) carry a significant risk of cardiometabolic side effects including insulin resistance. It is thought that the insulin resistance resulting from the use of AAP may be associated with changes in DNA methylation. We aimed to identify and validate a candidate gene associated with AAP-induced insulin resistance by using a multi-step approach that included an epigenome-wide association study (EWAS) and validation with site-specific methylation and metabolomics data. Methods Bipolar subjects treated with AAPs or lithium monotherapy were recruited for a cross-sectional visit to analyze peripheral blood DNA methylation and insulin resistance. Epigenome-wide DNA methylation was analyzed in a discovery sample (n=48) using the Illumina 450K BeadChip. Validation analyses of the epigenome-wide findings occurred in a separate sample (n=72) using site-specific methylation with pyrosequencing and untargeted metabolomics data. Regression analyses were conducted controlling for known confounders in all analyses and a mediation analysis was performed to investigate if AAP-induced insulin resistance occurs through changes in DNA methylation. Results A differentially methylated probe associated with insulin resistance was discovered and validated in the Fatty Acyl CoA Reductase 2 (FAR2) gene of Chromosome 12. Functional associations of this DNA methylation site on untargeted phospholipid-related metabolites were also detected. Our results identified a mediating effect of this FAR2 methylation site on AAP-induced insulin resistance. Conclusions Going forward, prospective, longitudinal studies assessing comprehensive changes in FAR2 DNA methylation, expression, and lipid metabolism before and after AAP treatment are required to assess its potential role in the development of insulin resistance. PMID:27542345

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

    PubMed Central

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

    1986-01-01

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

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

    PubMed

    Ngernprasirtsiri, J; Kobayashi, H; Akazawa, T

    1988-09-01

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

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

    PubMed

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

    2016-01-19

    Until recently, an elevated disease risk has been ascribed to a genetic predisposition, however, exciting progress over the past years has discovered alternate elements of inheritance that involve epigenetic regulation. Epigenetic changes are heritably stable alterations that include DNA methylation, histone modifications and RNA-mediated silencing. Aberrant DNA methylation is a common molecular basis for a number of important human diseases, including breast cancer. Changes in DNA methylation profoundly affect global gene expression patterns. What is emerging is a more dynamic and complex association between DNA methylation and gene expression than previously believed. Although many tools have already been developed for analyzing genome-wide gene expression data, tools for analyzing genome-wide DNA methylation have not yet reached the same level of refinement. Here we provide an in-depth analysis of DNA methylation in parallel with gene expression data characteristics and describe the particularities of low-level and high-level analyses of DNA methylation data. Low-level analysis refers to pre-processing of methylation data (i.e. normalization, transformation and filtering), whereas high-level analysis is focused on illustrating the application of the widely used class comparison, class prediction and class discovery methods to DNA methylation data. Furthermore, we investigate the influence of DNA methylation on gene expression by measuring the correlation between the degree of CpG methylation and the level of expression and to explore the pattern of methylation as a function of the promoter region.

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

    PubMed

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

    2013-01-01

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

  11. Higher expression of somatic repair genes in long-lived ant queens than workers

    PubMed Central

    Lucas, Eric R.; Privman, Eyal; Keller, Laurent

    2016-01-01

    Understanding why organisms senesce is a fundamental question in biology. One common explanation is that senescence results from an increase in macromolecular damage with age. The tremendous variation in lifespan between genetically identical queen and worker ants, ranging over an order of magnitude, provides a unique system to study how investment into processes of somatic maintenance and macromolecular repair influence lifespan. Here we use RNAseq to compare patterns of expression of genes involved in DNA and protein repair of age-matched queens and workers. There was no difference between queens and workers in 1-day-old individuals, but the level of expression of these genes increased with age and this up-regulation was greater in queens than in workers, resulting in significantly queen-biased expression in 2-month-old individuals in both legs and brains. Overall, these differences are consistent with the hypothesis that higher longevity is associated with increased investment into somatic repair. PMID:27617474

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

    PubMed Central

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

    2016-01-01

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

  13. Global prevalence and distribution of genes and microorganisms involved in mercury methylation

    SciTech Connect

    Podar, Mircea; Gilmour, C. C.; Brandt, Craig C.; Soren, Allyson; Brown, Steven D.; Crable, Bryan R.; Palumbo, Anthony Vito; Somenahally, Anil C.; Elias, Dwayne A.

    2015-10-09

    Mercury methylation produces the neurotoxic, highly bioaccumulative methylmercury (MeHg). Recent identification of the methylation genes (hgcAB) provides the foundation for broadly evaluating microbial Hg-methylation potential in nature without making explicit rate measurements. We first queried hgcAB diversity and distribution in all available microbial metagenomes, encompassing most environments. The genes were found in nearly all anaerobic, but not in aerobic, environments including oxygenated layers of the open ocean. Critically, hgcAB was effectively absent in ~1500 human microbiomes, suggesting a low risk of endogenous MeHg production. New potential methylation habitats were identified, including invertebrate guts, thawing permafrost, coastal dead zones, soils, sediments, and extreme environments, suggesting multiple routes for MeHg entry into food webs. Several new taxonomic groups potentially capable of Hg-methylation emerged, including lineages having no cultured representatives. We then begin to address long-standing evolutionary questions about Hg-methylation and ancient carbon fixation mechanisms while generating a new global view of Hg-methylation potential.

  14. Global prevalence and distribution of genes and microorganisms involved in mercury methylation

    DOE PAGES

    Podar, Mircea; Gilmour, C. C.; Brandt, Craig C.; ...

    2015-10-09

    Mercury methylation produces the neurotoxic, highly bioaccumulative methylmercury (MeHg). Recent identification of the methylation genes (hgcAB) provides the foundation for broadly evaluating microbial Hg-methylation potential in nature without making explicit rate measurements. We first queried hgcAB diversity and distribution in all available microbial metagenomes, encompassing most environments. The genes were found in nearly all anaerobic, but not in aerobic, environments including oxygenated layers of the open ocean. Critically, hgcAB was effectively absent in ~1500 human microbiomes, suggesting a low risk of endogenous MeHg production. New potential methylation habitats were identified, including invertebrate guts, thawing permafrost, coastal dead zones, soils, sediments,more » and extreme environments, suggesting multiple routes for MeHg entry into food webs. Several new taxonomic groups potentially capable of Hg-methylation emerged, including lineages having no cultured representatives. We then begin to address long-standing evolutionary questions about Hg-methylation and ancient carbon fixation mechanisms while generating a new global view of Hg-methylation potential.« less

  15. Role of Morphological Growth State and Gene Expression in Desulfovibrio africanus strain Walvis Bay Mercury Methylation

    SciTech Connect

    Moberly, James G; Miller, Carrie L; Brown, Steven D; Biswas, Abir; Brandt, Craig C; Palumbo, Anthony Vito; Elias, Dwayne A

    2012-01-01

    The biogeochemical transformations of mercury are a complex process, with the production of methylmercury, a potent human neurotoxin, repeatedly demonstrated in sulfate- and Fe(III)- reducing as well as methanogenic bacteria. However, little is known regarding the morphology, genes or proteins involved in methylmercury generation. Desulfovibrio africanus strain Walvis Bay is a Hg-methylating -proteobacterium with a sequenced genome and has unusual pleomorphic forms. In this study, a relationship between the pleomorphism and Hg methylation was investigated. Proportional increases in the sigmoidal (regular) cell form corresponded with increased net MeHg production, but decreased when the pinched cocci (persister) form became the major morphotype. D. africanus microarrays indicated that the ferrous iron transport genes (feoAB), as well as ribosomal genes and several genes whose products are predicted to have metal binding domains (CxxC), were up-regulated during exposure to Hg in the exponential phase. While no specific methylation pathways were identified, the finding that Hg may interfere with iron transport and the correlation of growth-phase dependent morphology with MeHg production are notable. The identification of these relationships between differential gene expression, morphology, and the growth phase dependence of Hg transformations suggests that actively growing cells are primarily responsible for methylation, and so areas with ample carbon and electron-acceptor concentrations may also generate a higher proportion of methylmercury than more oligotrophic environments. The observation of increased iron transporter expression also suggests that Hg methylation may interfere with iron biogeochemical cycles.

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

    PubMed Central

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

    2013-01-01

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

  17. Characterization of the IGF2 Imprinted Gene Methylation Status in Bovine Oocytes during Folliculogenesis.

    PubMed

    Mendonça, Anelise dos Santos; Guimarães, Ana Luíza Silva; da Silva, Naiara Milagres Augusto; Caetano, Alexandre Rodrigues; Dode, Margot Alves Nunes; Franco, Maurício Machaim

    2015-01-01

    DNA methylation reprogramming occurs during mammalian gametogenesis and embryogenesis. Sex-specific DNA methylation patterns at specific CpG islands controlling imprinted genes are acquired during this window of development. Characterization of the DNA methylation dynamics of imprinted genes acquired by oocytes during folliculogenesis is essential for understanding the physiological and genetic aspects of female gametogenesis and to determine the parameters for oocyte competence. This knowledge can be used to improve in vitro embryo production (IVP), specifically because oocyte competence is one of the most important aspects determining the success of IVP. Imprinted genes, such as IGF2, play important roles in embryo development, placentation and fetal growth. The aim of this study was to characterize the DNA methylation profile of the CpG island located in IGF2 exon 10 in oocytes during bovine folliculogenesis. The methylation percentages in oocytes from primordial follicles, final secondary follicles, small antral follicles, large antral follicles, MII oocytes and spermatozoa were 73.74 ± 2.88%, 58.70 ± 7.46%, 56.00 ± 5.58%, 65.77 ± 5.10%, 56.35 ± 7.45% and 96.04 ± 0.78%, respectively. Oocytes from primordial follicles showed fewer hypomethylated alleles (15.5%) than MII oocytes (34.6%) (p = 0.039); spermatozoa showed only hypermethylated alleles. Moreover, MII oocytes were less methylated than spermatozoa (p<0.001). Our results showed that the methylation pattern of this region behaves differently between mature oocytes and spermatozoa. However, while this region has a classical imprinted pattern in spermatozoa that is fully methylated, it was variable in mature oocytes, showing hypermethylated and hypomethylated alleles. Furthermore, our results suggest that this CpG island may have received precocious reprogramming, considering that the hypermethylated pattern was already found in growing oocytes from primordial follicles. These results may contribute to

  18. Aging and chronic alcohol consumption are determinants of p16 gene expression, genomic DNA methylation and p16 promoter methylation in the mouse colon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elder age and chronic alcohol consumption are important risk factors for the development of colon cancer. Each factor can alter genomic and gene-specific DNA methylation. This study examined the effects of aging and chronic alcohol consumption on genomic and p16-specific methylation, and p16 express...

  19. Adaptation of the targeted capture Methyl-Seq platform for the mouse genome identifies novel tissue-specific DNA methylation patterns of genes involved in neurodevelopment

    PubMed Central

    Hing, Benjamin; Ramos, Enrique; Braun, Patricia; McKane, Melissa; Jancic, Dubravka; Tamashiro, Kellie L K; Lee, Richard S; Michaelson, Jacob J; Druley, Todd E; Potash, James B

    2015-01-01

    Methyl-Seq was recently developed as a targeted approach to assess DNA methylation (DNAm) at a genome-wide level in human. We adapted it for mouse and sought to examine DNAm differences across liver and 2 brain regions: cortex and hippocampus. A custom hybridization array was designed to isolate 99 Mb of CpG islands, shores, shelves, and regulatory elements in the mouse genome. This was followed by bisulfite conversion and sequencing on the Illumina HiSeq2000. The majority of differentially methylated cytosines (DMCs) were present at greater than expected frequency in introns, intergenic regions, near CpG islands, and transcriptional enhancers. Liver-specific enhancers were observed to be methylated in cortex, while cortex specific enhancers were methylated in the liver. Interestingly, commonly shared enhancers were differentially methylated between the liver and cortex. Gene ontology and pathway analysis showed that genes that were hypomethylated in the cortex and hippocampus were enriched for neuronal components and neuronal function. In contrast, genes that were hypomethylated in the liver were enriched for cellular components important for liver function. Bisulfite-pyrosequencing validation of 75 DMCs from 19 different loci showed a correlation of r = 0.87 with Methyl-Seq data. We also identified genes involved in neurodevelopment that were not previously reported to be differentially methylated across brain regions. This platform constitutes a valuable tool for future genome-wide studies involving mouse models of disease. PMID:25985232

  20. Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer

    PubMed Central

    Pritchard, C.C.; Mateo, J.; Walsh, M.F.; De Sarkar, N.; Abida, W.; Beltran, H.; Garofalo, A.; Gulati, R.; Carreira, S.; Eeles, R.; Elemento, O.; Rubin, M.A.; Robinson, D.; Lonigro, R.; Hussain, M.; Chinnaiyan, A.; Vinson, J.; Filipenko, J.; Garraway, L.; Taplin, M-E.; AlDubayan, S.; Han, G.C.; Beightol, M.; Morrissey, C.; Nghiem, B.; Cheng, H.H.; Montgomery, B.; Walsh, T.; Casadei, S.; Berger, M.; Zhang, L.; Zehir, A.; Vijai, J.; Scher, H.I.; Sawyers, C.; Schultz, N.; Kantoff, P.W.; Solit, D.; Robson, M.; Van Allen, E.M.; Offit, K.; de Bono, J.; Nelson, P.S.

    2016-01-01

    BACKGROUND Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. METHODS We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. RESULTS A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). CONCLUSIONS In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family

  1. Classification of breast cancer subtypes by combining gene expression and DNA methylation data.

    PubMed

    List, Markus; Hauschild, Anne-Christin; Tan, Qihua; Kruse, Torben A; Mollenhauer, Jan; Baumbach, Jan; Batra, Richa

    2014-06-13

    Selecting the most promising treatment strategy for breast cancer crucially depends on determining the correct subtype. In recent years, gene expression profiling has been investigated as an alternative to histochemical methods. Since databases like TCGA provide easy and unrestricted access to gene expression data for hundreds of patients, the challenge is to extract a minimal optimal set of genes with good prognostic properties from a large bulk of genes making a moderate contribution to classification. Several studies have successfully applied machine learning algorithms to solve this so-called gene selection problem. However, more diverse data from other OMICS technologies are available, including methylation. We hypothesize that combining methylation and gene expression data could already lead to a largely improved classification model, since the resulting model will reflect differences not only on the transcriptomic, but also on an epigenetic level. We compared so-called random forest derived classification models based on gene expression and methylation data alone, to a model based on the combined features and to a model based on the gold standard PAM50. We obtained bootstrap errors of 10-20% and classification error of 1-50%, depending on breast cancer subtype and model. The gene expression model was clearly superior to the methylation model, which was also reflected in the combined model, which mainly selected features from gene expression data. However, the methylation model was able to identify unique features not considered as relevant by the gene expression model, which might provide deeper insights into breast cancer subtype differentiation on an epigenetic level.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Identification of genes required for de novo DNA methylation in Arabidopsis

    PubMed Central

    Greenberg, Maxim VC; Ausin, Israel; Chan, Simon WL; Cokus, Shawn J; Cuperus, Josh T; Feng, Suhua; Law, Julie A; Chu, Carolyn; Pellegrini, Matteo; Carrington, James C

    2011-01-01

    De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation. PMID:21150311

  5. An essential gene, ESR1, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Kato, R; Ogawa, H

    1994-01-01

    A new mutant, which was sensitive to both methyl-methanesulfonate (MMS) and ultra-violet light (UV) and defective in meiotic recombination, was isolated from Saccharomyces cerevisiae. The gene, ESR1, was cloned by complementation of the MMS sensitivity of the mutant and found to be essential for cell growth, as the deleted haploid strain was lethal. The ESR1 gene was adjacent to the CKS1 gene on chromosome II and encoded a putative 2368-amino acid protein with a molecular weight of 273 k. The ESR1 transcript was 8.0 kb long and was induced during meiosis. The predicted Esr1 protein had a mosaic structure composed of homologous regions and showed amino acid sequence similarities to Schizosaccharomyces pombe rad3+ protein, which monitors completion of DNA repair synthesis, and cut1+ protein, which is required for spindle pole body (SPB) duplication. The Esr1 protein was also similar to phosphatidylinositol (PI) 3-kinases, including Saccharomyces cerevisiae TOR2 (and DRR1), which are involved in G1 progression. These results suggest that ESR1 is multi-functional throughout mitosis and meiosis. Images PMID:8065923

  6. The role of DNA repair genes in recombination between repeated sequences in yeast.

    PubMed

    Liefshitz, B; Parket, A; Maya, R; Kupiec, M

    1995-08-01

    The presence of repeated sequences in the genome represents a potential source of karyotypic instability. Genetic control of recombination is thus important to preserve the integrity of the genome. To investigate the genetic control of recombination between repeated sequences, we have created a series of isogenic strains in which we could assess the role of genes involved in DNA repair in two types of recombination: direct repeat recombination and ectopic gene conversion. Naturally occurring (Ty elements) and artificially constructed repeats could be compared in the same cell population. We have found that direct repeat recombination and gene conversion have different genetic requirements. The role of the RAD51, RAD52, RAD54, RAD55, and RAD57 genes, which are involved in recombinational repair, was investigated. Based on the phenotypes of single and double mutants, these genes can be divided into three functional subgroups: one composed of RAD52, a second one composed of RAD51 and RAD54, and a third one that includes the RAD55 and RAD57 genes. Among seven genes involved in excision repair tested, only RAD1 and RAD10 played a role in the types of recombination studied. We did not detect a differential effect of any rad mutation on Ty elements as compared to artificially constructed repeats.

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

    PubMed

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

    2016-10-01

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

  8. Global prevalence and distribution of genes and microorganisms involved in mercury methylation

    PubMed Central

    Podar, Mircea; Gilmour, Cynthia C.; Brandt, Craig C.; Soren, Allyson; Brown, Steven D.; Crable, Bryan R.; Palumbo, Anthony V.; Somenahally, Anil C.; Elias, Dwayne A.

    2015-01-01

    Mercury (Hg) methylation produces the neurotoxic, highly bioaccumulative methylmercury (MeHg). The highly conserved nature of the recently identified Hg methylation genes hgcAB provides a foundation for broadly evaluating spatial and niche-specific patterns of microbial Hg methylation potential in nature. We queried hgcAB diversity and distribution in >3500 publicly available microbial metagenomes, encompassing a broad range of environments and generating a new global view of Hg methylation potential. The hgcAB genes were found in nearly all anaerobic (but not aerobic) environments, including oxygenated layers of the open ocean. Critically, hgcAB was effectively absent in ~1500 human and mammalian microbiomes, suggesting a low risk of endogenous MeHg production. New potential methylation habitats were identified, including invertebrate digestive tracts, thawing permafrost soils, coastal “dead zones,” soils, sediments, and extreme environments, suggesting multiple routes for MeHg entry into food webs. Several new taxonomic groups capable of methylating Hg emerged, including lineages having no cultured representatives. Phylogenetic analysis points to an evolutionary relationship between hgcA and genes encoding corrinoid iron-sulfur proteins functioning in the ancient Wood-Ljungdahl carbon fixation pathway, suggesting that methanogenic Archaea may have been the first to perform these biotransformations. PMID:26601305

  9. DNA methylation at stress-related genes is associated with exposure to early life institutionalization

    PubMed Central

    Non, Amy L.; Hollister, Brittany M.; Humphreys, Kathryn L.; Childebayeva, Ainash; Esteves, Kyle; Zeanah, Charles H.; Fox, Nathan A.; Nelson, Charles A.; Drury, Stacy S.

    2017-01-01

    Objectives Differences in DNA methylation have been associated with early life adversity, suggesting that alterations in methylation function as one pathway through which adverse early environments are biologically embedded. This study examined associations between exposure to institutional care, quantified as the percent time in institutional care at specified follow-up assessment ages, and DNA methylation status in two stress-related genes: FKBP5 and SLC6A4. Materials and Methods We analyzed data from the Bucharest Early Intervention Project, which is a prospective study in which children reared in institutional settings were randomly assigned (mean age 22 months) to either newly created foster care or care as usual (to remain in their current placement) and prospectively followed. A group of children from the same geographic area, with no history of institutionalized caregiving, were also recruited. DNA methylation status was determined in DNA extracted from buccal epithelial cells of children at age 12. Results An inverse association was identified such that more time spent in institutional care was associated with lower DNA methylation at specific CpG sites within both genes. Discussion These results suggest a lasting impact of early severe social deprivation on methylation patterns in these genes, and contribute to a growing literature linking early adversity and epigenetic variation in children. PMID:27218411

  10. Global prevalence and distribution of genes and microorganisms involved in mercury methylation.

    PubMed

    Podar, Mircea; Gilmour, Cynthia C; Brandt, Craig C; Soren, Allyson; Brown, Steven D; Crable, Bryan R; Palumbo, Anthony V; Somenahally, Anil C; Elias, Dwayne A

    2015-10-01

    Mercury (Hg) methylation produces the neurotoxic, highly bioaccumulative methylmercury (MeHg). The highly conserved nature of the recently identified Hg methylation genes hgcAB provides a foundation for broadly evaluating spatial and niche-specific patterns of microbial Hg methylation potential in nature. We queried hgcAB diversity and distribution in >3500 publicly available microbial metagenomes, encompassing a broad range of environments and generating a new global view of Hg methylation potential. The hgcAB genes were found in nearly all anaerobic (but not aerobic) environments, including oxygenated layers of the open ocean. Critically, hgcAB was effectively absent in ~1500 human and mammalian microbiomes, suggesting a low risk of endogenous MeHg production. New potential methylation habitats were identified, including invertebrate digestive tracts, thawing permafrost soils, coastal "dead zones," soils, sediments, and extreme environments, suggesting multiple routes for MeHg entry into food webs. Several new taxonomic groups capable of methylating Hg emerged, including lineages having no cultured representatives. Phylogenetic analysis points to an evolutionary relationship between hgcA and genes encoding corrinoid iron-sulfur proteins functioning in the ancient Wood-Ljungdahl carbon fixation pathway, suggesting that methanogenic Archaea may have been the first to perform these biotransformations.

  11. Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast

    PubMed Central

    Conconi, Antonio; Bespalov, Vyacheslav A.; Smerdon, Michael J.

    2002-01-01

    Nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active and inactive ribosomal genes of yeast. Ribosomal genes (rDNA) are present in multiple copies, but only a fraction of them is actively transcribed. Restriction enzyme digestion was used to specifically release the transcriptionally active fraction from yeast nuclei, and selective psoralen crosslinking was used to distinguish between active and inactive rDNA chromatin. Removal of CPDs was followed in both rDNA populations, and the data clearly show that strand-specific repair occurs in transcriptionally active rDNA while being absent in the inactive rDNA fraction. Thus, transcription-coupled repair occurs in RNA polymerase I-transcribed genes in yeast. Moreover, the nontranscribed strand of active rDNA is repaired faster than either strand of inactive rDNA, implying that NER has preferred access to the active, non-nucleosomal rDNA chromatin. Finally, restriction enzyme accessibility to active rDNA varies during NER, suggesting that there is a change in ribosomal gene chromatin structure during or soon after CPD removal. PMID:11782531

  12. Semiconservative replication, genetic repair, and many-gened genomes: Extending the quasispecies paradigm to living systems

    NASA Astrophysics Data System (ADS)

    Tannenbaum, Emmanuel; Shakhnovich, Eugene I.

    2005-12-01

    Quasispecies theory has emerged as an important tool for modeling the evolutionary dynamics of biological systems. We review recent advances in the field, with an emphasis on the quasispecies dynamics of semiconservatively replicating genomes. Applications to cancer and adult stem cell growth are discussed. Additional topics, such as genetic repair and many-gene genomes, are covered as well.

  13. Defects in base excision repair combined with elevated intracellular dCTP levels dramatically reduce mutation induction in yeast by ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine.

    PubMed

    Kunz, B A; Henson, E S; Karthikeyan, R; Kuschak, T; McQueen, S A; Scott, C A; Xiao, W

    1998-01-01

    Previously, we determined that elimination of deoxycytidylate (dCMP) deaminase (DCD1) in the yeast Saccharomyces cerevisiae increases the intracellular dCTP:dTTP ratio and reduces the induction of G x C --> A x T transitions in the SUP4-o gene by ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Simultaneously, the G x C --> C x G transversion frequency rises substantially. We attributed the first response to dCTP outcompeting dTTP for incorporation opposite O6-alkylguanine, and the second outcome to the increased dCTP pool causing error-prone repair of apurinic (AP) sites resulting from the removal or lability of N7-alkylguanine. To test the latter hypothesis, we used isogenic dcd1 strains deleted for either of two genes (MAG1: 3-methyladenine glycosylase; APN1: apurinic endonuclease) involved in the repair of N7-alkylguanine. In these backgrounds, EMS or MNNG induction of total SUP4-o mutations, G x C --> A x T transitions and G x C --> C x G transversions were reduced by >98%, >97%, and >80%, respectively. Mutation frequencies in the dcd1 apn1 strain were close to those for spontaneous mutagenesis in the wild-type parent. These findings argue that misincorporation of dCTP during repair of alkylation-induced AP sites is responsible for the increased G x C --> C x G transversion frequency in the dcd1 strain treated with EMS or MNNG. The data also demonstrate that defective repair of AP sites coupled with an elevated dCTP:dTTP ratio eliminates most EMS and MNNG mutagenesis. In addition, the results point to a role for AP sites in the production of some EMS- and MNNG-induced G x C --> A x T transitions as well as other substitutions in the dcd1 strain.

  14. Germline Stem Cell Gene PIWIL2 Mediates DNA Repair through Relaxation of Chromatin

    PubMed Central

    Yin, De-Tao; Wang, Qien; Chen, Li; Liu, Meng-Yao; Han, Chunhua; Yan, Qingtao; Shen, Rulong; He, Gang; Duan, Wenrui; Li, Jian-Jian; Wani, Altaf; Gao, Jian-Xin

    2011-01-01

    DNA damage response (DDR) is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV) irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili-/- MEFs) were defective in cyclobutane pyrimidine dimers (CPD) repair after UV treatment. As a result, the UV-treated mili-/- MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose) polymerase (PARP) and Bik. The impaired DNA repair in the mili-/- MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine–guanine (Pt-[GG]) and double strand break (DSB) repair were also defective in the mili-/- MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR), respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis. PMID:22110608

  15. Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair.

    PubMed

    Reumann, Marie K; Strachna, Olga; Yagerman, Sarah; Torrecilla, Daniel; Kim, Jihye; Doty, Stephen B; Lukashova, Lyudmila; Boskey, Adele L; Mayer-Kuckuk, Philipp

    2011-10-01

    Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1(-/-) mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1(-/-) mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1(-/-) callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair.

  16. [Epigenetic heredity (deoxyribonucleic acid methylation): Clinical context in neurodegenerative disorders and ATXN2 gene].

    PubMed

    Laffita-Mesa, José Miguel; Bauer, Peter

    2014-10-21

    Epigenetics is the group of changes in the phenotype which are related with the process independently of the primary DNA sequence. These changes are intimately related with changes in the gene expression level and its profile across the body. These are mediated by histone tail modifications, DNA methylation, micro-RNAs, with chromatin remodeling remaining as the foundation of epigenetic changes. DNA methylation involves the covalent addition of methyl group to cytosine of the DNA, which is mediated by methyltransferases enzymes. DNA methylation regulates gene expression by repressing transcription, while de-methylation activates gene transcription. Several human diseases are related with the epigenetic process: cancer, Alzheimer disease, stroke, Parkinson disease, and diabetes. We present here the basis of epigenetic inheritance and show the pathogenic mechanisms relating epigenetics in human diseases, specifically with regard to neurodegeneration. We discuss current concepts aimed at understanding the contribution of epigenetics to human neurodegenerative diseases. We also discuss recent findings obtained in our and other centers regarding the ATXN2 gene that causes spinocerebellar ataxia 2 and amyotrophic lateral sclerosis. Epigenetics play a pivotal role in the pathogenesis of human diseases and in several neurodegenerative disorders, and this knowledge will illuminate the pathways in the diagnostic and therapeutic field, which ultimately will be translated into the clinic context of neurodegenerative diseases.

  17. Integrative analysis of DNA methylation and gene expression in butyrate-treated CHO cells.

    PubMed

    Wippermann, Anna; Rupp, Oliver; Brinkrolf, Karina; Hoffrogge, Raimund; Noll, Thomas

    2016-11-24

    The cellular mechanisms responsible for the versatile properties of CHO cells as the major production cell line for biopharmaceutical molecules are not entirely understood yet, although several 'omics' data facilitate the understanding of CHO cells and their reactions to environmental conditions. However, genome-wide studies of epigenetic processes such as DNA methylation are still limited. To prove the applicability and usefulness of integrating DNA methylation and gene expression data in a biotechnological context, we exemplarily analyzed the time course of cellular reactions upon butyrate addition in antibody-producing CHO cells by whole-genome bisulfite sequencing and CHO-specific cDNA microarrays. Gene expression and DNA methylation analyses showed that pathways known to be affected by butyrate, including cell cycle and apoptosis, as well as pathways potentially involved in butyrate-induced hyperproductivity such as central energy metabolism and protein biosynthesis were affected. Differentially methylated regions were furthermore found to contain binding-site motifs of specific transcription factors and were hypothesized to represent regulatory regions closely connected to the cellular response to butyrate. Generally, our experiment underlines the benefit of integrating DNA methylation and gene expression data, as it provided potential novel candidate genes for rational cell line development and allowed for new insights into the butyrate effect on CHO cells.

  18. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

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

    PubMed

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

    1998-06-01

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

  20. Specificities of human, rat and E. coli O6-methylguanine-DNA methyltransferases towards the repair of O6-methyl and O6-ethylguanine in DNA.

    PubMed Central

    Liem, L K; Lim, A; Li, B F

    1994-01-01

    The behaviour of highly purified bacterial expressed rat O6-methylguanine-DNA methyltransferase (MGMT) towards the repair of CGCm6GAGCTCGCG and CGCe6GAGCTCGCG (km6G/ke6G = 1.45, where k is the second order repair rate constant determined, m6G and e6G are O6-methyl and O6-ethylguanine) is similar to that of E. coli 39kD Ada protein (km6G/ke6G = 1.6). However, the human MGMT is very different (km6G/ke6G = 163). The preferential repair of O6-ethylguanine lesion by the rat MGMT appears not to be related to the lack of the initiator methionine in the expressed protein since similar results were obtained from N-terminal Glutathione-S-transferase (GST) fused protein (GSTMGMT) which retains the methionine. The possible relationship between these findings and the differences observed in the primary amino acid sequence of these proteins is discussed. In addition the preferential repair of O6-ethylguanine substrate by the 39kD Ada protein as compared to the catalytic C-terminus alone (different by 134 times) suggests that the N-terminus plays a crucial role in the repair of O6-ethylguanine. This is in contrast to the minor effects of the GST domain when fused to the N-terminus of mammalian MGMT. Images PMID:8202360

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

    PubMed

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

    2011-02-01

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

  2. Insights into the epigenetic mechanisms involving histone lysine methylation and demethylation in ischemia induced damage and repair has therapeutic implication.

    PubMed

    Chakravarty, Sumana; Jhelum, Priya; Bhat, Unis Ahmad; Rajan, Wenson D; Maitra, Swati; Pathak, Salil S; Patel, Anant B; Kumar, Arvind

    2017-01-01

    Cerebral ischemic stroke is one of the leading causes of death and disability worldwide. Therapeutic interventions to minimize ischemia-induced neural damage are limited due to poor understanding of molecular mechanisms mediating complex pathophysiology in stroke. Recently, epigenetic mechanisms mostly histone lysine (K) acetylation and deacetylation have been implicated in ischemic brain damage and have expanded the dimensions of potential therapeutic intervention to the systemic/local administration of histone deacetylase inhibitors. However, the role of other epigenetic mechanisms such as histone lysine methylation and demethylation in stroke-induced damage and subsequent recovery process is elusive. Here, we established an Internal Carotid Artery Occlusion (ICAO) model in CD1 mouse that resulted in mild to moderate level of ischemic damage to the striatum, as suggested by magnetic resonance imaging (MRI), TUNEL and histopathological staining along with an evaluation of neurological deficit score (NDS), grip strength and rotarod performance. The molecular investigations show dysregulation of a number of histone lysine methylases (KMTs) and few of histone lysine demethylases (KDMs) post-ICAO with significant global attenuation in the transcriptionally repressive epigenetic mark H3K9me2 in the striatum. Administration of Dimethyloxalylglycine (DMOG), an inhibitor of KDM4 or JMJD2 class of histone lysine demethylases, significantly ameliorated stroke-induced NDS by restoring perturbed H3K9me2 levels in the ischemia-affected striatum. Overall, these results highlight the novel role of epigenetic regulatory mechanisms controlling the epigenetic mark H3K9me2 in mediating the stroke-induced striatal damage and subsequent repair following mild to moderate cerebral ischemia.

  3. Molecular cloning and characterization of a Streptococcus sanguis DNase necessary for repair of DNA damage induced by UV light and methyl methanesulfonate

    SciTech Connect

    Lindler, L.E.; Macrina, F.L.

    1987-07-01

    We developed a method for cloning cellular nucleases from streptococci. Recombinant lambda gt11 bacteriophage containing streptococcal nuclease determinants were identified by the production of pink plaques on toluidine blue O DNase plates. We used this technique to clone a 3.2-kilobase-pair EcoRI fragment with DNase activity from the chromosome of Streptococcus sanguis. The locus was designated don (DNase one) and could be subcloned and stably maintained on plasmid vectors in Escherichia coli. Minicell analyses of various subclones of the don locus allowed us to determine the coding region and size of the Don nuclease in E. coli. The don gene product had an apparent molecular mass of 34 kilodaltons and degraded native DNA most efficiently, with lesser activity against denatured DNA and no detectable activity against RNA. S. sanguis don deletion mutants were constructed by transformation of competent cells with in vitro-prepared plasmid constructs. S. sanguis don deletion mutants retained normal transformation frequencies for exogenously added donor DNA. However, when compared with Don+ wild-type cells, these mutants were hypersensitive to DNA damage induced by UV light and methyl methanesulfonate. An S. sanguis don-specific DNA probe detected homology to chromosomal DNA isolated from Streptococcus pneumoniae and Streptococcus mutans Bratthall serogroups d and g. Our results suggested that the don locus was the S. sanguis allele of the previously described S. pneumoniae major exonuclease and was involved in repair of DNA damage. Furthermore, hybridization studies suggested that the don locus was conserved among species of oral streptococci.

  4. Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

    PubMed

    da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

  5. Glucocorticoid receptor gene methylation and HPA-axis regulation in adolescents. The TRAILS study.

    PubMed

    van der Knaap, Lisette J; Oldehinkel, Albertine J; Verhulst, Frank C; van Oort, Floor V A; Riese, Harriëtte

    2015-08-01

    Early life adversity and psychopathology are thought to be linked through HPA-axis deregulation. Changes in methylation levels of stress reactivity genes such as the glucocorticoid receptor gene (NR3C1) can be induced by adversity. Higher NR3C1 methylation levels have been associated with a reduced NR3C1 expression, possibly leading to impaired negative feedback regulation of the HPA-axis. In this study we tested whether methylation levels of NR3C1 were associated with HPA-axis regulation, operationalized as cortisol responses. In 361 adolescents (mean age 16.1, SD=0.6), salivary cortisol samples were collected before, during, and after a social stress task, from which response measures (cortisol activation and recovery) were calculated. Higher NR3C1 methylation levels were associated with a flattened cortisol recovery slope, indicating a delayed recovery time. Cortisol response activation was not associated with NR3C1 methylation. These results suggest that methylation of NR3C1 may impair negative feedback of the HPA-axis in adolescents.

  6. Active Repression of Methylated Genes by the Chromosomal Protein MBD1

    PubMed Central

    Ng, Huck-Hui; Jeppesen, Peter; Bird, Adrian

    2000-01-01

    MBD1 belongs to a family of mammalian proteins that share a methyl-CpG binding domain. Previous work has shown that MBD1 binds to methylated sites in vivo and in vitro and can repress transcription from methylated templates in transcription extracts and in cultured cells. In the present study we established by several experimental criteria that, contrary to a previous report, MBD1 is not a component of the MeCP1 repressor complex. We identified a powerful transcriptional repression domain (TRD) at the C terminus of MBD1 that can actively repress transcription at a distance. Methylation-dependent repression in vivo depends on the presence of both the TRD and the methyl-CpG binding domain. The mechanism is likely to involve deacetylation, since the deacetylase inhibitor trichostatin A can overcome MBD1-mediated repression. Accordingly, we found that endogenous MBD1 is particularly concentrated at sites of centromeric heterochromatin, where acetylated histone H4 is deficient. Unlike MBD2 and MeCP2, MBD1 is not depleted by antibodies to the histone deacetylase HDAC1. Thus, the deacetylase-dependent pathway by which MBD1 actively silences methylated genes is likely to be different from that utilized by the methylation-dependent repressors MeCP1 and MeCP2. PMID:10648624

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

    PubMed Central

    2009-01-01

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

  8. Gene-specific repair of benzo[a]pyrene diol epoxide DNA damage in human cells

    SciTech Connect

    Denisenko, M.F.; Venkatachalam, S.; Wani, A.A.

    1995-11-01

    Gene-specific preferential repair of UV damage has been well documented in a variety of organisms. Less is known about many other types of critical DNA lesions, the data available being not numerous and contradictory. To date, the majority of observations with UV were obtained by using T4 endonuclease V system. Recent report questions the applicability of UvrABC nuclease incision method for detecting gene-specific repair. This has stimulated our search for simple and sensitive approach based on a different principle. We have employed the idea of detection by the Southern hybridization of restriction cleavage inhibition at rare sites and developed a method for the analysis of benzo[a]pyrene diol epoxide (anti-BPDE) DNA damage in human H-ras proto-oncogene. Damage-dependent induction of individual facultative bands resulting from cleavage inhibition was observed in in vitro modified (4-50 adducts/10{sup 3}kb) p220-ras plasmid DNA digested with EcoRI/NotI, Xhol/Xbal/PstI, and SstI/XbaI/Pst/I. In vivo lesion formation and removal was monitored at several PstI sites distributed along the 6.4 kb single copy ras sequence. Rapid gene-specific repair was seen in primary culture of normal human fibroblasts and in SV40 transformed GM00637 cells. Surprisingly, SV40 transformed XP12BE (complementation group A) GM4429 fibroblasts also repaired anti-BPDE DNA damage at comparable levels. All investigated sites within ras sequence were repaired faster than the genome overall. The results show the utility of the above approach for fine mapping of anti-BPDE DNA lesions. Data suggests that the xeroderma pigmentosum (group A) fibroblasts have a capacity of removing these bulky adducts at least from the active genes.

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

    PubMed

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

    2014-08-01

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

  10. Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer.

    PubMed

    Perry, Antoinette S; O'Hurley, Gillian; Raheem, Omer A; Brennan, Kevin; Wong, Simon; O'Grady, Anthony; Kennedy, Anne-Marie; Marignol, Laure; Murphy, Therese M; Sullivan, Linda; Barrett, Ciara; Loftus, Barbara; Thornhill, John; Hewitt, Stephen M; Lawler, Mark; Kay, Elaine; Lynch, Thomas; Hollywood, Donal

    2013-04-15

    Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

  11. Imprinted Genes and Satellite Loci Are Differentially Methylated in Bovine Somatic Cell Nuclear Transfer Clones

    PubMed Central

    Shen, Chih-Jie; Lin, Chiao-Chieh; Shen, Perng-Chih; Cheng, Winston T.K.; Chen, Hsiao-Ling; Chang, Tsung-Chou; Liu, Shyh-Shyan

    2013-01-01

    Abstract In mammals, genome-wide epigenetic reprogramming systems exist in primordial germ cells and zygotes. These reprogramming systems play crucial roles in regulating genome functions during critical stages of embryonic development, and they confer the stability of gene expression during mammalian development. The frequent unexpected loss of progeny from somatic cell nuclear transfer (SCNT) is an ongoing problem. In this study, we used six cloned bovines (named NT-1 to NT-6), which were created by ear fibroblast nuclear transfer and displayed short life spans with multiple organ defects, as an experimental model. We focus here on three imprinted genes (IGF2, H19, and XIST) and four satellite loci (Satellite I, Satellite II, Art2, and VNTR) to investigate their methylation changes. The results revealed that aberrant methylation frequently occurred in the analyzed imprinted genes, but not in the satellite loci, of the cloned bovines. After the bovine fibroblast cells were treated with the 5-aza-2(′)-deoxycytidine (5-Aza-dc) demethylation agent, the methylation percentages of the XIST and H19 putative differentially methylated region (DMR) were significantly decreased (XIST, p<0.01; H19, p<0.05) followed by an increase in their mRNA expression levels (p<0.01). Furthermore, we found that five short-lived cloned bovines (NT-1 to NT-5) exhibited more severe aberrant methylation changes in the three imprinted genes examined than the little longer-lived clone (NT-6) compared with wild-type (WT) cows. Our data suggest that the reprogramming of the methylation-controlled regions between the imprinted genes and satellite loci are differences and may be involved with additional mechanisms that need further elucidation. PMID:23961768

  12. The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 Gene Codes for an S-Adenosyl-l-Homocysteine Hydrolase Required for DNA Methylation-Dependent Gene Silencing

    PubMed Central

    Rocha, Pedro S.C.F.; Sheikh, Mazhar; Melchiorre, Rosalba; Fagard, Mathilde; Boutet, Stéphanie; Loach, Rebecca; Moffatt, Barbara; Wagner, Conrad; Vaucheret, Hervé; Furner, Ian

    2005-01-01

    Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silencing of homologous genes at unlinked sites (homology-dependent gene silencing or HDG silencing). Mutations of the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) locus relieve transcriptional gene silencing and methylation-dependent HDG silencing and result in genome-wide demethylation. The hog1 mutant plants also grow slowly and have low fertility and reduced seed germination. Three independent mutants of HOG1 were each found to have point mutations at the 3′ end of a gene coding for S-adenosyl-l-homocysteine (SAH) hydrolase, and hog1-1 plants show reduced SAH hydrolase activity. A transposon (hog1-4) and a T-DNA tag (hog1-5) in the HOG1 gene each behaved as zygotic embryo lethal mutants and could not be made homozygous. The results suggest that the homozygous hog1 point mutants are leaky and result in genome demethylation and poor growth and that homozygous insertion mutations result in zygotic lethality. Complementation of the hog1-1 point mutation with a T-DNA containing the gene coding for SAH hydrolase restored gene silencing, HDG silencing, DNA methylation, fast growth, and normal seed viability. The same T-DNA also complemented the zygotic embryo lethal phenotype of the hog1-4 tagged mutant. A model relating the HOG1 gene, DNA methylation, and methylation-dependent HDG silencing is presented. PMID:15659630

  13. Dopamine transporter gene susceptibility to methylation is associated with impulsivity in nonhuman primates

    PubMed Central

    Rajala, Abigail Z.; Zaitoun, Ismail; Henriques, Jeffrey B.; Converse, Alexander K.; Murali, Dhanabalan; Epstein, Miles L.

    2014-01-01

    Impulsivity, the predisposition to act without regard for negative consequences, is a characteristic of several psychiatric disorders and is thought to result in part from genetic variation in the untranslated region of the dopamine transporter (DAT) gene. As the exact link between genetic mutations and impulsivity has not been established, we used oculomotor behavior to characterize rhesus monkeys as impulsive or calm and genetic/epigenetic analysis and positron emission tomography (PET) to correlate phenotype to DAT genotype, DAT gene methylation, and DAT availability. We found three single nucleotide polymorphisms (SNPs) in the 3′-UTR of the DAT gene, one of which provided a potential site for methylation in the impulsive group. Bisulfite analysis showed that the DNA of the impulsive but not the calm subjects was methylated at one SNP. Because genetic/epigenetic modifications could lead to differences in protein expression, we measured DAT availability using [18F]2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)-nortropane ([18F]FECNT) PET and found higher DAT availability in the internal globus pallidus, an output nucleus of the basal ganglia, of the impulsive group. Higher DAT availability lowers dopamine levels, potentially altering neuronal circuits involved in the initiation of action, thus contributing to the impulsive phenotype. The association between increased methylation in the DAT gene and greater DAT availability suggests that mutations to the regulatory portion of the DAT gene lead to a susceptibility to epigenetic modification resulting in a discrete behavioral phenotype. PMID:25122707

  14. [Analysis of gene expression and DNA methylation patterns in childhood acute lymphoblastic leukemia].

    PubMed

    Iijima, Kazutoshi; Kiyokawa, Nobutaka

    2016-04-01

    The 5-year survival rate of patients with childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) now exceeds 90%, though there are still patients who fail to achieve remission or relapse early. To improve the outcomes of these cases, new diagnostic markers for stratification of those with unfavorable outcomes and novel targets for treatment have been investigated based on data from the OMICs analysis. We performed gene expression analysis of leukemic cells from 91 near-diploid BCP-ALL cases without specific fusion genes enrolled in Tokyo Children's Cancer Study Group (TCCSG)-L0416 & L0616 clinical trials employing the Affymetrix Human Genome U133 Plus 2.0 Array. Among them, DNA methylation status was analyzed in 24 cases by using the Infinium HumanMethylation450 BeadChip. Herein, initially, the current situations of gene expression analysis and DNA methylation analysis of childhood BCP-ALL are reviewed. Then, our analyses of gene expressions and DNA methylation related to the prognosis of childhood ALL without fusion genes are presented.

  15. Methylation impact analysis of erythropoietin (EPO) Gene to hypoxia inducible factor-1α (HIF-1α) activity.

    PubMed

    Dewi, Firli Rahmah Primula; Fatchiyah, Fatchiyah

    2013-01-01

    Erythropoietin (EPO) is a glycoprotein hormone that play a role as key regulator in the production of red blood cells. The promoter region of EPO is methylated in normoxic (non-hypoxia) condition, but not in hypoxic condition. Methylation of the EPO enhancer region decline the transcription activity of EPO gene. The aim of this study is to investigate how different methylation percentage affected on the regulation and transcriptional activity of EPO gene. The DNA sequence of erythropoietin gene and protein sequence was retrieved from the sequence database of NCBI. DNA structure was constructed using 3D-DART web server and modeling structure of HIF1 predicted using SWISS-MODEL web server. Methylated DNA sequence of EPO gene using performed with YASARA View software and docking of EPO gene and transcription factor HIF1 analyzed by using HADDOCK webserver. Our result showed that binding energy in 46% methylated DNA was higher (-161,45 kcal/mol) than in unmethylated DNA (-194,16 kcal/mol) and 8% methylated DNA (-175,94 kcal/mol). So, we presume that a silencing mechanism of the Epo gene by methylation is correlated with the binding energy, which is required for interaction. A higher methylation percentage correlates with a higher binding energy which can cause an unstable interaction between DNA and transcription factor. In conclution, methylation of promoter and enhancer region of Epo gene leads to silencing.

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2013-11-21

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

  18. Methyl jasmonate affects phenolic metabolism and gene expression in blueberry (Vaccinium corymbosum).

    PubMed

    Cocetta, Giacomo; Rossoni, Mara; Gardana, Claudio; Mignani, Ilaria; Ferrante, Antonio; Spinardi, Anna

    2015-02-01

    Blueberry (Vaccinium corymbosum) is a fruit very much appreciated by consumers for its antioxidant potential and health-promoting traits. Its beneficial potential properties are mainly due to a high content of anthocyanins and their amount can change after elicitation with methyl jasmonate. The aim of this work is to evaluate the changes in expression of several genes, accumulation of phenolic compounds and alterations in antioxidant potential in two different blueberry cultivars ('Duke' and 'Blueray') in response to methyl jasmonate (0.1 mM). Results showed that 9 h after treatment, the expression of phenylalanine ammonium lyase, chalcone synthase and anthocyanidin synthase genes was stimulated more in the 'Blueray' variety. Among the phenols measured an increase was recorded also for epicatechin and anthocyanin concentrations. 'Duke' is a richer sourche of anthocyanins compared to 'Blueray', treatment with methyl jasmonate promoted in 'Blueray' an increase in pigments as well as in the antioxidant potential, especially in fully ripe berries, but treated 'Duke' berries had greater levels, which were not induced by methyl jasmonate treatment. In conclusion, methyl jasmonate was, in some cases, an effective elicitor of phenolic metabolism and gene expression in blueberry, though with different intensity between cultivars.

  19. DNA Methylation of Regulatory Regions of Imprinted Genes at Birth and Its Relation to Infant Temperament

    PubMed Central

    Fuemmeler, Bernard F.; Lee, Chien-Ti; Soubry, Adelheid; Iversen, Edwin S.; Huang, Zhiqing; Murtha, Amy P.; Schildkraut, Joellen M.; Jirtle, Randy L.; Murphy, Susan K.; Hoyo, Cathrine

    2016-01-01

    BACKGROUND DNA methylation of the differentially methylated regions (DMRs) of imprinted genes is relevant to neurodevelopment. METHODS DNA methylation status of the DMRs of nine imprinted genes in umbilical cord blood leukocytes was analyzed in relation to infant behaviors and temperament (n = 158). RESULTS MEG3 DMR levels were positively associated with internalizing (β = 0.15, P = 0.044) and surgency (β = 0.19, P = 0.018) behaviors, after adjusting for birth weight, gender, gestational age at birth, maternal age at delivery, race/ethnicity, education level, smoking status, parity, and a history of anxiety or depression. Higher methylation levels at the intergenic MEG3-IG methylation regions were associated with surgency (β = 0.28, P = 0.0003) and PEG3 was positively related to externalizing (β = 0.20, P = 0.01) and negative affectivity (β = 0.18, P = 0.02). CONCLUSION While the small sample size limits inference, these pilot data support gene-specific associations between epigenetic differences in regulatory regions of imprinted domains at birth and later infant temperament. PMID:27920589

  20. Putting DNA methylation in context: from genomes to gene expression in plants.

    PubMed

    Niederhuth, Chad E; Schmitz, Robert J

    2017-01-01

    Plant DNA methylation is its own language, interpreted by the cell to maintain silencing of transposons, facilitate chromatin structure, and to ensure proper expression of some genes. Just as in any language, context is important. Rather than being a simple "on-off switch", DNA methylation has a range of "meanings" dependent upon the underlying sequence and its location in the genome. Differences in the sequence context of individual sites are established, maintained, and interpreted by differing molecular pathways. Varying patterns of methylation within genes and surrounding sequences are associated with a continuous range of expression differences, from silencing to constitutive expression. These often-subtle differences have been pieced together from years of effort, but have taken off with the advent of methods for assessing methylation across entire genomes. Recognizing these patterns and identifying underlying causes is essential for understanding the function of DNA methylation and its systems-wide contribution to a range of processes in plant genomes. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

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

    PubMed Central

    2011-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  3. A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.

    PubMed Central

    Sebastian, J; Sancar, G B

    1991-01-01

    The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. We report here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 upstream repression sequence, that together regulate induction of PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. A prominent feature of the foot-printed region is a 22-base-pair palindrome. Deletion of the PHR1 upstream repression sequence increased the basal level expression of PHR1 in vivo and decreased induction after exposure of cells to UV radiation or methyl methanesulfonate, whereas insertion of the PRP binding site between the CYC1 upstream activation sequence and "TATA" sequence reduced basal level expression and conferred damage responsiveness upon a reporter gene. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription. Images PMID:1763039

  4. Expression of the osteoarthritis-associated gene GDF5 is modulated epigenetically by DNA methylation.

    PubMed

    Reynard, Louise N; Bui, Catherine; Canty-Laird, Elizabeth G; Young, David A; Loughlin, John

    2011-09-01

    GDF5 is involved in synovial joint development, maintenance and repair, and the rs143383 C/T single nucleotide polymorphism (SNP) located in the 5'UTR of GDF5 is associated, at the genome-wide significance level, with osteoarthritis susceptibility, and with other musculoskeletal phenotypes including height, congenital hip dysplasia and Achilles tendinopathy. There is a significant reduction in the expression of the disease-associated T allele relative to the C allele in synovial joint tissues, an effect influenced by a second SNP (rs143384, C/T) also within the 5'UTR. The differential allelic expression (DAE) imbalance of the C and T alleles of rs143383 varies intra- and inter-individually, suggesting that DAE may be modulated epigenetically. The C alleles of both SNPs form CpG dinucleotides that are potentially amenable to regulation by methylation. Here, we have examined whether DNA methylation regulates GDF5 expression and the allelic imbalance caused by rs143383. We observed methylation of the GDF5 promoter and 5'UTR in cell lines and joint tissues, with demethylation correlating with increased GDF5 expression. The CpG sites created by the C alleles at rs143383 and rs143384 were variably methylated, and treatment of a heterozygous cell line with a demethylating agent further increased the allelic expression imbalance between the C and T alleles. This demonstrates that the genetic effect of the rs143383 SNP on GDF5 expression is modulated epigenetically by DNA methylation. The variability in DAE of rs143383 is therefore partly accounted for by differences in DNA methylation that could influence the penetrance of this allele in susceptibility to common musculoskeletal diseases.

  5. Chromosomal localization of three repair genes: The xeroderma pigmentosum group C gene and two human homologs of yeast RAD23

    SciTech Connect

    Spek, P.J. van der; Smit, E.M.E.; Beverloo, H.B.

    1994-10-01

    The nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) is characterized by sun (UV) sensitivity, predisposition to skin cancer, and extensive genetic heterogeneity. Recently, we reported the cloning and analysis of three human NER genes, XPC, HHR23A, and HHR23B. The previously cloned XPC gene is involved in the common XP complementation group C, which is defective in excision repair of nontranscribed sequences in the genome. The XPC protein was found to be complexed with the product of HHR23B, one of the two human homologs of the Saccharomyes cerevisiae NER gene RAD23. Here we present the chromosomal localization by in situ hybridization using haptenized probes of all three genes. The HHR23A gene was assigned to chromosome 19p13.2. Interestingly, the HHR23B and XPC genes, the product of which forms a tight complex, were found to colocalize on band 3p25.1. Pulsed-field gel electrophoresis revealed that the HHR23B and XPC genes possibly share a MluI restriction fragment of about 625 kb. Potential involvement of the HHR23 genes in human genetic disorders is discussed. 53 refs., 4 figs., 2 tabs.

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

    PubMed Central

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

    2017-01-01

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

  7. Differential introduction of DNA damage and repair in mammalian genes transcribed by RNA polymerase I and II

    SciTech Connect

    Vos, J.H.; Wauthier, E.L. )

    1991-04-01

    The authors have developed a general quantitative method for comparing the levels of drug-induced DNA crosslinking in specific mammalian genes. They observed a dramatic difference between the efficiency of the removal of both psoralen monoadducts and interstrand crosslinks from the rRNA genes and the efficiency of their removal from the dihydrofolate reductase (DHFR) gene in cultured human and hamster cells. While 90% of the interstrand crosslinks were removed from the human DHFR gene in 48 h, less than 25% repair occurred in the rRNA genes. Similarly, in Chinese hamster ovary cells, 85% repair of interstrand crosslinks within 8 h in the DHFR gene versus only 20% repair in the rRNA genes. The preferential repair of the DHFR gene relative to that of the rRNA genes was also observed for psoralen monoadducts in cells from both mammalian species. In human-mouse hybrid cells, the active mouse rRNA genes were five times more susceptible to psoralen modification than are the silent rRNA human genes, but adduct removal was similarly inefficient for both classes. They conclude that the repair of chemical damage such as psoralen photadducts in an expressed mammalian gene may depend upon the class of transcription to which it belongs.

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

    PubMed Central

    Kudo, Shinichi

    1998-01-01

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

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

    SciTech Connect

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

    2006-02-03

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

  10. Constructing gene-enriched plant genomic libraries using methylation filtration technology.

    PubMed

    Rabinowicz, Pablo D

    2003-01-01

    Full genome sequencing in higher plants is a very difficult task, because their genomes are often very large and repetitive. For this reason, gene targeted partial genomic sequencing becomes a realistic option. The method reported here is a simple approach to generate gene-enriched plant genomic libraries called methylation filtration. This technique takes advantage of the fact that repetitive DNA is heavily methylated and genes are hypomethylated. Then, by simply using an Escherichia coli host strain harboring a wild-type modified cytosine restriction (McrBC) system, which cuts DNA containing methylcytosine, repetitive DNA is eliminated from these genomic libraries, while low copy DNA (i.e., genes) is recovered. To prevent cloning significant proportions of organelle DNA, a crude nuclear preparation must be performed prior to purifying genomic DNA. Adaptor-mediated cloning and DNA size fractionation are necessary for optimal results.

  11. Glial kon/NG2 gene network for central nervous system repair

    PubMed Central

    Losada-Perez, Maria; Harrison, Neale; Hidalgo, Alicia

    2017-01-01

    The glial regenerative response to central nervous system (CNS) injury, although limited, can be harnessed to promote regeneration and repair. Injury provokes the proliferation of ensheathing glial cells, which can differentiate to remyelinate axons, and partially restore function. This response is evolutionarily conserved, strongly implying an underlying genetic mechanism. In mammals, it is elicited by NG2 glia, but most often newly generated cells fail to differentiate. Thus an important goal had been to find out how to promote glial differentiation following the proliferative response. A gene network involving Notch and prospero (pros) controls the balance between glial proliferation and differentiation in flies and mice, and promotes CNS repair at least in fruit-flies. A key missing link had been how to relate the function of NG2 to this gene network. Recent findings by Losada-Perez et al., published in JCB, demonstrated that the Drosophila NG2 homologue kon-tiki (kon) is functionally linked to Notch and pros in glia. By engaging in two feedback loops with Notch and Pros, in response to injury, Kon can regulate both glial cell number and glial shape homeostasis, essential for repair. Drosophila offers powerful genetics to unravel the control of stem and progenitor cells for regeneration and repair. PMID:28250735

  12. Low intensity infrared laser affects expression of oxidative DNA repair genes in mitochondria and nucleus

    NASA Astrophysics Data System (ADS)

    Fonseca, A. S.; Magalhães, L. A. G.; Mencalha, A. L.; Geller, M.; Paoli, F.

    2014-11-01

    Practical properties and physical characteristics of low intensity lasers have made possible their application to treat soft tissue diseases. Excitation of intracellular chromophores by red and infrared radiation at low energy fluences with increase of mitochondrial metabolism is the basis of the biostimulation effect but free radicals can be produced. DNA lesions induced by free radicals are repaired by the base excision repair pathway. In this work, we evaluate the expression of POLγ and APEX2 genes related to repair of mitochondrial and nuclear DNA, respectively. Skin and muscle tissue of Wistar rats were exposed to low intensity infrared laser at different fluences. One hour and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of POLγ and APEX2 mRNA expression by real time quantitative polymerase chain reaction. Skin and muscle tissue of Wistar rats exposed to laser radiation show different expression of POLγ and APEX2 mRNA depending of the fluence and time after exposure. Our study suggests that a low intensity infrared laser affects expression of genes involved in repair of oxidative lesions in mitochondrial and nuclear DNA.

  13. Overexpression of DNA repair genes is associated with metastasis: a new hypothesis.

    PubMed

    Sarasin, Alain; Kauffmann, Audrey

    2008-01-01

    Tumorigenesis is a multistep process, where it is believed that the transformation of normal cells into tumoral cells needs a succession of genetic and epigenetic changes, such as point mutations, chromosomal rearrangements, and changes in gene expression level. All these modifications are supposed to confer a selective advantage and to generate highly malignant cancer cells. Until recently, the same selection procedure of rare cells in the tumour mass was believed to be necessary for the metastatic process. Using gene expression profiling, several recent publications report that a gene expression signature could discriminate between primary tumours with high metastatic potentiality and poor clinical outcome, and primary tumours that are not going to metastasize. Analysis of the biological pathways associated with metastatic potential points to cell adhesion, angiogenesis, cell cycle regulation, initiation of DNA synthesis, and DNA repair. Analysing human primary malignant melanoma and various biological processes, we have shown that the overexpression of DNA repair pathways, particularly those involved in double-stand break repair and surveillance of the DNA replication forks, is associated with metastasis and poor patient survival [V. Winnepenninckx, V. Lazar, S. Michiels, P. Dessen, M. Stas, S.R. Alonso, M.F. Avril, P.L. Ortiz Romero, T. Robert, O. Balacescu, A.M. Eggermont, G. Lenoir, A. Sarasin, T. Tursz, J.J. van den Oord, A. Spatz, Gene expression profiling of primary cutaneous melanoma and clinical outcome, J. Natl. Cancer Inst. 98 (2006) 472-482]. These results, also found by analysing other types of human tumours, such as breast or bladder cancers, would clearly explain the high resistance of metastasis towards chemo- and radiotherapies. Our hypothesis is that genetic instability is absolutely necessary to go from normal cells to tumoral cells, but one needs some type of genetic stabilization, which can be obtained by overexpressing specific DNA repair

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA methylation is important for gene regulation and is vulnerable to early-life exposure to environmental contaminants. We found that direct waterborne benzo[a]pyrene (BaP) exposure at 24 'g/L from 2.5 to 96 hours post fertilization (hpf) to zebrafish embryos significantly decreased global cytosine...

  15. Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes

    PubMed Central

    Atilano, Shari R.; Malik, Deepika; Chwa, Marilyn; Cáceres-Del-Carpio, Javier; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin; Kenney, M. Cristina

    2015-01-01

    Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2′-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases. PMID:25964427

  16. CDKL5 is a brain MeCP2 target gene regulated by DNA methylation.

    PubMed

    Carouge, Delphine; Host, Lionel; Aunis, Dominique; Zwiller, Jean; Anglard, Patrick

    2010-06-01

    Rett syndrome and its "early-onset seizure" variant are severe neurodevelopmental disorders associated with mutations within the MECP2 and the CDKL5 genes. Antidepressants and drugs of abuse induce the expression of the epigenetic factor MeCP2, thereby influencing chromatin remodeling. We show that increased MeCP2 levels resulted in the repression of Cdkl5 in rat brain structures in response to cocaine, as well as in cells exposed to serotonin, or overexpressing MeCP2. In contrast, Cdkl5 was induced by siRNA-mediated knockdown of Mecp2 and by DNA-methyltransferase inhibitors, demonstrating its regulation by MeCP2 and by DNA methylation. Cdkl5 gene methylation and its methylation-dependent binding to MeCP2 were increased in the striatum of cocaine-treated rats. Our data demonstrate that Cdkl5 is a MeCP2-repressed target gene providing a link between genes the mutation of which generates overlapping symptoms. They highlight DNA methylation changes as a potential mechanism participating in the long-term plasticity triggered by pharmacological agents.

  17. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems.

  18. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  19. TET1 modulates H4K16 acetylation by controlling auto-acetylation of hMOF to affect gene regulation and DNA repair function

    PubMed Central

    Zhong, Jianing; Li, Xianfeng; Cai, Wanshi; Wang, Yan; Dong, Shanshan; Yang, Jie; Zhang, Jian'an; Wu, Nana; Li, Yuanyuan; Mao, Fengbiao; Zeng, Cheng; Wu, Jinyu; Xu, Xingzhi; Sun, Zhong Sheng

    2017-01-01

    The Ten Eleven Translocation 1 (TET1) protein is a DNA demethylase that regulates gene expression through altering statue of DNA methylation. However, recent studies have demonstrated that TET1 could modulate transcriptional expression independent of its DNA demethylation activity; yet, the detailed mechanisms underlying TET1's role in such transcriptional regulation remain not well understood. Here, we uncovered that Tet1 formed a chromatin complex with histone acetyltransferase Mof and scaffold protein Sin3a in mouse embryonic stem cells by integrative genomic analysis using publicly available ChIP-seq data sets and a series of in vitro biochemical studies in human cell lines. Mechanistically, the TET1 facilitated chromatin affinity and enzymatic activity of hMOF against acetylation of histone H4 at lysine 16 via preventing auto-acetylation of hMOF, to regulate expression of the downstream genes, including DNA repair genes. We found that Tet1 knockout MEF cells exhibited an accumulation of DNA damage and genomic instability and Tet1 deficient mice were more sensitive to x-ray exposure. Taken together, our findings reveal that TET1 forms a complex with hMOF to modulate its function and the level of H4K16Ac ultimately affect gene expression and DNA repair. PMID:27733505

  20. Expression of DNA repair genes in porcine oocytes before and after fertilization by ICSI using freeze-dried sperm.

    PubMed

    Men, Nguyen Thi; Kikuchi, Kazuhiro; Furusawa, Tadashi; Dang-Nguyen, Thanh Quang; Nakai, Michiko; Fukuda, Atsunori; Noguchi, Junko; Kaneko, Hiroyuki; Viet Linh, Nguyen; Xuan Nguyen, Bui; Tajima, Atsushi

    2016-11-01

    Boar sperm freeze-dried with trehalose showed a protective effect against sperm DNA fragmentation. However, normal fertilization and embryonic development were not improved. Damaged sperm may activate maternal DNA repair genes when injected into oocytes. Therefore, we investigated the expression profile of some DNA repair genes in porcine oocytes after intra-cytoplasmic sperm injection. First, the expression levels of MGMT, UDG, XPC, MSH2, XRCC6 and RAD51 genes that are concerned with different types of DNA repair were examined in in vitro mature (IVM) oocytes injected with ejaculated sperm, or freeze-dried sperm with or without trehalose. Quantitative reverse transcription polymerase chain reaction revealed that expression of six DNA repair genes in the oocytes at 4 h after injection did not differ among the four groups. Next, we investigated the gene expression levels of these genes at different stages of maturation. The relative expression levels of UDG and XPC were significantly up-regulated in mature oocytes compared with earlier stages. Furthermore, there was an increased tendency in relative expression of MSH2 and RAD51. These results suggested two possible mechanisms that messenger RNA of DNA repair genes are either accumulated during IVM to be ready for fertilization or increased expression levels of DNA repair genes in oocytes caused by suboptimal IVM conditions.

  1. Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA.

    PubMed

    Burghout, Peter; Bootsma, Hester J; Kloosterman, Tomas G; Bijlsma, Jetta J E; de Jongh, Christa E; Kuipers, Oscar P; Hermans, Peter W M

    2007-09-01

    We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.

  2. SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation

    PubMed Central

    Li, Shaofang; Liu, Lin; Li, Shengben; Gao, Lei; Zhao, Yuanyuan; Kim, Yun Ju; Chen, Xuemei

    2016-01-01

    Transposable elements are found throughout the genomes of all organisms. Repressive marks such as DNA methylation and histone H3 lysine 9 (H3K9) methylation silence these elements and maintain genome integrity. However, how silencing mechanisms are themselves regulated to avoid the silencing of genes remains unclear. Here, an anti-silencing factor was identified using a forward genetic screen on a reporter line that harbors a LUCIFERASE (LUC) gene driven by a promoter that undergoes DNA methylation. SUVH1, a Su(var)3–9 homolog, was identified as a factor promoting the expression of the LUC gene. Treatment with a cytosine methylation inhibitor completely suppressed the LUC expression defects of suvh1, indicating that SUVH1 is dispensable for LUC expression in the absence of DNA methylation. SUVH1 also promotes the expression of several endogenous genes with promoter DNA methylation. However, the suvh1 mutation did not alter DNA methylation levels at the LUC transgene or on a genome-wide scale; thus, SUVH1 functions downstream of DNA methylation. Histone H3 lysine 4 (H3K4) trimethylation was reduced in suvh1; in contrast, H3K9 methylation levels remained unchanged. This work has uncovered a novel, anti-silencing function for a member of the Su(var)3–9 family that has previously been associated with silencing through H3K9 methylation. PMID:26400170

  3. DNA methylation of the oxytocin receptor gene predicts neural response to ambiguous social stimuli

    PubMed Central

    Jack, Allison; Connelly, Jessica J.; Morris, James P.

    2012-01-01

    Oxytocin and its receptor (OXTR) play an important role in a variety of social perceptual and affiliative processes. Individual variability in social information processing likely has a strong heritable component, and as such, many investigations have established an association between common genetic variants of OXTR and variability in the social phenotype. However, to date, these investigations have primarily focused only on changes in the sequence of DNA without considering the role of epigenetic factors. DNA methylation is an epigenetic mechanism by which cells control transcription through modification of chromatin structure. DNA methylation of OXTR decreases expression of the gene and high levels of methylation have been associated with autism spectrum disorders (ASD). This link between epigenetic variability and social phenotype allows for the possibility that social processes are under epigenetic control. We hypothesized that the level of DNA methylation of OXTR would predict individual variability in social perception. Using the brain's sensitivity to displays of animacy as a neural endophenotype of social perception, we found significant associations between the degree of OXTR methylation and brain activity evoked by the perception of animacy. Our results suggest that consideration of DNA methylation may substantially improve our ability to explain individual differences in imaging genetic association studies. PMID:23087634

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

    PubMed Central

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

    2012-01-01

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

  5. DNA Methylation Changes in the IGF1R Gene in Birth Weight Discordant Adult Monozygotic Twins.

    PubMed

    Tsai, Pei-Chien; Van Dongen, Jenny; Tan, Qihua; Willemsen, Gonneke; Christiansen, Lene; Boomsma, Dorret I; Spector, Tim D; Valdes, Ana M; Bell, Jordana T

    2015-12-01

    Low birth weight (LBW) can have an impact on health outcomes in later life, especially in relation to pre-disposition to metabolic disease. Several studies suggest that LBW resulting from restricted intrauterine growth leaves a footprint on DNA methylation in utero, and this influence likely persists into adulthood. To investigate this further, we performed epigenome-wide association analyses of blood DNA methylation using Infinium HumanMethylation450 BeadChip profiles in 71 adult monozygotic (MZ) twin pairs who were extremely discordant for birth weight. A signal mapping to the IGF1R gene (cg12562232, p = 2.62 × 10(-8)), was significantly associated with birth weight discordance at a genome-wide false-discovery rate (FDR) of 0.05. We pursued replication in three additional independent datasets of birth weight discordant MZ pairs and observed the same direction of association, but the results were not significant. However, a meta-analysis across the four independent samples, in total 216 birth-weight discordant MZ twin pairs, showed a significant positive association between birth weight and DNA methylation differences at IGF1R (random-effects meta-analysis p = .04), and the effect was particularly pronounced in older twins (random-effects meta-analysis p = .008, 98 older birth-weight discordant MZ twin pairs). The results suggest that severe intra-uterine growth differences (birth weight discordance >20%) are associated with methylation changes in the IGF1R gene in adulthood, independent of genetic effects.

  6. PRMT1 mediated methylation of TAF15 is required for its positive gene regulatory function

    SciTech Connect

    Jobert, Laure; Argentini, Manuela; Tora, Laszlo

    2009-04-15

    TAF15 (formerly TAF{sub II}68) is a nuclear RNA-binding protein that is associated with a distinct population of TFIID and RNA polymerase II complexes. TAF15 harbours an N-terminal activation domain, an RNA recognition motif (RRM) and many Arg-Gly-Gly (RGG) repeats at its C-terminal end. The N-terminus of TAF15 serves as an essential transforming domain in the fusion oncoprotein created by chromosomal translocation in certain human chondrosarcomas. Post-transcriptional modifications (PTMs) of proteins are known to regulate their activity, however, nothing is known on how PTMs affect TAF15 function. Here we demonstrate that endogenous human TAF15 is methylated in vivo at its numerous RGG repeats. Furthermore, we identify protein arginine N-methyltransferase 1 (PRMT1) as a TAF15 interactor and the major PRMT responsible for its methylation. In addition, the RGG repeat-containing C-terminus of TAF15 is responsible for the shuttling between the nucleus and the cytoplasm and the methylation of RGG repeats affects the subcellular localization of TAF15. The methylation of TAF15 by PRMT1 is required for the ability of TAF15 to positively regulate the expression of the studied endogenous TAF15-target genes. Our findings demonstrate that arginine methylation of TAF15 by PRMT1 is a crucial event determining its proper localization and gene regulatory function.

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

    PubMed Central

    Turecki, Gustavo; Meaney, Michael

    2015-01-01

    The early-life social environment can induce stable changes that influence neurodevelopment and mental health. Research focused on early-life adversity revealed that early-life experiences have a persistent impact on gene expression and behaviour through epigenetic mechanisms. The hypothalamus-pituitary-adrenal (HPA) axis is sensitive to changes in the early-life environment that associate with DNA methylation of a neuron-specific exon 17 promoter of the glucocorticoid receptor (GR; NR3C1). Since Weaver et al published the initial findings in 2004, numerous reports have investigated GR gene methylation in relationship to early-life experience, parental stress and psychopathology. We conducted a systematic review of this growing literature, which identified 40 articles (13 animal and 27 human studies) published since 2004. The majority of these examined the GR exon variant 1F in humans or the GR17 in rats, and 89% of human studies and 70% of animal studies of early-life adversity reported increased methylation at this exon variant. All the studies investigating exon 1F/17 methylation in conditions of parental stress (one animal study and 7 human studies) also reported increased methylation. Studies examining psychosocial stress and psychopathology had less consistent results, with 67% of animal studies reporting increased exon 17 methylation and 17% of human studies reporting increased exon 1F methylation. We found great consistency among studies investigating early life adversity and the effect of parental stress, even if the precise phenotype and measures of social environment adversity varied among studies. These results are encouraging and warrant further investigation to better understand correlates and characteristics of these associations. PMID:25687413

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

    PubMed

    Turecki, Gustavo; Meaney, Michael J

    2016-01-15

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

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

    PubMed Central

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

    2001-01-01

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

  10. Polymorphisms in DNA repair genes, medical exposure to ionizing radiation, and breast cancer risk.

    PubMed

    Millikan, Robert C; Player, Jon S; Decotret, Allan Rene; Tse, Chiu-Kit; Keku, Temitope

    2005-10-01

    An epidemiologic study was conducted to determine whether polymorphisms in DNA repair genes modify the association between breast cancer risk and exposure to ionizing radiation. Self-reported exposure to ionizing radiation from medical sources was evaluated as part of a population-based, case-control study of breast cancer in African-American (894 cases and 788 controls) and White (1,417 cases and 1,234 controls) women. Genotyping was conducted for polymorphisms in four genes involved in repair of radiation-induced DNA damage, the double-strand break repair pathway: X-ray cross-complementing group 3 (XRCC3) codon 241 Thr/Met, Nijmegen breakage syndrome 1 (NBS1) codon 185 Glu/Gln, X-ray cross-complementing group 2 (XRCC2) codon 188 Arg/His, and breast cancer susceptibility gene 2 (BRCH2) codon 372 Asn/His. Allele and genotype frequencies were not significantly different in cases compared with controls for all four genetic polymorphisms, and odds ratios for breast cancer were close to the null. Combining women with two, three, and four variant genotypes, a positive association was observed between breast cancer and number of lifetime mammograms (P(trend) < 0.0001). No association was observed among women with zero or one variant genotype (P = 0.86). Odds ratios for radiation treatments to the chest and number of lifetime chest X-rays were slightly elevated but not statistically significant among women with two to four variant genotypes. The study has several limitations, including inability to distinguish between diagnostic and screening mammograms or reliably classify prediagnostic mammograms and chest X-rays in cases. Prospective studies are needed to address whether common polymorphisms in DNA repair genes modify the effects of low-dose radiation exposure from medical sources.

  11. Genes Involved in DNA Double-Strand Break Repair: Implications for Breast Cancer.

    DTIC Science & Technology

    1996-10-01

    locus . The disruption of one of 3 the two Ku alleles might lead to a phenotype, since Chinese hamster ovary cells are functionally...significant in-frame deletions. This is the substantial part of the genome in Chinese hamster cell lines is first direct evidence that mutant cells in group 5...Department of the Army, by grant A135763 from DNA repair gene in Chinese hamster ovary cells . Mol. Cell . Biol. 6:2944- the National

  12. DNA Repair Gene Expression and Risk of Locoregional Relapse in Breast Cancer Patients

    SciTech Connect

    Le Scodan, Romuald; Cizeron-Clairac, Geraldine

    2010-10-01

    Purpose: Radiation therapy appears to kill cells mainly by inducing DNA double-strand breaks. We investigated whether the DNA repair gene expression status might influence the risk of locoregional recurrence (LRR) in breast cancer patients. Methods and Materials: We used a quantitative reverse transcriptase PCR-based approach to measure messenger RNA levels of 20 selected DNA repair genes in tumor samples from 97 breast cancer patients enrolled in a phase III trial (Centre Rene Huguenin cohort). Normalized mRNA levels were tested for an association with LRR-free survival (LRR-FS) and overall survival (OS). The findings were validated in comparison with those of an independent cohort (Netherlands Cancer Institute (NKI) cohort). Multivariate analysis encompassing known prognostic factors was used to assess the association between DNA repair gene expression and patient outcome. Results: RAD51 was the only gene associated with LRR in both cohorts. With a median follow-up of 126 months in the CRH cohort, the 5-year LRR-FS and OS rates were 100% and 95% in the 61 patients with low RAD51 expression, compared with 70% and 69% in the 36 patients with high RAD51 expression, respectively (p < 0.001). RAD51 overexpression was associated with a higher risk of LRR (hazard ratio [HR], 12.83; 95% confidence interval [CI], 3.6-45.6) and death (HR, 4.10; 95% CI, 1.7-9.7). RAD51 overexpression was also significantly associated with shorter LRR-FS and OS in the NKI cohort. Conclusions: Overexpression of RAD51, a key component of the homologous DNA repair pathway, is associated with poor breast cancer outcome. This finding warrants prospective studies of RAD51 as a prognosticator and therapeutic target.

  13. Role of DNA methylation at the placental RTL1 gene locus in type 1 diabetes.

    PubMed

    Belot, Marie-Pierre; Nadéri, Kambiz; Mille, Clémence; Boëlle, Pierre-Yves; Benachi, Alexandra; Bougnères, Pierre; Fradin, Delphine

    2016-05-13

    Genome-wide association studies (GWAS) have identified more than 40 T1D loci associated with type 1 diabetes (T1D). How these polymorphisms interact with environmental factors to trigger T1D is unknown, but recent evidence suggests that epigenetic mechanisms could play a role. To begin to explore the contribution of epigenetics to T1D, we have examined DNA methylation in a pilot study of whole blood cells DNA from 10 young T1D patients and 10 young controls. Through the study of >900 000 CG loci across a diverse set of functionally relevant genomic regions using a custom DNA methylation array, we identified 250 T1D-differentially methylated region (DMR) at p < 0.05 and 1 DMR using next a permutation-based multiple testing correction method. This DMR is located in an imprinted region previously associated with T1D on the chromosome 14 that encompasses RTL1 gene and 2 miRNAs (miR136 and miR432). Using pyrosequencing-based bisulfite PCR, we replicated this association in a different and larger set of T1D patients and controls. DNA methylation at this DMR was inversely correlated with RTL1 gene expression and positively correlated with miR136 expression in human placentas. The DMR identified in this study presents suggestive evidence for altered methylation site in T1D and provide a promising new candidate gene. RTL1 is essential for placental permeability function in the mid-to-late fetal stages. We suggest that hypo-methylation could increase the fetal exposure to environmental factors in T1D susceptibility.

  14. Methylation of HPA axis related genes in men with hypersexual disorder.

    PubMed

    Jokinen, Jussi; Boström, Adrian E; Chatzittofis, Andreas; Ciuculete, Diana M; Öberg, Katarina Görts; Flanagan, John N; Arver, Stefan; Schiöth, Helgi B

    2017-03-10

    Hypersexual Disorder (HD) defined as non-paraphilic sexual desire disorder with components of compulsivity, impulsivity and behavioral addiction, and proposed as a diagnosis in the DSM 5, shares some overlapping features with substance use disorder including common neurotransmitter systems and dysregulated hypothalamic-pituitary-adrenal (HPA) axis function. In this study, comprising 67 HD male patients and 39 male healthy volunteers, we aimed to identify HPA-axis coupled CpG-sites, in which modifications of the epigenetic profile are associated with hypersexuality. The genome-wide methylation pattern was measured in whole blood using the Illumina Infinium Methylation EPIC BeadChip, measuring the methylation state of over 850K CpG sites. Prior to analysis, the global DNA methylation pattern was pre-processed according to standard protocols and adjusted for white blood cell type heterogeneity. We included CpG sites located within 2000bp of the transcriptional start site of the following HPA-axis coupled genes: Corticotropin releasing hormone (CRH), corticotropin releasing hormone binding protein (CRHBP), corticotropin releasing hormone receptor 1 (CRHR1), corticotropin releasing hormone receptor 2 (CRHR2), FKBP5 and the glucocorticoid receptor (NR3C1). We performed multiple linear regression models of methylation M-values to a categorical variable of hypersexuality, adjusting for depression, dexamethasone non-suppression status, Childhood Trauma Questionnaire total score and plasma levels of TNF-alpha and IL-6. Of 76 tested individual CpG sites, four were nominally significant (p<0.05), associated with the genes CRH, CRHR2 and NR3C1. Cg23409074-located 48bp upstream of the transcription start site of the CRH gene - was significantly hypomethylated in hypersexual patients after corrections for multiple testing using the FDR-method. Methylation levels of cg23409074 were positively correlated with gene expression of the CRH gene in an independent cohort of 11 healthy

  15. Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient ``wasted`` mice

    SciTech Connect

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M.; Libertin, C.R.

    1992-11-01

    Mice recessive for the autosomal gene ``wasted`` (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{sm_bullet} mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/{sm_bullet} and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  16. Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient wasted'' mice

    SciTech Connect

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M. ); Libertin, C.R. )

    1992-01-01

    Mice recessive for the autosomal gene wasted'' (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/[sm bullet] mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/[sm bullet] and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

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

    PubMed

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

    2016-03-15

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

  18. Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair

    PubMed Central

    Sone, Kenbun; Piao, Lianhua; Nakakido, Makoto; Ueda, Koji; Jenuwein, Thomas; Nakamura, Yusuke; Hamamoto, Ryuji

    2014-01-01

    The presence of phosphorylated histone H2AX (γ-H2AX) is associated with the local activation of DNA-damage repair pathways. Although γ-H2AX deregulation in cancer has previously been reported, the molecular mechanism involved and its relationship with other histone modifications remain largely unknown. Here we find that the histone methyltransferase SUV39H2 methylates histone H2AX on lysine 134. When H2AX was mutated to abolish K134 methylation, the level of γ-H2AX became significantly reduced. We also found lower γ-H2AX activity following the introduction of double-strand breaks in Suv39h2 knockout cells or on SUV39H2 knockdown. Tissue microarray analyses of clinical lung and bladder tissues also revealed a positive correlation between H2AX K134 methylation and γ-H2AX levels. Furthermore, introduction of K134-substituted histone H2AX enhanced radio- and chemosensitivity of cancer cells. Overall, our results suggest that H2AX methylation plays a role in the regulation of γ-H2AX abundance in cancer. PMID:25487737

  19. Sequence variation and methylation of the flax 5S RNA genes.

    PubMed Central

    Goldsbrough, P B; Ellis, T H; Lomonossoff, G P

    1982-01-01

    The complete sequence of the flax 5S DNA repeat is presented. Length heterogeneity is the consequence of the presence or absence of a single direct repeat and the majority of single base changes are transition mutations. No sequence variation has been found in the coding sequence. The extent of methylation of cytosines has been measured at one location in the gene and one in the spacer. The relationship between the observed sequence heterogeneity and the level of methylation is discussed in the context of the operation of a correction mechanism. Images PMID:6290983

  20. DNA methylation map of mouse and human brain identifies target genes in Alzheimer’s disease

    PubMed Central

    Sanchez-Mut, Jose V.; Aso, Ester; Panayotis, Nicolas; Lott, Ira; Dierssen, Mara; Rabano, Alberto; Urdinguio, Rocio G.; Fernandez, Agustin F.; Astudillo, Aurora; Martin-Subero, Jose I.; Balint, Balazs; Fraga, Mario F.; Gomez, Antonio; Gurnot, Cecile; Roux, Jean-Christophe; Avila, Jesus; Hensch, Takao K.; Ferrer, Isidre

    2013-01-01

    The central nervous system has a pattern of gene expression that is closely regulated with respect to functional and anatomical regions. DNA methylation is a major regulator of transcriptional activity, and aberrations in the distribution of this epigenetic mark may be involved in many neurological disorders, such as Alzheimer’s disease. Herein, we have analysed 12 distinct mouse brain regions according to their CpG 5’-end gene methylation patterns and observed their unique epigenetic landscapes. The DNA methylomes obtained from the cerebral cortex were used to identify aberrant DNA methylation changes that occurred in two mouse models of Alzheimer’s disease. We were able to translate these findings to patients with Alzheimer’s disease, identifying DNA methylation-associated silencing of three targets genes: thromboxane A2 receptor (TBXA2R), sorbin and SH3 domain containing 3 (SORBS3) and spectrin beta 4 (SPTBN4). These hypermethylation targets indicate that the cyclic AMP response element-binding protein (CREB) activation pathway and the axon initial segment could contribute to the disease. PMID:24030951

  1. DNA methylation map of mouse and human brain identifies target genes in Alzheimer's disease.

    PubMed

    Sanchez-Mut, Jose V; Aso, Ester; Panayotis, Nicolas; Lott, Ira; Dierssen, Mara; Rabano, Alberto; Urdinguio, Rocio G; Fernandez, Agustin F; Astudillo, Aurora; Martin-Subero, Jose I; Balint, Balazs; Fraga, Mario F; Gomez, Antonio; Gurnot, Cecile; Roux, Jean-Christophe; Avila, Jesus; Hensch, Takao K; Ferrer, Isidre; Esteller, Manel

    2013-10-01

    The central nervous system has a pattern of gene expression that is closely regulated with respect to functional and anatomical regions. DNA methylation is a major regulator of transcriptional activity, and aberrations in the distribution of this epigenetic mark may be involved in many neurological disorders, such as Alzheimer's disease. Herein, we have analysed 12 distinct mouse brain regions according to their CpG 5'-end gene methylation patterns and observed their unique epigenetic landscapes. The DNA methylomes obtained from the cerebral cortex were used to identify aberrant DNA methylation changes that occurred in two mouse models of Alzheimer's disease. We were able to translate these findings to patients with Alzheimer's disease, identifying DNA methylation-associated silencing of three targets genes: thromboxane A2 receptor (TBXA2R), sorbin and SH3 domain containing 3 (SORBS3) and spectrin beta 4 (SPTBN4). These hypermethylation targets indicate that the cyclic AMP response element-binding protein (CREB) activation pathway and the axon initial segment could contribute to the disease.

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

    PubMed Central

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

    2007-01-01

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

  3. Effect of developmental dioxin exposure on methylation and expression of specific imprinted genes in mice.

    PubMed

    Somm, Emmanuel; Stouder, Christelle; Paoloni-Giacobino, Ariane

    2013-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disruptor affecting the reproductive system in humans. The aim of this study was to evaluate the effects of TCDD administered to pregnant mice at two different doses (2-10 ng/kg/day), on imprinted genes in the male offspring. The degree of methylation and the mRNA expression of Snrpn, Peg3 and Igf2r were analyzed in the sperm, skeletal muscle and liver. TCDD administration (10 ng/kg/day) decreased the sperm count in the male offspring. It did not affect methylation but increased mRNA expression of Snrpn, Peg3, Igf2r and Air ncRNA. In muscle and liver, TCDD (10 ng/kg/day) induced increases in methylation and decreases in mRNA expression of Igf2r. These results show that the robust effects of TCDD on the mRNA expression of Snrpn, Peg3 and Igf2r genes in the sperm and of Igf2r in the muscle and liver are unrelated to changes in methylation in their respective genes.

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

    PubMed

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

    2016-08-02

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

  5. Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts.

    PubMed Central

    Chen, R H; Maher, V M; Brouwer, J; van de Putte, P; McCormick, J J

    1992-01-01

    If excision repair-proficient human cells are allowed time for repair before onset of S phase, the premutagenic lesions formed by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, BPDE) are lost from the transcribed strand of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene faster than from the nontranscribed strand. No change in strand distribution is seen with repair-deficient cells. These results suggest strand-specific repair of BPDE-induced DNA damage in human cells. To test this, we measured the initial number of BPDE adducts formed in each strand of the actively transcribed HPRT gene and the rate of repair, using UvrABC excinuclease in conjunction with Southern hybridization and strand-specific probes. We also measured the rate of loss of BPDE adducts from the inactive 754 locus. The frequencies of adducts formed by exposure to BPDE (1.0 or 1.2 microM) in either strand of a 20-kilobase fragment that lies entirely within the transcription unit of the HPRT gene were similar; the frequency in the 14-kilobase 754 fragment was approximately 20% lower. The rates of repair in the two strands of the HPRT fragment differed significantly. Within 7 hr after treatment with 1.2 microM BPDE, 53% of the adducts had been removed from the transcribed strand, but only 26% from the nontranscribed strand; after 20 hr, these values were 87% and 58%, respectively. In contrast, only approximately 14% of the BPDE adducts were lost from the 754 locus in 20 hr, a value even lower than the rate of loss from the overall genome (i.e., 38%). These results demonstrate strand-specific and preferential repair of BPDE adducts in human cells. They suggest that the heterogeneous repair of BPDE adducts in the human genome cannot be accounted for merely by the greatly increased rate of the repair specific to the transcribed strand of the active genes, and they point to a role for the chromatin structure. Images

  6. On the origin and evolutionary consequences of gene body DNA methylation

    PubMed Central

    Bewick, Adam J.; Ji, Lexiang; Niederhuth, Chad E.; Willing, Eva-Maria; Hofmeister, Brigitte T.; Shi, Xiuling; Wang, Li; Lu, Zefu; Rohr, Nicholas A.; Hartwig, Benjamin; Kiefer, Christiane; Deal, Roger B.; Schmutz, Jeremy; Grimwood, Jane; Stroud, Hume; Jacobsen, Steven E.; Schneeberger, Korbinian; Zhang, Xiaoyu; Schmitz, Robert J.

    2016-01-01

    In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum. Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales. PMID:27457936

  7. DNA adenine methylation of sams1 gene in symbiont-bearing Amoeba proteus.

    PubMed

    Jeon, Taeck J

    2008-10-01

    The expression of amoeba sams genes is switched from sams1 to sams2 when amoebae are infected with Legionella jeonii. To elucidate the mechanism for the inactivation of host sams1 gene by endosymbiotic bacteria, methylation states of the sams1 gene of D and xD amoebae was compared in this study. The sams1 gene of amoebae was methylated at an internal adenine residue of GATC site in symbiont-bearing xD amoebae but not in symbiont-free D amoebae, suggesting that the modification might have caused the inactivation of sams1 in xD amoebae. The sams1 gene of xD amoebae was inactivated at the transcriptional level. Analysis of DNA showed that adenine residues in L. jeonii sams were also methylated, implying that L. jeonii bacteria belong to a Dam methylase-positive strain. In addition, both SAM and Met appeared to act as negative regulators for the expression of sams1 whereas the expression of sams2 was not affected in amoebae.

  8. Integrating DNA Methylation and Gene Expression Data in the Development of the Soybean-Bradyrhizobium N2-Fixing Symbiosis

    PubMed Central

    Davis-Richardson, Austin G.; Russell, Jordan T.; Dias, Raquel; McKinlay, Andrew J.; Canepa, Ronald; Fagen, Jennie R.; Rusoff, Kristin T.; Drew, Jennifer C.; Kolaczkowski, Bryan; Emerich, David W.; Triplett, Eric W.

    2016-01-01

    Very little is known about the role of epigenetics in the differentiation of a bacterium from the free-living to the symbiotic state. Here genome-wide analysis of DNA methylation changes between these states is described using the model of symbiosis between soybean and its root nodule-forming, nitrogen-fixing symbiont, Bradyrhizobium diazoefficiens. PacBio resequencing of the B. diazoefficiens genome from both states revealed 43,061 sites recognized by five motifs with the potential to be methylated genome-wide. Of those sites, 3276 changed methylation states in 2921 genes or 35.5% of all genes in the genome. Over 10% of the methylation changes occurred within the symbiosis island that comprises 7.4% of the genome. The CCTTGAG motif was methylated only during symbiosis with 1361 adenosines methylated among the 1700 possible sites. Another 89 genes within the symbiotic island and 768 genes throughout the genome were found to have methylation and significant expression changes during symbiotic development. Of those, nine known symbiosis genes involved in all phases of symbiotic development including early infection events, nodule development, and nitrogenase production. These associations between methylation and expression changes in many B. diazoefficiens genes suggest an important role of the epigenome in bacterial differentiation to the symbiotic state. PMID:27148207

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

    PubMed Central

    Chater-Diehl, Eric J.; Castellani, Christina A.; Alberry, Bonnie L.; Singh, Shiva M.

    2016-01-01

    The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse’s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as “Free radical scavenging”. We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was “Peroxisome biogenesis”; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD. PMID:27136348

  10. Microsatellites in the Eukaryotic DNA Mismatch Repair Genes as Modulators of Evolutionary Mutation Rate

    NASA Technical Reports Server (NTRS)

    Chang, Dong Kyung; Metzgar, David; Wills, Christopher; Boland, C. Richard

    2003-01-01

    All "minor" components of the human DNA mismatch repair (MMR) system-MSH3, MSH6, PMS2, and the recently discovered MLH3-contain mononucleotide microsatellites in their coding sequences. This intriguing finding contrasts with the situation found in the major components of the DNA MMR system-MSH2 and MLH1-and, in fact, most human genes. Although eukaryotic genomes are rich in microsatellites, non-triplet microsatellites are rare in coding regions. The recurring presence of exonal mononucleotide repeat sequences within a single family of human genes would therefore be considered exceptional.

  11. A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

    PubMed Central

    Słabicki, Mikołaj; Theis, Mirko; Krastev, Dragomir B.; Samsonov, Sergey; Mundwiller, Emeline; Junqueira, Magno; Paszkowski-Rogacz, Maciej; Teyra, Joan; Heninger, Anne-Kristin; Poser, Ina; Prieur, Fabienne; Truchetto, Jérémy; Confavreux, Christian; Marelli, Cécilia; Durr, Alexandra; Camdessanche, Jean Philippe; Brice, Alexis; Shevchenko, Andrej; Pisabarro, M. Teresa; Stevanin, Giovanni; Buchholz, Frank

    2010-01-01

    DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair. PMID:20613862

  12. Aberrant methylation of imprinted genes is associated with negative hormone receptor status in invasive breast cancer

    PubMed Central

    Barrow, Timothy M; Barault, Ludovic; Ellsworth, Rachel E; Harris, Holly R; Binder, Alexandra M; Valente, Allyson L; Shriver, Craig D; Michels, Karin B

    2015-01-01

    Epigenetic regulation of imprinted genes enables monoallelic expression according to parental origin, and its disruption is implicated in many cancers and developmental disorders. The expression of hormone receptors is significant in breast cancer as they are indicators of cancer cell growth rate and determine response to endocrine therapies. We investigated the frequency of aberrant events and variation in DNA methylation at nine imprinted sites in invasive breast cancer and examined the association with estrogen and progesterone receptor status. Breast tissue and blood from patients with invasive breast cancer (n=38) and benign breast disease (n=30) were compared to those from healthy individuals (n=36), matched to the cancer patients by age at diagnosis, ethnicity, BMI, menopausal status, and familial history of cancer. DNA methylation and allele-specific expression were analyzed by pyrosequencing. Tumor-specific methylation changes at IGF2 DMR2 were observed in 59% of cancer patients, IGF2 DMR0 in 38%, DIRAS3 DMR in 36%, GRB10 ICR in 23%, PEG3 DMR in 21%, MEST ICR in 19%, H19 ICR in 18%, KvDMR in 8%, and SNRPN/SNURF ICR in 4%. Variation of methylation was significantly greater in breast tissue from cancer patients than healthy individuals and benign breast disease. Aberrant methylation of three or more sites was significantly associated with negative estrogen-alpha (Fisher’s Exact Test, p=0.02) and progesterone-A (p=0.02) receptor status. Aberrant events and increased variation of imprinted gene DNA methylation therefore appear to be frequent in invasive breast cancer and are associated with negative estrogen and progesterone receptor status, without loss of monoallelic expression. PMID:25560175

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2008-12-16

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

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

    PubMed

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

    2016-10-25

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

  16. EGFR gene methylation is not involved in Royalactin controlled phenotypic polymorphism in honey bees

    PubMed Central

    Kucharski, R.; Foret, S.; Maleszka, R.

    2015-01-01

    The 2011 highly publicised Nature paper by Kamakura on honeybee phenotypic dimorphism, (also using Drosophila as an experimental surrogate), claims that a single protein in royal jelly, Royalactin, essentially acts as a master “on-off” switch in development via the epidermal growth factor receptor (AmEGFR), to seal the fate of queen or worker. One mechanism proposed in that study as important for the action of Royalactin is differential amegfr methylation in alternate organismal outcomes. According to the author differential methylation of amegfr was experimentally confirmed and shown in a supportive figure. Here we have conducted an extensive analysis of the honeybee egfr locus and show that this gene is never methylated. We discuss several lines of evidence casting serious doubts on the amegfr methylation result in the 2011 paper and consider possible origins of the author’s statement. In a broader context, we discuss the implication of our findings for contrasting context-dependent regulation of EGFR in three insect species, Apis mellifera, D. melanogaster and the carpenter ant, Camponotus floridanus, and argue that more adequate methylation data scrutiny measures are needed to avoid unwarranted conclusions. PMID:26358539

  17. EGFR gene methylation is not involved in Royalactin controlled phenotypic polymorphism in honey bees.

    PubMed

    Kucharski, R; Foret, S; Maleszka, R

    2015-09-11

    The 2011 highly publicised Nature paper by Kamakura on honeybee phenotypic dimorphism, (also using Drosophila as an experimental surrogate), claims that a single protein in royal jelly, Royalactin, essentially acts as a master "on-off" switch in development via the epidermal growth factor receptor (AmEGFR), to seal the fate of queen or worker. One mechanism proposed in that study as important for the action of Royalactin is differential amegfr methylation in alternate organismal outcomes. According to the author differential methylation of amegfr was experimentally confirmed and shown in a supportive figure. Here we have conducted an extensive analysis of the honeybee egfr locus and show that this gene is never methylated. We discuss several lines of evidence casting serious doubts on the amegfr methylation result in the 2011 paper and consider possible origins of the author's statement. In a broader context, we discuss the implication of our findings for contrasting context-dependent regulation of EGFR in three insect species, Apis mellifera, D. melanogaster and the carpenter ant, Camponotus floridanus, and argue that more adequate methylation data scrutiny measures are needed to avoid unwarranted conclusions.

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

    PubMed

    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.

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed Central

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

    1999-01-01

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

  1. Influence of DNA-repair gene variants on the micronucleus frequency in thyroid cancer patients.

    PubMed

    García-Quispes, W A; Pastor, S; Galofré, P; Biarnés, F; Castell, J; Velázquez, A; Marcos, R

    2013-01-20

    The role of different DNA-repair genes (OGG1, XRCC1, XRCC2 and XRCC3) on both the spontaneous and the induced frequency of micronuclei (MN) has been studied in the lymphocytes of a group of 114 patients with differentiated thyroid cancer (DTC). Induction of MN was achieved by treatment of the lymphocytes with 0.5Gy of gamma-radiation. The selected genes are involved in base-excision repair (BER) (OGG1, Ser326Cys; XRCC1, Arg280His and Arg399Gln), and in homologous recombination repair (HRR) (XRCC2, Arg188His and XRCC3, IVS5-14G). Genotyping was carried out by use of the iPLEX (Sequenom) technique. Results indicate that only the OGG1-Ser326Cys polymorphism was able to modulate the MN frequency. This effect was only observed in the spontaneous MN frequency (P=0.016), but not in the MN frequency induced after irradiation. In addition, a strong correlation was observed between spontaneous and induced MN frequency, which would suggest an underlying genetic background.

  2. Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

    SciTech Connect

    Sun Hong; Zhou Xue; Chen Haobin; Li Qin; Costa, Max

    2009-06-15

    Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1.

  3. Widespread differences in cortex DNA methylation of the "language gene" CNTNAP2 between humans and chimpanzees.

    PubMed

    Schneider, Eberhard; El Hajj, Nady; Richter, Steven; Roche-Santiago, Justin; Nanda, Indrajit; Schempp, Werner; Riederer, Peter; Navarro, Bianca; Bontrop, Ronald E; Kondova, Ivanela; Scholz, Claus Jürgen; Haaf, Thomas

    2014-04-01

    CNTNAP2, one of the largest genes in the human genome, has been linked to human-specific language abilities and neurodevelopmental disorders. Our hypothesis is that epigenetic rather than genetic changes have accelerated the evolution of the human brain. To compare the cortex DNA methylation patterns of human and chimpanzee CNTNAP2 at ultra-high resolution, we combined methylated DNA immunoprecipitation (MeDIP) with NimbleGen tiling arrays for the orthologous gene and flanking sequences. Approximately 1.59 Mb of the 2.51 Mb target region could be aligned and analyzed with a customized algorithm in both species. More than one fifth (0.34 Mb) of the analyzed sequence throughout the entire gene displayed significant methylation differences between six human and five chimpanzee cortices. One of the most striking interspecies differences with 28% methylation in human and 59% in chimpanzee cortex (by bisulfite pyrosequencing) lies in a region 300 bp upstream of human SNP rs7794745 which has been associated with autism and parent-of-origin effects. Quantitative real-time RT PCR revealed that the protein-coding splice variant CNTNAP2-201 is 1.6-fold upregulated in human cortex, compared with the chimpanzee. Transcripts CNTNAP2-001, -002, and -003 did not show skewed allelic expression, which argues against CNTNAP2 imprinting, at least in adult human brain. Collectively, our results suggest widespread cortex DNA methylation changes in CNTNAP2 since the human-chimpanzee split, supporting a role for CNTNAP2 fine-regulation in human-specific language and communication traits.

  4. Arsenic Methylation in Arabidopsis thaliana Expressing an Algal Arsenite Methyltransferase Gene Increases Arsenic Phytotoxicity

    PubMed Central

    Tang, Zhong; Lv, Yanling; Chen, Fei; Zhang, Wenwen; Rosen, Barry P.; Zhao, Fang-Jie

    2016-01-01

    Arsenic (As) contamination in soil can lead to elevated transfer of As to the food chain. One potential mitigation strategy is to genetically engineer plants to enable them to transform inorganic As to methylated and volatile As species. In this study, we genetically engineered two ecotypes of Arabidopsis thaliana with the arsenite (As(III)) S-adenosylmethyltransferase (arsM) gene from the eukaryotic alga Chlamydomonas reinhardtii. The transgenic A. thaliana plants gained a strong ability to methylate As, converting most of the inorganic As into dimethylarsenate [DMA(V)] in the shoots. Small amounts of volatile As were detected from the transgenic plants. However, the transgenic plants became more sensitive to As(III) in the medium, suggesting that DMA(V) is more phytotoxic than inorganic As. The study demonstrates a negative consequence of engineered As methylation in plants and points to a need for arsM genes with a strong ability to methylate As to volatile species. PMID:26998776

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-08-01

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

  7. Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: Involvement of the human ERCC2 DNA repair gene

    SciTech Connect

    Flejter, W.L.; McDaniel, L.D.; Johns, D.; Schultz, R.A. ); Friedberg, E.C. )

    1992-01-01

    Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. The authors now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2) gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.

  8. Polymorphisms in DNA repair genes, traffic-related polycyclic aromatic hydrocarbon exposure and breast cancer incidence.

    PubMed

    Mordukhovich, Irina; Beyea, Jan; Herring, Amy H; Hatch, Maureen; Stellman, Steven D; Teitelbaum, Susan L; Richardson, David B; Millikan, Robert C; Engel, Lawrence S; Shantakumar, Sumitra; Steck, Susan E; Neugut, Alfred I; Rossner, Pavel; Santella, Regina M; Gammon, Marilie D

    2016-07-15

    Vehicular traffic polycyclic aromatic hydrocarbons (PAHs) have been associated with breast cancer incidence in epidemiologic studies, including our own. Because PAHs damage DNA by forming adducts and oxidative lesions, genetic polymorphisms that alter DNA repair capacity may modify associations between PAH-related exposures and breast cancer risk. Our goal was to examine the association between vehicular traffic exposure and breast cancer incidence within strata of a panel of nine biologically plausible nucleotide excision repair (NER) and base excision repair (BER) genotypes. Residential histories of 1,508 cases and 1,556 controls were assessed in the Long Island Breast Cancer Study Project between 1996 and 1997 and used to reconstruct residential traffic exposures to benzo[a]pyrene, as a proxy for traffic-related PAHs. Likelihood ratio tests from adjusted unconditional logistic regression models were used to assess multiplicative interactions. A gene-traffic interaction was evident (p = 0.04) for ERCC2 (Lys751); when comparing the upper and lower tertiles of 1995 traffic exposure estimates, the odds ratio (95% confidence interval) was 2.09 (1.13, 3.90) among women with homozygous variant alleles. Corresponding odds ratios for 1960-1990 traffic were also elevated nearly 2-3-fold for XRCC1(Arg194Trp), XRCC1(Arg399Gln) and OGG1(Ser326Cys), but formal multiplicative interaction was not evident. When DNA repair variants for ERCC2, XRCC1 and OGG1 were combined, among women with 4-6 variants, the odds ratios were 2.32 (1.22, 4.49) for 1995 traffic and 2.96 (1.06, 8.21) for 1960-1990 traffic. Our study is first to report positive associations between traffic-related PAH exposure and breast cancer incidence among women with select biologically plausible DNA repair genotypes.

  9. DNA Repair Genes XRCC1, XRCC3, XPD, and OGG1 Polymorphisms among the Central Region Population of Saudi Arabia.

    PubMed

    Alanazi, Mohammad; Pathan, Akbar Ali Khan; Ajaj, Sana Abdulla; Khan, Wajahatullah; Shaik, Jilani P; Al Tassan, Nada; Parine, Narasimha Reddy

    2013-01-01

    DNA repair is one of the central defense mechanisms against mutagenic exposures. Inherited SNPs of DNA repair genes may contribute to variations in DNA repair capacity and susceptibility to cancer. Due to the presence of these variants, inter-individual and ethnic differences in DNA repair capacity have been established in various populations. Saudi Arabia harbors enormous genetic and cultural diversity. In the present study we aimed to determine the genotype and allele frequencies of XRCC1 Arg399Gln (rs25487), XRCC3 Thr241Met (rs861539), XPD Lys751Gln (rs13181), and OGG1 Ser326Cys (rs1052133) gene polymorphisms in 386 healthy individuals residing in the central region of Saudi Arabia and compare them with HapMap and other populations. The genotype and allele frequencies of the four DNA repair gene loci in central Saudi population showed a distinctive pattern. Furthermore, comparison of polymorphisms in these genes with other populations also showed a unique pattern for the central Saudi population. To the best of our knowledge, this is the first report that deals with these DNA repair gene polymorphisms among the central Saudi population.

  10. The Preference for Error-Free or Error-Prone Postreplication Repair in Saccharomyces cerevisiae Exposed to Low-Dose Methyl Methanesulfonate Is Cell Cycle Dependent

    PubMed Central

    Huang, Dongqing; Piening, Brian D.

    2013-01-01

    Cells employ error-free or error-prone postreplication repair (PRR) processes to tolerate DNA damage. Here, we present a genome-wide screen for sensitivity to 0.001% methyl methanesulfonate (MMS). This relatively low dose is of particular interest because wild-type cells exhibit no discernible phenotypes in response to treatment, yet PRR mutants are unique among repair mutants in their exquisite sensitivity to 0.001% MMS; thus, low-dose MMS treatment provides a distinctive opportunity to study postreplication repair processes. We show that upon exposure to low-dose MMS, a PRR-defective rad18Δ mutant stalls into a lengthy G2 arrest associated with the accumulation of single-stranded DNA (ssDNA) gaps. Consistent with previous results following UV-induced damage, reactivation of Rad18, even after prolonged G2 arrest, restores viability and genome integrity. We further show that PRR pathway preference in 0.001% MMS depends on timing and context; cells preferentially employ the error-free pathway in S phase and do not require MEC1-dependent checkpoint activation for survival. However, when PRR is restricted to the G2 phase, cells utilize REV3-dependent translesion synthesis, which requires a MEC1-dependent delay and results in significant hypermutability. PMID:23382077

  11. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells.

    PubMed

    Guo, Ge; Wang, Wei; Bradley, Allan

    2004-06-24

    Phenotype-driven recessive genetic screens in diploid organisms require a strategy to render the mutation homozygous. Although homozygous mutant mice can be generated by breeding, a reliable method to make homozygous mutations in cultured cells has not been available, limiting recessive screens in culture. Cultured embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Here we have exploited the high rate of mitotic recombination in Bloom's syndrome protein (Blm)-deficient ES cells to generate a genome-wide library of homozygous mutant cells from heterozygous mutations induced with a revertible gene trap retrovirus. We have screened this library for cells with defects in DNA mismatch repair (MMR), a system that detects and repairs base-base mismatches. We demonstrate the recovery of cells with homozygous mutations in known and novel MMR genes. We identified Dnmt1(ref. 5) as a novel MMR gene and confirmed that Dnmt1-deficient ES cells exhibit micro-satellite instability, providing a mechanistic explanation for the role of Dnmt1 in cancer. The combination of insertional mutagenesis in Blm-deficient ES cells establishes a new approach for phenotype-based recessive genetic screens in ES cells.

  12. Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair

    PubMed Central

    Alcalay, Myriam; Meani, Natalia; Gelmetti, Vania; Fantozzi, Anna; Fagioli, Marta; Orleth, Annette; Riganelli, Daniela; Sebastiani, Carla; Cappelli, Enrico; Casciari, Cristina; Sciurpi, Maria Teresa; Mariano, Angela Rosa; Minardi, Simone Paolo; Luzi, Lucilla; Muller, Heiko; Di Fiore, Pier Paolo; Frosina, Guido; Pelicci, Pier Giuseppe

    2003-01-01

    Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins. PMID:14660751

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

    PubMed Central

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

    2016-01-01

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

  14. Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

    SciTech Connect

    Oorschot, Bregje van; Hovingh, Suzanne E.; Moerland, Perry D.; Medema, Jan Paul; Stalpers, Lukas J.A.; Vrieling, Harry; Franken, Nicolaas A.P.

    2014-03-01

    Purpose: To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Methods and Materials: Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Results: Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Conclusions: Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

  15. Risk of childhood leukemia associated with diagnostic irradiation and polymorphisms in DNA repair genes.

    PubMed Central

    Infante-Rivard, C; Mathonnet, G; Sinnett, D

    2000-01-01

    The purpose of the study was to measure risk of childhood acute lymphoblastic leukemia associated with reported postnatal diagnostic X rays and to determine if it was modified in the presence of variants in genes involved in DNA repair. We conducted a population-based case-control study with 491 cases and 491 healthy controls among children 0-9 years of age at diagnosis. To evaluate gene-environment interaction, we used a subgroup of 129 cases. The adjusted odds ratio (OR) for one reported postnatal child X ray versus none was 1.04 [95% confidence interval (CI), 0.72-1.49], whereas the OR for two or more X rays was 1.61 (CI, 1.13-2.28). Among girls, the former ORs were 1.14 (CI, 0.66-1.96) and 2.26 (1. 20-4.23), respectively. Among girls who carried the hMSH3 [exon (ex) 23] variant, the ORs were 3.33 (CI, 0.75-14.82) for one X ray and 0. 27 (CI, 0.05-1.57) for two or more X rays, whereas among those who carried the XRCCI (ex 6) variant, the ORs were 1.45 (0.11-19.08) and 6.66 (0.78-56.63), respectively. On the other hand, at low levels of exposure, boys seemed protected by the variant hMLH1 (ex 8). The latter results must be interpreted with caution but suggest that the effect of diagnostic X rays could be modified by variants in repair genes according to sex. Few studies have evaluated the risk of postnatal diagnostic irradiation, which was moderately strong here; we are not aware of any studies that also considered the effect of polymorphisms in DNA repair genes. Based on the present results, both aspects deserve further study. PMID:10856021

  16. DNA methylation changes in plasticity genes accompany the formation and maintenance of memory.

    PubMed

    Halder, Rashi; Hennion, Magali; Vidal, Ramon O; Shomroni, Orr; Rahman, Raza-Ur; Rajput, Ashish; Centeno, Tonatiuh Pena; van Bebber, Frauke; Capece, Vincenzo; Garcia Vizcaino, Julio C; Schuetz, Anna-Lena; Burkhardt, Susanne; Benito, Eva; Navarro Sala, Magdalena; Javan, Sanaz Bahari; Haass, Christian; Schmid, Bettina; Fischer, Andre; Bonn, Stefan

    2016-01-01

    The ability to form memories is a prerequisite for an organism's behavioral adaptation to environmental changes. At the molecular level, the acquisition and maintenance of memory requires changes in chromatin modifications. In an effort to unravel the epigenetic network underlying both short- and long-term memory, we examined chromatin modification changes in two distinct mouse brain regions, two cell types and three time points before and after contextual learning. We found that histone modifications predominantly changed during memory acquisition and correlated surprisingly little with changes in gene expression. Although long-lasting changes were almost exclusive to neurons, learning-related histone modification and DNA methylation changes also occurred in non-neuronal cell types, suggesting a functional role for non-neuronal cells in epigenetic learning. Finally, our data provide evidence for a molecular framework of memory acquisition and maintenance, wherein DNA methylation could alter the expression and splicing of genes involved in functional plasticity and synaptic wiring.

  17. Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

    PubMed

    Creelman, R A; Tierney, M L; Mullet, J E

    1992-06-01

    Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.

  18. Isolation of novel human and mouse genes of the recA/RAD51 recombination-repair gene family.

    PubMed Central

    Cartwright, R; Dunn, A M; Simpson, P J; Tambini, C E; Thacker, J

    1998-01-01

    Genes from the recA/RAD51 family play essential roles in homologous recombination in all organisms. Using sequence homologies from eukaryotic members of this family we have identified fragments of two additional mammalian genes with homology to RAD51. Cloning the full-length cDNAs for both human and mouse genes showed that the sequences are highly conserved, and that the predicted proteins have characteristic features of this gene family. One of the novel genes (R51H2) occurs in two forms in human cDNA, differing extensively at the 3' end, probably due to an unusual form of alternative splicing. The new genes (R51H2 and R51H3) were mapped to human chromosomes 14q23-24 and 17q1.2, respectively. Expression studies showed that R51H2 is expressed at lower levels than R51H3 , but that expression of both genes occurs at elevated levels in the testis compared with other tissues. The combination of gene structure conservation and the transcript expression patterns suggests that these new members of the recA/RAD51 family may also function in homologous recombination-repair pathways. PMID:9512535

  19. Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

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

    PubMed Central

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

    2015-01-01

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

  1. Ets-1 regulates its target genes mainly by DNA methylation in human ovarian cancer.

    PubMed

    Wan, S M; Peng, P; Guan, T

    2013-11-01

    Ovarian cancer is the second most common gynaecological cancer worldwide, and its molecular mechanism has not been completely understood. Ets-1 is a member of the Ets transcription family and can play important roles in the regulation of extracellular matrix remodelling, invasion, angiogenesis and drug resistance in several malignancies, including ovarian cancer. In the current study, we downloaded two datasets from Gene Expression Omnibus database and sought to explore the regulation mechanism of Ets-1 in ovarian cancer by computational analysis of gene expression profiles. Microarray analysis identified a total of 548 genes that were regulated by Ets-1 in ovarian cancer. Functional annotation of these genes revealed that Ets-1 may be involved in several biological processes, both physiological and pathological, such as system development, response to stimulus, vascular endothelial growth factor (VEGF) production, morphogenesis, cell proliferation, cell adhesion and signal transduction. Further, DNA methylation analysis of the DEGs found that 26.5% (145) of them were differentially methylated genes in ovarian cancer. Our results provide insight into the mechanism of Ets-1 regulating the transcription of its target genes in the complex and multistep process of ovarian cancer progression.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  4. Genome wide analysis of DNA methylation and gene expression changes in the mouse lung following subchronic arsenate exposure

    EPA Science Inventory

    Alterations in DNA methylation have been proposed as a mechanism for the complex toxicological effects of arsenic. In this study, whole genome DNA methylation and gene expression changes were evaluated in lungs from female mice exposed for 90 days to 50 ppm arsenate (As) in drink...

  5. Dietary selenium intake increases exon-specific DNA methylation of p53 gene in rat liver and colon mucosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The regulation of site-specific DNA methylation of tumor suppressor genes has been considered as a leading mechanism by which certain nutrients exert their anticancer property. Our previous studies suggest that dietary selenium (Se) may alter DNA methylation, and the purpose of this study was to inv...

  6. Dietary selenomethionine intake increases exon-specific DNA methylation of p53 gene in rat liver and colon mucosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The regulation of site-specific DNA methylation of tumor suppressor genes has been considered as a leading mechanism by which certain nutrients exert their anticancer property. Our previous studies suggest that dietary selenium (Se) may alter DNA methylation, and the purpose of this study was to inv...

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Barrachina, Marta; Ferrer, Isidre

    2009-08-01

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

  9. Oxytocin receptor gene methylation: converging multilevel evidence for a role in social anxiety.

    PubMed

    Ziegler, Christiane; Dannlowski, Udo; Bräuer, David; Stevens, Stephan; Laeger, Inga; Wittmann, Hannah; Kugel, Harald; Dobel, Christian; Hurlemann, René; Reif, Andreas; Lesch, Klaus-Peter; Heindel, Walter; Kirschbaum, Clemens; Arolt, Volker; Gerlach, Alexander L; Hoyer, Jürgen; Deckert, Jürgen; Zwanzger, Peter; Domschke, Katharina

    2015-05-01

    Social anxiety disorder (SAD) is a commonly occurring and highly disabling disorder. The neuropeptide oxytocin and its receptor (OXTR) have been implicated in social cognition and behavior. This study-for the first time applying a multilevel epigenetic approach-investigates the role of OXTR gene methylation in categorical, dimensional, and intermediate neuroendocrinological/neural network phenotypes of social anxiety. A total of 110 unmedicated patients with SAD and matched 110 controls were analyzed for OXTR methylation by direct sequencing of sodium bisulfite-converted DNA extracted from whole blood. Furthermore, OXTR methylation was investigated regarding SAD-related traits (Social Phobia Scale (SPS) and Social Interaction Anxiety Scale (SIAS)), salivary cortisol response during the Trier social stress test (TSST), and amygdala responsiveness to social phobia related verbal stimuli using fMRI. Significantly decreased OXTR methylation particularly at CpG Chr3: 8 809 437 was associated with (1) the categorical phenotype of SAD (p<0.001, Cohen's d=0.535), (2) increased SPS and SIAS scores (p<0.001), (3) increased cortisol response to the TSST (p=0.02), and (4) increased amygdala responsiveness during social phobia-related word processing (right: p(corr)<0.001; left: p(corr)=0.005). Assuming that decreased OXTR methylation confers increased OXTR expression, the present finding may reflect a compensatory upregulation for pathologically reduced oxytocin levels or a causally relevant increased OXTR activation in SAD and related traits. OXTR methylation patterns might thus serve as peripheral surrogates of oxytocin tone and aid in establishing accessible biomarkers of SAD risk allowing for indicated preventive interventions and personalized treatment approaches targeting the oxytocin system.

  10. DNA Methylation of Lipid-Related Genes Affects Blood Lipid Levels

    PubMed Central

    Pfeiffer, Liliane; Wahl, Simone; Pilling, Luke C.; Reischl, Eva; Sandling, Johanna K.; Kunze, Sonja; Holdt, Lesca M.; Kretschmer, Anja; Schramm, Katharina; Adamski, Jerzy; Klopp, Norman; Illig, Thomas; Hedman, Åsa K.; Roden, Michael; Hernandez, Dena G.; Singleton, Andrew B.; Thasler, Wolfgang E.; Grallert, Harald; Gieger, Christian; Herder, Christian; Teupser, Daniel; Meisinger, Christa; Spector, Timothy D.; Kronenberg, Florian; Prokisch, Holger; Melzer, David; Peters, Annette; Deloukas, Panos; Ferrucci, Luigi; Waldenberger, Melanie

    2016-01-01

    Background Epigenetic mechanisms might be involved in the regulation of interindividual lipid level variability and thus may contribute to the cardiovascular risk profile. The aim of this study was to investigate the association between genome-wide DNA methylation and blood lipid levels high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol. Observed DNA methylation changes were also further analyzed to examine their relationship with previous hospitalized myocardial infarction. Methods and Results Genome-wide DNA methylation patterns were determined in whole blood samples of 1776 subjects of the Cooperative Health Research in the Region of Augsburg F4 cohort using the Infinium HumanMethylation450 BeadChip (Illumina). Ten novel lipid-related CpG sites annotated to various genes including ABCG1, MIR33B/SREBF1, and TNIP1 were identified. CpG cg06500161, located in ABCG1, was associated in opposite directions with both high-density lipoprotein cholesterol (β coefficient=−0.049; P=8.26E-17) and triglyceride levels (β=0.070; P=1.21E-27). Eight associations were confirmed by replication in the Cooperative Health Research in the Region of Augsburg F3 study (n=499) and in the Invecchiare in Chianti, Aging in the Chianti Area study (n=472). Associations between triglyceride levels and SREBF1 and ABCG1 were also found in adipose tissue of the Multiple Tissue Human Expression Resource cohort (n=634). Expression analysis revealed an association between ABCG1 methylation and lipid levels that might be partly mediated by ABCG1 expression. DNA methylation of ABCG1 might also play a role in previous hospitalized myocardial infarction (odds ratio, 1.15; 95% confidence interval=1.06–1.25). Conclusions Epigenetic modifications of the newly identified loci might regulate disturbed blood lipid levels and thus contribute to the development of complex lipid-related diseases. PMID:25583993

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

    PubMed

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

    2014-07-01

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

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

    PubMed

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

    2016-03-03

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

  13. [Phenylhexyl isothiocyanate induces gene p15 re-expression by regulating histone methylation and DNA demethylation in Molt-4 cells].

    PubMed

    Ma, Xu-Dong; Huang, Yi-Qun; Jiang, Shao-Hong; Zheng, Rui-Ji

    2010-06-01

    This study was aimed to investigate the regulatory effect of phenylhexyl isothiocyanate (PHI) on methylation of histone H3K4, H3K9 and demethylation of p15 gene in acute leukemia cell line Molt-4, and to explore the possible mechanism inducing re-expression of silent gene. The methylation status of histone H3K4, H3K9 and the expression of P15 protein in the Molt-4 cells treated with PHI were detected by Western blot; the methylation status of p15 gene in the Molt-4 cells before and after treatment with PHI was determined by methylation specific polymerase chain reaction (MSP); the expression level of p15 gene mRNA in Molt-4 cells treated with PHI was assayed by semiquantitative reverse transcription-PCR. The results indicated that the PHI could increase methylation of histone H3K4 and decrease methylation of histone H3K9 in concentration-and time-dependent manners. After treatment of Molt-4 cells with PHI for 5 days, the methylation of p15 gene was reduced, the significant hypermethylation of p15 gene was reversed, the silenced p15 gene re-expressed; the expressions of p15 mRNA and P15 protein were enhanced in concentration-dependent manner. It is concluded that probably through specifically regulating the methylation level of histone H3K4 and H3K9, the PHI causes the changes of chromosome space structure and results in the demethylation of CPG island in p15 gene, thereby induces the re-expression of p15 gene which was silenced.

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

    PubMed

    Song, Min-Ae; Brasky, Theodore M; Marian, Catalin; Weng, Daniel Y; Taslim, Cenny; Dumitrescu, Ramona G; Llanos, Adana A; Freudenheim, Jo L; Shields, Peter G

    2015-01-01

    Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer.

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

    PubMed Central

    Song, Min-Ae; Brasky, Theodore M; Marian, Catalin; Weng, Daniel Y; Taslim, Cenny; Dumitrescu, Ramona G; Llanos, Adana A; Freudenheim, Jo L; Shields, Peter G

    2015-01-01

    Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer. PMID:26680018

  16. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    PubMed

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

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

    PubMed

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

    2016-11-01

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

  18. Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability.

    PubMed

    Nowosielska, Anetta

    2007-01-01

    Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.

  19. Comprehensive interrogation of CpG island methylation in the gene encoding COMT, a key estrogen and catecholamine regulator

    PubMed Central

    2014-01-01

    Background The catechol-O-methyltransferase (COMT) enzyme has been widely studied due to its multiple roles in neurological functioning, estrogen biology, and methylation metabolic pathways. Numerous studies have investigated variation in the large COMT gene, with the majority focusing on single nucleotide polymorphisms (SNPs). This body of work has linked COMT genetic variation with a vast array of conditions, including several neurobehavioral disorders, pain sensitivity, and multiple human cancers. Based on COMT’s numerous biological roles and recent studies suggesting that methylation of the COMT gene impacts COMT gene expression, we comprehensively interrogated methylation in over 200 CpG dinucleotide sequences spanning the length of the COMT gene. Methods Using saliva-derived DNA from a non-clinical sample of human subjects, we tested for associations between COMT CpG methylation and factors reported to interact with COMT genetic effects, including demographic factors and alcohol use. Finally, we tested associations between COMT CpG methylation state and COMT gene expression in breast cancer cell lines. We interrogated >200 CpGs in 13 amplicons spanning the 5’ UTR to the last exon of the CpG dinucleotide-rich COMT gene in n = 48 subjects, n = 11 cell lines and 1 endogenous 18S rRNA control. Results With the exception of the CpG island in the 5’UTR and 1st exon, all other CpG islands were strongly methylated with typical dynamic ranges between 50-90%. In the saliva samples, methylation of multiple COMT loci was associated with socioeconomic status or ethnicity. We found associations between methylation at numerous loci and genotype at the functional Val 158 Met SNP (rs4680), and most of the correlations between methylation and demographic and alcohol use factors were Val 158 Met allele-specific. Methylation at several of these loci also associated with COMT gene expression in breast cancer cell lines. Conclusions We report the first comprehensive

  20. Rearrangement of RAG-1 recombinase gene in DNA-repair deficient ``wasted`` mice

    SciTech Connect

    Woloschak, G.E.; Libertin, C.R.; Weaver, P.; Churchill, M.; Chang-Liu, C.M.

    1993-11-01

    Mice recessive for the autosomal gene ``wasted`` wst display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (RAG-l/RAG-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed expression of RAG-1 mRNA in spinal cord (but not brain) of control mice; no expression of RAG-1 mRNA was detected in spinal cord or brain from wst/wst mice or their normal littermates (wst/{center_dot}mice). In thymus tissue, a small RAG-1 transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{center_dot}mice, a two-fold increase in RAG-1 mRNA was evident in thymus tissue. RAG-2 mRNA could only be detected in thymus tissue from wst/{center_dot} and not from wst/wst or parental control BCF{sub 1} mice. Southern blots revealed a rearrangement/deletion within the RAG-1 gene of affected wasted mice, not evident in known strain-specific parental or littermate controls. These results support the idea that the RAG-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  1. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    PubMed

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

  2. Identifying Significant Features in Cancer Methylation Data Using Gene Pathway Segmentation

    PubMed Central

    Hira, Zena M.; Gillies, Duncan F.

    2016-01-01

    In order to provide the most effective therapy for cancer, it is important to be able to diagnose whether a patient’s cancer will respond to a proposed treatment. Methylation profiling could contain information from which such predictions could be made. Currently, hypothesis testing is used to determine whether possible biomarkers for cancer progression produce statistically significant results. However, this approach requires the identification of individual genes, or sets of genes, as candidate hypotheses, and with the increasing size of modern microarrays, this task is becoming progressively harder. Exhaustive testing of small sets of genes is computationally infeasible, and so hypothesis generation depends either on the use of established biological knowledge or on heuristic methods. As an alternative machine learning, methods can be used to identify groups of genes that are acting together within sets of cancer data and associate their behaviors with cancer progression. These methods have the advantage of being multivariate and unbiased but unfortunately also rapidly become computationally infeasible as the number of gene probes and datasets increases. To address this problem, we have investigated a way of utilizing prior knowledge to segment microarray datasets in such a way that machine learning can be used to identify candidate sets of genes for hypothesis testing. A methylation dataset is divided into subsets, where each subset contains only the probes that relate to a known gene pathway. Each of these pathway subsets is used independently for classification. The classification method is AdaBoost with decision trees as weak classifiers. Since each pathway subset contains a relatively small number of gene probes, it is possible to train and test its classification accuracy quickly and determine whether it has valuable diagnostic information. Finally, genes from successful pathway subsets can be combined to create a classifier of high accuracy. PMID

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

    PubMed

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

    2014-07-01

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

  4. Diabetes causes multiple genetic alterations and downregulates expression of DNA repair genes in the prostate.

    PubMed

    Ye, Chunwei; Li, Xiaojuan; Wang, Yu; Zhang, Yuying; Cai, Mengyin; Zhu, Baoyi; Mu, Panwei; Xia, Xuan; Zhao, Yi; Weng, Jianping; Gao, Xin; Wen, Xingqiao

    2011-09-01

    The molecular impact of diabetes mellitus on prostate gland has not been elucidated. In this study, we performed a whole-genome cDNA microarray analysis using a streptozotocin-induced diabetic rat model to identify the effects of diabetes on the gene expression profiles in prostate. Our study shows that diabetes causes changes in the expression of multiple genes, particularly those related to cell proliferation and differentiation, oxidative stress, DNA damage repair, cell cycle checkpoints, angiogenesis and apoptosis. These findings were confirmed by real-time polymerase chain reaction and immunohistochemical staining using rat and human prostate tissue. We also used a cell culture model (human normal prostatic RWPE-1 cell line) to study the direct effect of high glucose. We found that high glucose caused increased intracellular oxidative stress and DNA damage, as well as downregulation of anti-oxidative enzymes and DNA damage repair genes MRE11 and XRCC3. Our findings provide important insights into understanding the pathogenesis of the diabetes-induced changes in prostate as well as identifying potential therapeutic targets for future studies.

  5. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    PubMed

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

  6. DNA mismatch repair gene MSH6 implicated in determining age at natural menopause

    PubMed Central

    Perry, John R.B.; Hsu, Yi-Hsiang; Chasman, Daniel I.; Johnson, Andrew D.; Elks, Cathy; Albrecht, Eva; Andrulis, Irene L.; Beesley, Jonathan; Berenson, Gerald S.; Bergmann, Sven; Bojesen, Stig E.; Bolla, Manjeet K.; Brown, Judith; Buring, Julie E.; Campbell, Harry; Chang-Claude, Jenny; Chenevix-Trench, Georgia; Corre, Tanguy; Couch, Fergus J.; Cox, Angela; Czene, Kamila; D'adamo, Adamo Pio; Davies, Gail; Deary, Ian J.; Dennis, Joe; Easton, Douglas F.; Engelhardt, Ellen G.; Eriksson, Johan G.; Esko, Tõnu; Fasching, Peter A.; Figueroa, Jonine D.; Flyger, Henrik; Fraser, Abigail; Garcia-Closas, Montse; Gasparini, Paolo; Gieger, Christian; Giles, Graham; Guenel, Pascal; Hägg, Sara; Hall, Per; Hayward, Caroline; Hopper, John; Ingelsson, Erik; Kardia, Sharon L.R.; Kasiman, Katherine; Knight, Julia A.; Lahti, Jari; Lawlor, Debbie A.; Magnusson, Patrik K.E.; Margolin, Sara; Marsh, Julie A.; Metspalu, Andres; Olson, Janet E.; Pennell, Craig E.; Polasek, Ozren; Rahman, Iffat; Ridker, Paul M.; Robino, Antonietta; Rudan, Igor; Rudolph, Anja; Salumets, Andres; Schmidt, Marjanka K.; Schoemaker, Minouk J.; Smith, Erin N.; Smith, Jennifer A.; Southey, Melissa; Stöckl, Doris; Swerdlow, Anthony J.; Thompson, Deborah J.; Truong, Therese; Ulivi, Sheila; Waldenberger, Melanie; Wang, Qin; Wild, Sarah; Wilson, James F; Wright, Alan F.; Zgaga, Lina; Ong, Ken K.; Murabito, Joanne M.; Karasik, David; Murray, Anna

    2014-01-01

    The length of female reproductive lifespan is associated with multiple adverse outcomes, including breast cancer, cardiovascular disease and infertility. The biological processes that govern the timing of the beginning and end of reproductive life are not well understood. Genetic variants are known to contribute to ∼50% of the variation in both age at menarche and menopause, but to date the known genes explain <15% of the genetic component. We have used genome-wide association in a bivariate meta-analysis of both traits to identify genes involved in determining reproductive lifespan. We observed significant genetic correlation between the two traits using genome-wide complex trait analysis. However, we found no robust statistical evidence for individual variants with an effect on both traits. A novel association with age at menopause was detected for a variant rs1800932 in the mismatch repair gene MSH6 (P = 1.9 × 10−9), which was also associated with altered expression levels of MSH6 mRNA in multiple tissues. This study contributes to the growing evidence that DNA repair processes play a key role in ovarian ageing and could be an important therapeutic target for infertility. PMID:24357391

  7. Are SNP-Smoking Association Studies Needed in Controls? DNA Repair Gene Polymorphisms and Smoking Intensity.

    PubMed

    Verde, Zoraida; Reinoso, Luis; Chicharro, Luis Miguel; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando; Gómez-Gallego, Félix; Santiago, Catalina

    2015-01-01

    Variations in tobacco-related cancers, incidence and prevalence reflect differences in tobacco consumption in addition to genetic factors. Besides, genes related to lung cancer risk could be related to smoking behavior. Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Common problems in genetic association studies, such as presence of gene-by-environment (G x E) correlation in the population, may reduce the validity of these designs. The main purpose of this study was to evaluate the independence assumption for selected SNPs and smoking behaviour in a cohort of 320 healthy Spanish smokers. We found an association between the wild type alleles of XRCC3 Thr241Met or KLC3 Lys751Gln and greater smoking intensity (OR = 12.98, 95% CI = 2.86-58.82 and OR=16.90, 95% CI=2.09-142.8; respectively). Although preliminary, the results of our study provide evidence that genetic variations in DNA-repair genes may influence both smoking habits and the development of lung cancer. Population-specific G x E studies should be carried out when genetic and environmental factors interact to cause the disease.

  8. Cloning and sequencing of the PIF gene involved in repair and recombination of yeast mitochondrial DNA.

    PubMed Central

    Foury, F; Lahaye, A

    1987-01-01

    The nuclear gene PIF of Saccharomyces cerevisiae is required for both repair of mitochondrial DNA (mtDNA) and recognition of a recombinogenic signal characterized by a 26-bp palindromic AT sequence in the ery region of mtDNA. This gene has been cloned in yeast by genetic complementation of pif mutants. Its chromosomal disruption does not destroy the genetic function of mitochondria. The nucleotide sequence of the 3.5-kb insert from a complementing plasmid reveals an open reading frame encoding a potential protein of 857 amino acids and Mr = 97,500. An ATP-binding domain is present in the central part of the gene and in the carboxy-terminal region a putative DNA-binding site is present. Its alpha helix-turn-alpha helix motif is found in DNA-binding proteins such as lambda and lactose repressors which recognize symmetric sequences. Significant amino acid homology is observed with yeast RAD3 and E. coli UvrD (helicase II) proteins which are required for excision repair of damaged DNA. Images Fig. 1. Fig. 2. PMID:3038524

  9. Antagonism of ultraviolet-light mutagenesis by the methyl-directed mismatch-repair system of Escherichia coli.

    PubMed Central

    Liu, H; Hewitt, S R; Hays, J B

    2000-01-01

    Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2.MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to "matched" photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC --> CTC and CTT --> CTC transitions. F' lacZ targets were mated from mut(+) donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu(+) mut(+) recipients, a range of UV fluences induced lac(+) revertant frequencies of 4-25 x 10(-8); these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd(-) defect, it appears not to involve transcription-coupled excision repair. In mut(+) umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m(2)) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5-10 x 10(-8). Thus, at UV doses too low to induce SOS functions, such as Umu(2)'D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial. PMID:10655206

  10. Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome.

    PubMed

    Rabinowicz, P D; Schutz, K; Dedhia, N; Yordan, C; Parnell, L D; Stein, L; McCombie, W R; Martienssen, R A

    1999-11-01

    The genomes of higher plants and animals are highly differentiated, and are composed of a relatively small number of genes and a large fraction of repetitive DNA. The bulk of this repetitive DNA constitutes transposable, and especially retrotransposable, elements. It has been hypothesized that most of these elements are heavily methylated relative to genes, but the evidence for this is controversial. We show here that repeat sequences in maize are largely excluded from genomic shotgun libraries by the selection of an appropriate host strain because of their sensitivity to bacterial restriction-modification systems. In contrast, unmethylated genic regions are preserved in these genetically filtered libraries if the insert size is less than the average size of genes. The representation of unique maize sequences not found in plant reference genomes is also greatly enriched. This demonstrates that repeats, and not genes, are the primary targets of methylation in maize. The use of restrictive libraries in genome shotgun sequencing in plant genomes should allow significant representation of genes, reducing the number of reactions required.

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

    PubMed Central

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

    2017-01-01

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

  12. Gene expression, methylation and neuropathology correlations at progressive supranuclear palsy risk loci.

    PubMed

    Allen, Mariet; Burgess, Jeremy D; Ballard, Travis; Serie, Daniel; Wang, Xue; Younkin, Curtis S; Sun, Zhifu; Kouri, Naomi; Baheti, Saurabh; Wang, Chen; Carrasquillo, Minerva M; Nguyen, Thuy; Lincoln, Sarah; Malphrus, Kimberly; Murray, Melissa; Golde, Todd E; Price, Nathan D; Younkin, Steven G; Schellenberg, Gerard D; Asmann, Yan; Ordog, Tamas; Crook, Julia; Dickson, Dennis; Ertekin-Taner, Nilüfer

    2016-08-01

    To determine the effects of single nucleotide polymorphisms (SNPs) identified in a genome-wide association study of progressive supranuclear palsy (PSP), we tested their association with brain gene expression, CpG methylation and neuropathology. In 175 autopsied PSP subjects, we performed associations between seven PSP risk variants and temporal cortex levels of 20 genes in-cis, within ±100 kb. Methylation measures were collected using reduced representation bisulfite sequencing in 43 PSP brains. To determine whether SNP/expression associations are due to epigenetic modifications, CpG methylation levels of associated genes were tested against relevant variants. Quantitative neuropathology endophenotypes were tested for SNP associations in 422 PSP subjects. Brain levels of LRRC37A4 and ARL17B were associated with rs8070723; MOBP with rs1768208 and both ARL17A and ARL17B with rs242557. Expression associations for LRRC37A4 and MOBP were available in an additional 100 PSP subjects. Meta-analysis revealed highly significant associations for PSP risk alleles of rs8070723 and rs1768208 with higher LRRC37A4 and MOBP brain levels, respectively. Methylation levels of one CpG in the 3' region of ARL17B associated with rs242557 and rs8070723. Additionally, methylation levels of an intronic ARL17A CpG associated with rs242557 and that of an intronic MOBP CpG with rs1768208. MAPT and MOBP region risk alleles also associated with higher levels of neuropathology. Strongest associations were observed for rs242557/coiled bodies and tufted astrocytes; and for rs1768208/coiled bodies and tau threads. These findings suggest that PSP variants at MAPT and MOBP loci may confer PSP risk via influencing gene expression and tau neuropathology. MOBP, LRRC37A4, ARL17A and ARL17B warrant further assessment as candidate PSP risk genes. Our findings have implications for the mechanism of action of variants at some of the top PSP risk loci.

  13. Polymorphisms in DNA repair genes and therapeutic outcomes of AML patients from SWOG clinical trials.

    PubMed

    Kuptsova, Nataliya; Kopecky, Kenneth J; Godwin, John; Anderson, Jeanne; Hoque, Ashraful; Willman, Cheryl L; Slovak, Marilyn L; Ambrosone, Christine B

    2007-05-01

    Repair of damage to DNA resulting from chemotherapy may influence drug toxicity and survival in response to treatment. We evaluated the role of polymorphisms in DNA repair genes APE1, XRCC1, ERCC1, XPD, and XRCC3 in predicting therapeutic outcomes of older adults with acute myeloid leukemia (AML) from 2 Southwest Oncology Group (SWOG) clinical trials. All patients received standard chemotherapy induction regimens. Using logistic and proportional hazards regression models, relationships between genotypes, haplotypes, and toxicities, response to induction therapy, and overall survival were evaluated. Patients with XPD Gln751C/Asp312G ('D') haplotype were more likely to have complete response (OR = 3.06; 95% CI, 1.44-6.70) and less likely to have resistant disease (OR = 0.32; 95%CI, 0.14-0.72) than patients with other haplotypes. ERCC1 polymorphisms were significantly associated with lung (P = .037) and metabolic (P = .041) toxicities, and patients with the XRCC3 241Met variant had reduced risk of liver toxicity (OR = 0.32; 95%CI, 0.11-0.95). Significant associations with other toxicities were also found for variant XPD genotypes/haplotypes. These data from clinical trials of older patients treated for AML indicate that variants in DNA repair pathways may have an impact on both outcomes of patients and toxicities associated with treatments. With validation of results in larger samples, these findings could lead to optimizing individual chemotherapy options.

  14. Identification of Methyl Halide-Utilizing Genes in the Methyl Bromide-Utilizing Bacterial Strain IMB-1 Suggests a High Degree of Conservation of Methyl Halide-Specific Genes in Gram-Negative Bacteria

    USGS Publications Warehouse

    Woodall, C.A.; Warner, K.L.; Oremland, R.S.; Murrell, J.C.; McDonald, I.R.

    2001-01-01

    Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source. A single cmu gene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partial metF. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyl-transferase motif. However, cmuB, identified in M. chloromethanicum and H. chloromethanicum, was not detected in IMB-1.

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

    PubMed

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

    2008-11-01

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

  16. Histone Methylation Dynamics and Gene Regulation Occur through the Sensing of One-Carbon Metabolism.

    PubMed

    Mentch, Samantha J; Mehrmohamadi, Mahya; Huang, Lei; Liu, Xiaojing; Gupta, Diwakar; Mattocks, Dwight; Gómez Padilla, Paola; Ables, Gene; Bamman, Marcas M; Thalacker-Mercer, Anna E; Nichenametla, Sailendra N; Locasale, Jason W

    2015-11-03

    S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) link one-carbon metabolism to methylation status. However, it is unknown whether regulation of SAM and SAH by nutrient availability can be directly sensed to alter the kinetics of key histone methylation marks. We provide evidence that the status of methionine metabolism is sufficient to determine levels of histone methylation by modulating SAM and SAH. This dynamic interaction led to rapid changes in H3K4me3, altered gene transcription, provided feedback regulation to one-carbon metabolism, and could be fully recovered upon restoration of methionine. Modulation of methionine in diet led to changes in metabolism and histone methylation in the liver. In humans, methionine variability in fasting serum was commensurate with concentrations needed for these dynamics and could be partly explained by diet. Together these findings demonstrate that flux through methionine metabolism and the sensing of methionine availability may allow direct communication to the chromatin state in cells.

  17. A systematic gene-gene and gene-environment interaction analysis of DNA repair genes XRCC1, XRCC2, XRCC3, XRCC4, and oral cancer risk.

    PubMed

    Yang, Cheng-Hong; Lin, Yu-Da; Yen, Ching-Yui; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2015-04-01

    Oral cancer is the sixth most common cancer worldwide with a high mortality rate. Biomarkers that anticipate susceptibility, prognosis, or response to treatments are much needed. Oral cancer is a polygenic disease involving complex interactions among genetic and environmental factors, which require multifaceted analyses. Here, we examined in a dataset of 103 oral cancer cases and 98 controls from Taiwan the association between oral cancer risk and the DNA repair genes X-ray repair cross-complementing group (XRCCs) 1-4, and the environmental factors of smoking, alcohol drinking, and betel quid (BQ) chewing. We employed logistic regression, multifactor dimensionality reduction (MDR), and hierarchical interaction graphs for analyzing gene-gene (G×G) and gene-environment (G×E) interactions. We identified a significantly elevated risk of the XRCC2 rs2040639 heterozygous variant among smokers [adjusted odds ratio (OR) 3.7, 95% confidence interval (CI)=1.1-12.1] and alcohol drinkers [adjusted OR=5.7, 95% CI=1.4-23.2]. The best two-factor based G×G interaction of oral cancer included the XRCC1 rs1799782 and XRCC2 rs2040639 [OR=3.13, 95% CI=1.66-6.13]. For the G×E interaction, the estimated OR of oral cancer for two (drinking-BQ chewing), three (XRCC1-XRCC2-BQ chewing), four (XRCC1-XRCC2-age-BQ chewing), and five factors (XRCC1-XRCC2-age-drinking-BQ chewing) were 32.9 [95% CI=14.1-76.9], 31.0 [95% CI=14.0-64.7], 49.8 [95% CI=21.0-117.7] and 82.9 [95% CI=31.0-221.5], respectively. Taken together, the genotypes of XRCC1 rs1799782 and XRCC2 rs2040639 DNA repair genes appear to be significantly associated with oral cancer. These were enhanced by exposure to certain environmental factors. The observations presented here warrant further research in larger study samples to examine their relevance for routine clinical care in oncology.

  18. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium

    SciTech Connect

    Pelch, Katherine E.; Tokar, Erik J.; Merrick, B. Alex; Waalkes, Michael P.

    2015-08-01

    Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular

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

    PubMed

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

    2015-06-04

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2008-12-31

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

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

    PubMed

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

    2016-04-01

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

  5. Smad gene expression in pulmonary fibroblasts: indications for defective ECM repair in COPD

    PubMed Central

    Zandvoort, Andre; Postma, Dirkje S; Jonker, Marnix R; Noordhoek, Jacobien A; Vos, Johannes TWM; Timens, Wim

    2008-01-01

    Background Chronic Obstructive Pulmonary Disease (COPD) is characterized by defective extracellular matrix (ECM) turnover as a result of prolonged cigarette smoking. Fibroblasts have a central role in ECM turnover. The TGFβ induced Smad pathway provides intracellular signals to regulate ECM production. We address the following hypothesis: fibroblasts have abnormal expression of genes in the Smad pathway in COPD, resulting in abnormal proteoglycan modulation, the ground substance of ECM. Methods We compared gene expression of the Smad pathway at different time points after stimulation with TGFβ, TNF or cigarette smoke extract (CSE) in pulmonary fibroblasts of GOLD stage II and IV COPD patients, and controls. Results Without stimulation, all genes were similarly expressed in control and COPD fibroblasts. TGFβ stimulation: downregulation of Smad3 and upregulation of Smad7 occurred in COPD and control fibroblasts, indicating a negative feedback loop upon TGFβ stimulation. CSE hardly influenced gene expression of the TGFβ-Smad pathway in control fibroblasts, whereas it reduced Smad3 and enhanced Smad7 gene expression in COPD fibroblasts. Furthermore, decorin gene expression decreased by all stimulations in COPD but not in control fibroblasts. Conclusion Fibroblasts of COPD patients and controls differ in their regulation of the Smad pathway, the contrast being most pronounced under CSE exposure. This aberrant responsiveness of COPD fibroblasts to CSE might result in an impaired tissue repair capability and is likely important with regard to the question why only a subset of smokers demonstrates an excess ECM destruction under influence of cigarette smoking. PMID:19087346

  6. Damage, Repair, and Mutagenesis in Nuclear Genes after Mouse Forebrain Ischemia–Reperfusion

    PubMed Central

    Liu, Philip K.; Hsu, Chung Y.; Dizdaroglu, Miral; Floyd, Robert A.; Kow, Yoke W.; Karakaya, Asuman; Rabow, Lois E.; Cui, Jian-K.

    2009-01-01

    To determine whether oxidative stress after cerebral ischemia–reperfusion affects genetic stability in the brain, we studied mutagenesis after forebrain ischemia–reperfusion in Big Blue transgenic mice (male C57BL/6 strain) containing a reporter lacI gene, which allows detection of mutation frequency. The frequency of mutation in this reporter lacI gene increased from 1.5 to 7.7 (per 100,000) in cortical DNA after 30 min of forebrain ischemia and 8 hr of reperfusion and remained elevated at 24 hr reperfusion. Eight DNA lesions that are characteristic of DNA damage mediated by free radicals were detected. Four mutagenic lesions (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, 5-hydroxycytosine, and 8-hydroxyguanine) examined by gas chromatography/mass spectrometry and one corresponding 8-hydroxy-2′-deoxyguanosine by a method of HPLC with electrochemical detection increased in cortical DNA two- to fourfold (p < 0.05) during 10–20 min of reperfusion. The damage to γ-actin and DNA polymerase-β genes was detected within 20 min of reperfusion based on the presence of formamidopyrimidine DNA N-glycosylase-sensitive sites. These genes became resistant to the glycosylase within 4–6 hr of reperfusion, suggesting a reduction in DNA damage and presence of DNA repair in nuclear genes. These results suggest that nuclear genes could be targets of free radicals. PMID:8824320

  7. Functional role of DNA mismatch repair gene PMS2 in prostate cancer cells.

    PubMed

    Fukuhara, Shinichiro; Chang, Inik; Mitsui, Yozo; Chiyomaru, Takeshi; Yamamura, Soichiro; Majid, Shahana; Saini, Sharanjot; Deng, Guoren; Gill, Ankurpreet; Wong, Darryn K; Shiina, Hiroaki; Nonomura, Norio; Lau, Yun-Fai C; Dahiya, Rajvir; Tanaka, Yuichiro

    2015-06-30

    DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells.

  8. Histone H1 Is Dispensable for Methylation-Associated Gene Silencing in Ascobolus immersus and Essential for Long Life Span

    PubMed Central

    Barra, Jose L.; Rhounim, Laïla; Rossignol, Jean-Luc; Faugeron, Godeleine

    2000-01-01

    A gene encoding a protein that shows sequence similarity with the histone H1 family only was cloned in Ascobolus immersus. The deduced peptide sequence presents the characteristic three-domain structure of metazoan linker histones, with a central globular region, an N-terminal tail, and a long positively charged C-terminal tail. By constructing an artificial duplication of this gene, named H1, it was possible to methylate and silence it by the MIP (methylation induced premeiotically) process. This resulted in the complete loss of the Ascobolus H1 histone. Mutant strains lacking H1 displayed normal methylation-associated gene silencing, underwent MIP, and showed the same methylation-associated chromatin modifications as did wild-type strains. However, they displayed an increased accessibility of micrococcal nuclease to chromatin, whether DNA was methylated or not, and exhibited a hypermethylation of the methylated genome compartment. These features are taken to imply that Ascobolus H1 histone is a ubiquitous component of chromatin which plays no role in methylation-associated gene silencing. Mutant strains lacking histone H1 reproduced normally through sexual crosses and displayed normal early vegetative growth. However, between 6 and 13 days after germination, they abruptly and consistently stopped growing, indicating that Ascobolus H1 histone is necessary for long life span. This constitutes the first observation of a physiologically important phenotype associated with the loss of H1. PMID:10594009

  9. Methylation of multiple genes in hepatitis C virus associated hepatocellular carcinoma

    PubMed Central

    Zekri, Abdel-Rahman N.; Bahnasy, Abeer A.; Shoeab, Fatma elzahraa M.; Mohamed, Waleed S.; El-Dahshan, Dina H.; Ali, Fahmey T.; Sabry, Gilane M.; Dasgupta, Nairajana; Daoud, Sayed S.

    2013-01-01

    We studied promoter methylation (PM) of 11 genes in Peripheral Blood Lymphocytes (PBLs) and tissues of hepatitis C virus (HCV) associated hepatocellular carcinoma (HCC) and chronic hepatitis (CH) Egyptian patients. The present study included 31 HCC with their ANT, 38 CH and 13 normal hepatic tissue (NHT) samples. In all groups, PM of APC, FHIT, p15, p73, p14, p16, DAPK1, CDH1, RARβ, RASSF1A, O6MGMT was assessed by methylation-specific PCR (MSP). APC and O6-MGMT protein expression was assessed by immunohistochemistry (IHC) in the studied HCC and CH (20 samples each) as well as in a different HCC and CH set for confirmation of MSP results. PM was associated with progression from CH to HCC. Most genes showed high methylation frequency (MF) and the methylation index (MI) increased with disease progression. MF of p14, p73, RASSF1A, CDH1 and O6MGMT was significantly higher in HCC and their ANT. MF of APC was higher in CH. We reported high concordance between MF in HCC and their ANT, MF in PBL and CH tissues as well as between PM and protein expression of APC and O6MGMT. A panel of 4 genes (APC, p73, p14, O6MGMT) classifies the cases independently into HCC and CH with high accuracy (89.9%), sensitivity (83.9%) and specificity (94.7%). HCV infection may contribute to hepatocarcinogenesis through enhancing PM of multiple genes. PM of APC occurs early in the cascade while PM of p14, p73, RASSF1A, RARB, CDH1 and O6MGMT are late changes. A panel of APC, p73, p14, O6-MGMT could be used in monitoring CH patients for early detection of HCC. Also, we found that, the methylation status is not significantly affected by whether the tissue was from the liver or PBL, indicating the possibility of use PBL as indicator to genetic profile instead of liver tissue regardless the stage of disease. PMID:25685469

  10. Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    PubMed Central

    Stephenson, Stacy Ann-Marie; Brown, Paul D.

    2016-01-01

    Urinary tract infections (UTI) are among the most frequently encountered infections in clinical practice globally. Predominantly a burden among female adults and infants, UTIs primarily caused by uropathogenic Escherichia coli (UPEC) results in high morbidity and fiscal health strains. During pathogenesis, colonization of the urinary tract via fimbrial adhesion to mucosal cells is the most critical point in infection and has been linked to DNA methylation. Furthermore, with continuous exposure to antibiotics as the standard therapeutic strategy, UPEC has evolved to become highly adaptable in circumventing the effect of antimicrobial agents and host defenses. Hence, the need for alternative treatment strategies arises. Since differential DNA methylation is observed as a critical precursor to virulence in various pathogenic bacteria, this body of work sought to assess the influence of the DNA adenine methylase (dam) gene on gene expression and cellular adhesion in UPEC and its potential as a therapeutic target. To monitor the influence of dam on attachment and FQ resistance, selected UPEC dam mutants created via one-step allelic exchange were transformed with cloned qnrA and dam complement plasmid for comparative analysis of growth rate, antimicrobial susceptibility, biofilm formation, gene expression, and mammalian cell attachment. The absence of DNA methylation among dam mutants was apparent. Varying deficiencies in cell growth, antimicrobial resistance and biofilm formation, alongside low-level increases in gene expression (recA and papI), and adherence to HEK-293 and HTB-9 mammalian cells were also detected as a factor of SOS i