Development and application of a single mouse embryo DNA methylation-detection assayKwan, Chun-kit, Peter, 關駿傑 January 2014 (has links)
During preimplantation embryonic development, imprinting genes are susceptible to methylation changes by artificial manipulation, which may lead to developmental abnormalities. In addition, environmental endocrine disruptors (EDs) in everyday household products are also found to perturb fertility development and cause epigenetic aberrations. While embryo supply is scarce and conventional epigenetic studies require embryos in vast amount, an assay was developed in this study to examine the methylation statuses of imprinting genes using DNA from single mouse blastocysts cultured in-vitro or exposed to EDs. Promoter CpG methylation patterns of three imprinting genes, small nuclear ribonucleoprotein polypeptide N (SNRPN), paternally expressed 3 (Peg3), and potassium voltage-gated channel 1 overlapping transcript 1 (Kcnq1ot1), were examined from genomic DNA of a single mouse blastocyst. The genomic DNA was isolated and treated with bisulfite modification to preserve the methylation statuses. Afterwards, the DNA was subjected to whole genome amplification (WGA). Methylation-specific polymerase chain reaction (methyl-PCR) was performed with allele-specific primers; the amplicons were cloned and sequenced. CpG methylations in SNRPN, Peg3 and Kcnq1ot1 showed no statistical significant difference (P>0.05; Mann Whitney U test) in both parental alleles between a single genomic-amplified blastocyst and 20 non-amplified blastocysts, indicating no artifact was being introduced during the WGA procedure. Using the assay, it was revealed that blastocysts cultured in-vitro expressed slight but nonsignificant deviation in methylation rates to both parental alleles of SNRPN and Kcnq1ot1 except in single blastocysts, which displayed significant loss in maternal methylation on SNRPN upon culturing. On the other hand, paternal methylation profile of Peg3 appeared unaffected, suggesting resistance to methylation perturbations induced by in-vitro culturing. Despite that there was no significant difference in overall methylation rates between in-vivo or in-vitro developed blastocysts, certain CpG residues appeared to displayed significant loss of methylation (LOM) or gain of methylation (GOM) induced by in-vitro culture in all three genes being studied. Furthermore, using the developed, assay the epigenetic effects of three endocrine disruptors, simazine, propiconazole, and cadmium chloride (CdCl2) on in-vitro cultured single blastocysts were revealed. When compared to blastocysts cultured with KSOM+AA medium as controls, CdCl2-treated blastocysts displayed the most methylation aberrations in both alleles and within particular CpG residues, possibly due to its dual effect in both hypermethylation and hypomethylation across the methylome. Both simazine- and propiconazole -treated blastocysts displayed overall methylation significant defects were observed within particular CpG residues. Overall, the assay used in this study allowed the comprehensive investigation of methylome from the DNA extracted from a single blastocyst.defects resembled to those blastocysts cultured with KSOM+AA medium alone but / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy
Contrôle épigénétique du risque de montaison chez une plante de grande culture : la betterave sucrière : mise au point d'une stratégie de caractérisation d'épiallèles associés à la sensibilité à la montaison en vue de l'élaboration d'un test de sélection / Epigenetic control of the bolting risk in a crop plant : sugar-beet : the development of a strategy to characterize epialleles associated with bolting sensivity, with a view to implementation as a selection toolGentil, Marie-Véronique 27 January 2009 (has links)
Chez les plantes, les processus de développement global et de plasticité développementale sont contrôlés par des mécanismes épigénétiques. La méthylation de l’ADN peut présenter un polymorphisme (épiallèles) qui est une source possible de biomarqueurs pour la sélection de génotypes d’intérêt agronomique. Pourtant, la recherche de tels biomarqueurs n’a pas encore été initiée. Dans ce contexte, nos objectifs ont concerné l'élaboration d'une stratégie pour la mise en évidence d’un contrôle épigénétique lors d’un processus développemental chez la betterave sucrière (Beta vulgaris altissima), ainsi que la recherche des biomarqueurs épigénétiques associés. Cette stratégie a d’abord été appliquée à la morphogenèse in vitro, sur trois lignées cellulaires de betterave sucrière. Une relation a pu être établie entre le niveau de méthylation de l’ADN et les propriétés morphogénétiques des lignées. Des biomarqueurs de morphogenèse in vitro ont ainsi été identifiés. La même stratégie a ensuite été appliquée in planta à la même espèce. L'existence d'un contrôle épigénétique lors de la vernalisation et de la dévernalisation chez plusieurs hybrides de betterave sucrière, avec des sensibilités à la montaison différentes, a été démontrée. Nous suggérons que l’amplitude et la cinétique des variations épigénétiques contrôlent l’induction de la montaison et sa rapidité, confirmant ainsi le rôle de la méthylation de l’ADN dans ce processus. Les loci cibles de ces remaniements de la méthylation de l'ADN lors de la vernalisation ont été définis. Un criblage a enfin permis d’identifier de potentiels biomarqueurs épigénétiques de la sensibilité à la montaison en vue de la mise au point d’un futur test de sélection agronomique. / In plants, the processes of global development and of developmental plasticity are controlled by epigenetic mechanisms. Polymorphism in DNA methylation (leading to epialleles) is a possible source of biomarkers for the selection of genotypes of agronomic interest. Until now, however, the search for such biomarkers has not been undertaken. Against this background, our objectives were to develop a strategy to investigate the existence of epigenetic control during a developmental process in sugar-beet (Beta vulgaris altissima) and to search for associated epigenetic biomarkers. The strategy was first applied to three sugar-beet cell lines, where we were able to established a relationship between the level of DNA methylation and the morphogenetic statue of the lines, and thus to identified a number of biomarkers for in vitro morphogenesis. We then applied the same strategy in planta in the same species and demonstrated the existence of epigenetic control (DNA methylation) during vernalization and devernalization in several sugar-beet hybrids that differed for bolting susceptibility. We propose that the scale and kinetics of epigenetic modifications control the induction and the rapidity of bolting, confirming the role of DNA methylation in this process. We have identified a number of target loci for these changes in DNA methylation during vernalization, and by screening these have been able to select several potential epigenetic biomarkers for bolting susceptibility, which may prove useful in future beet improvement programmes.
Genome defence in hypomethylated developmental contextsPlayfoot, Christopher James January 2017 (has links)
Retrotransposons constitute around 40% of the mammalian genome and their aberrant activation can have wide ranging detrimental consequences, both throughout development and into somatic lineages. DNA methylation is one of the major epigenetic mechanisms in mammals, and is essential in repressing retrotransposons throughout mammalian development. Yet during normal mouse embryonic development some cell lineages become extensively DNA hypomethylated and it is not clear how these cells maintain retrotransposon silencing in a globally hypomethylated genomic context. In this thesis I determine that hypomethylation in multiple contexts results in the consistent activation of only one gene in the mouse genome - Tex19.1. Thus if a generic compensatory mechanism for loss of DNA methylation exists in mice, it must function through this gene. Tex19.1-/- mice de-repress retrotransposons in the hypomethylated component of the placenta and in the mouse germline, and have developmental defects in these tissues. In this thesis I examine the mechanism of TEX19.1 mediated genome defence and the developmental consequences upon its removal. I show that TEX19.1 functions in repressing retrotransposons, at least in part, through physically interacting with the transcriptional co-repressor, KAP1. Tex19.1-/- ES cells have reduced levels of KAP1 bound retrotransposon chromatin and reduced levels of the repressive H3K9me3 modification at these loci. Furthermore, these subsets of retrotransposon loci are de-repressed in Tex19.1-/- placentas. Thus, my data indicates that mouse cells respond to hypomethylation by activating expression of Tex19.1, which in turn augments compensatory, repressive histone modifications at retrotransposon sequences, thereby helping developmentally hypomethylated cells to maintain genome stability. I next aimed to further elucidate the role of Tex19.1 in the developing hypomethylated placenta. I determine that Tex19.1-/- placental defects precede intrauterine growth restriction of the embryo and that alterations in mRNA abundance in E12.5 Tex19.1-/- placentas is likely in part due to genic transcriptional changes. De-repression of LINE- 1 is evident in these placentas and elements of the de-repressed subfamily are associated with significantly downregulated genes. If retrotransposon de-repression is contributing to developmental defects by interfering with gene expression remains to be determined, however I identify a further possible mechanism leading to placental developmental defects. I determine that Tex19.1-/- placentas have an increased innate immune response and I propose that this is contributing to the developmental defects observed. Developmental defects and retrotransposon de-repression are also observed in spermatogenesis in Tex19.1-/- testes, the molecular basis for which is unclear. I therefore investigate the possibility that the TEX19.1 interacting partners, the E3 ubiquitin ligase proteins, may be contributing to the phenotypes observed in Tex19.1- /- testes. I show that repression of MMERVK10C in the testes is dependent on UBR2, alongside TEX19.1. Furthermore, I have identified a novel role for the TEX19.1 interacting partner, UBR5, in spermatogenesis, whose roles are distinct from those of TEX19.1. The work carried out during the course of this thesis provides mechanistic insights into TEX19.1 mediated genome defence and highlights the importance of protecting the genome from aberrant retrotransposon expression.
A study of DNA methylation in adult gliomasMalley, Deborah Sarah January 2012 (has links)
No description available.
The effect of alcohol on the methylation status of the imprinting control regions contained within three developmentally significant lociKnezovich, Jaysen Gregory 25 February 2010 (has links)
MSc(Med), Human Genetics, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Imprinted loci are critical in foetal development and most are regulated by the methylation-specific CTCF binding protein which binds imprinting control regions (ICRs). The ICR is located between two genes that comprise imprinted loci, which are reciprocally expressed in a parent-of-origin specific manner. Maternally hypomethylated ICRs allow CTCF binding, creating a boundary element which prevents downstream enhancers from acting on the paternally expressed gene upstream of the ICR. Conversely, the hypermethylated (imprinted) paternal ICR prevents CTCF binding, allowing downstream enhancers to act on the gene upstream of the ICR, while suppressing the downstream maternally expressed gene. Alcohol and its metabolites are able to reach the testes via the blood supply and are known to reduce global DNA methylation by disrupting the folate, methyl group and homocysteine pathway. This may therefore affect gene expression at imprinted loci, whose parental alleles are discriminated by the imprinting status at the ICR. The effect of pre-conception paternal alcohol exposure on the DNA methylation of three paternally imprinted ICRs (H19, Rasgrf1, IG-DMR) as well as the maternally imprinted Snrpn ICR was examined in mouse sperm and their offspring. Male mice were gavaged with ethanol or sucrose. DNA was extracted from sperm of treated males and tail biopsies from offspring. Samples were bisulphite modified and the ICRs PCR amplified. DNA methylation patterns of ICRs were analysed by sequencing and quantitatively via pyrosequencing. Sperm samples of ethanol treated males did not show significant demethylation when compared to sucrose treated mice, with the exception of H19 CpG 7, Rasgrf1 CpG 26 and Snrpn CpG 10 (p=0.024, 0.014 and
Epigenetic inheritance of aberrant DNA methylation signatures as a consequence of chronic paternal alcohol exposure and the effect on embryonic gene expression in miceIsmail, Ayesha January 2015 (has links)
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree in Master of Science (Medicine) in the Division of Human Genetics / Epigenetic mechanisms regulate gene expression, a particularly important activity during foetal development. DNA methylation contained within promoter and regulatory intergenic regions influence gene activity. In utero alcohol exposure as a result of maternal consumption during pregnancy has been associated with disruption of foetal DNA methylation and gene expression, leading to neurological dysfunction, growth retardation and facial anomalies. While similar phenotypes in offspring have been associated with chronic preconception paternal alcohol exposure, the mechanisms underlying these effects remain largely unexplored. This study aimed to: (1) validate significant changes in sperm DNA methylation in a list of ten candidate genes in male mice chronically exposed for ten weeks to ethanol (n=10) compared to a calorie-equivalent sucrose solution (n=10); (2) validate significant changes in gene expression in candidate genes in the brain, liver and placenta of E16.5 embryos sired by ethanol (n=24) compared to sucrose (n=24) treated male mice; (3) quantify DNA methylation changes in candidate genes in the three embryonic tissues. (4) Lastly, previously generated microarray data were reanalysed using bioinformatics tools to generate a top ranked candidate differentially expressed gene list that was used to identify and analyse biological functions or pathways significantly over represented among these genes using PANTHER and DAVID. This study was unable to provide validation for most of the significant differences observed in the sperm DNA methylome in the original study, most likely because of the low sperm DNA concentration. Significant methylation differences were however observed at individual CpG sites in three candidate genes (Igf1r, Odc1, Depdc1b) in specific tissues of embryos sired by ethanol-exposed males relative to embryos sired by sucrose-treated males. There was concordance in the direction of altered gene expression between the cases and controls using the microarray and real-time PCR approaches for two genes in the brain (Grm7 and Zfp317), three genes in the liver (Igf1r, Vwf and Depdc1b) and one gene in the placenta vii (Vwf). However, none of the candidate genes selected for validation showed statistically significant changes. This may be a result of the modest fold changes observed in the microarray experiment that as shown in many cases, often do not replicate. The remainder of the genes showed no changes in expression in the test embryos relative to the control. The functional enrichment analysis revealed biological processes that were over represented in the brain and liver indicating that they may be more vulnerable to the effects of alcohol, compared to the placenta. Overall, the study could not provide a statistically significant correlation between methylation changes in the sperm that were inherited by the offspring which subsequently dysregulated gene expression in the embryo. However, as trends toward significance and significant DNA methylation changes were observed in the embryonic tissues, this study supports the idea that preconception paternal alcohol exposure can induce epigenetic alterations in a locus and organ specific manner within offspring. / MT2016
Identifying DNA Methylation Patterns as Novel Urinary Biomarkers for Kidney FunctionHasso, Ranya 11 1900 (has links)
Chronic kidney disease (CKD) is a major public health concern, characterized by an irreversible reduction in renal function. Currently, creatinine-based GFR estimation is predominantly used clinically to characterize CKD. However, this method is known to be an insensitive test for early losses of kidney function. Since patient prognosis relies heavily on slowing further decline of kidney function, uncovering novel biomarkers for kidney function, in conjunction with eGFR, will help improve patient outcome. Epigenetic-based biomarkers have been identified in numerous cancers, as DNA methylation changes alter cellular function. Thus, the objective of this study is to determine novel DNA methylation patterns reflecting altered kidney function. Five healthy participants that have undergone a nephrectomy have donated urine samples before and after their surgery, and global DNA methylation changes were analyzed through the 450K HumanMethylation microarray. Site- and region-level analyses were conducted to determine significant differentially methylated probes post-nephrectomy. The differential associations observed post-nephrectomy are statistically significant in both the site-level and regional analyses. Nineteen significant candidate probes have been systematically selected for validation, based on involvement in kidney function and consistent direction of methylation. Pyrosequencing assays have also been successfully designed and tested with control DNA, however replication of the microarray findings in participant DNA was unsuccessful. The inability to validate these candidate probes may be attributed to many influencing factors, and with this in mind, uncovering novel methylation patterns is still a promising biomarker for evaluating kidney function. / Thesis / Master of Science (MSc)
The effect of cytosine methylation on DNA structureVargason, Jeffrey M. 26 February 2002 (has links)
DNA methylation is common in prokaryotes and eukaryotes and has been implicated in various biological roles including gene silencing, X-chromosome inactivation, and genomic imprinting. 5-methylcytosine the "fifth base" of the genetic code comprises 1-3% of the human genome and is primarily found on cytosines within the context of the CpG sequence. Although progress has been made in understanding the biological roles of 5-methylcytosine, we are only beginning to uncover how it changes the local structure and global conformation of DNA. This thesis deals with the local perturbations in structure and hydration and the global conformational changes induced by the presence of 5-methylcytosine in DNA as determined by single crystal x-ray diffraction. 5-methylcytosine induces a novel conformation in the structure of duplex DNA. This conformation has characteristics of both the A-DNA and B-DNA conformations as well as some unique defining characteristics. This distinct duplex provides a structural rationale for the increased rate of deamination in 5-methylcytosine relative to cytosine. In addition to this novel conformation, 5-methylcytosine stabilizes intermediates within the B-DNA to A-DNA transition pathway, thus providing a crystallographic map of the transition from B-DNA to A-DNA. 5-methylcytosine was also used as a tool to probe the stabilizing features of the DNA four-way junction (known as the Holliday junction). The first crystal structures of Holliday junctions were found serendipitously while studying duplex DNA. The DNA four-way junction formation in these crystals was thought to be stabilized by a network of sequence dependent hydrogen bonds at the junction crossover. In this thesis, 5-methylcytosine was used to perturb these hydrogen bonds; however, the junction persisted, suggesting that there is flexibility in the types of sequences that can accommodate junction formation in the crystal, as well as, flexibility in the global structure of the junction. Overall, this work describes the effects of 5-methylcytosine on the local and global structure and hydration of DNA structure, as well as raising some interesting questions regarding the biological impact of methylation induced DNA structure. / Graduation date: 2002
Expression of the bovine DNA (cytosine 5) methyltransferase family during preimplantation development and aberrations induced by somatic cell nuclear transferGolding, Michael Cameron 17 February 2005 (has links)
Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos. Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.
Expression, purification and characterisation of the novel type I 1/2 methyltransferase M.AhdIMarks, Philip M. H. January 2002 (has links)
No description available.
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