DNA methylation in early mammalian development

In mammalian genomes, the base cytosine is frequently modified to&#13;\nS-methylcytosine by the action of DNA methyltransferases. DNA methylation may&#13;\naffect gene expression within the cell by acting to repress transcription, and has been&#13;\nsuggested to be involved in numerous cellular processes including tissue-specific gene&#13;\ncontrol, repression of transposon activity, X-chromosome inactivation, genomic&#13;\nimprinting and tumourigenesis. While the pattern of mammalian DNA methylation is&#13;\ntypically stable in the adult, widespread changes occur to genomic methylation levels&#13;\nduring embryonic development, with a generalised demethylation of the genome at&#13;\nimplantation followed by extensive de novo methylation in the postimplantation&#13;\nembryo. Embryonic changes to genomic methylation levels are important in&#13;\ndetermining the adult methylation state, however limitations in the methods used to&#13;\ndetect DNA methylation have prevented a detailed study of DNA methylation in the&#13;\nembryo.&#13;\n&#13;\nBisulfite sequencing is a technique for the detection of 5—methylcytosine that&#13;\noffers considerable advantages over previously developed methods, and may be used to&#13;\ncharacterise DNA methylation in detail. However, bisulfite sequencing has not&#13;\npreviously been used for the detection of DNA methylation in embryonic samples. In&#13;\nthis project, the bisulfite sequencing technique is adapted for the detection of DNA&#13;\nmethylation from mouse embryos, and the methylation state of single—copy genes&#13;\ndetermined throughout embryonic development. The various parameters affecting DNA&#13;\nmethylation analysis in mouse embryos are examined.&#13;\n&#13;\nThe CpG island within the promoter of the Rb gene was found to be completely&#13;\nunmethylated through all stages of development, supporting a model in which the CpG&#13;\nisland is protected from de novo methylation.&#13;\n&#13;\nThe methylation state of an allele-specific methylation imprint located 5‘ to the&#13;\nimprinted H19 gene was determined, and found to be methylated only at the paternal&#13;\nallele for all developmental stages examined. However, unlike the Rb CpG island, the&#13;\ncontrol of allele—specific methylation was not absolute, with a low level of&#13;\ndemethylation at the paternal allele and a low level of methylation at the maternal allele&#13;\nobserved within the area bounded by the methylation imprint. The methylation of a&#13;\nboundary region for the H19 methylation imprint was also characterised.&#13;\n&#13;\nThe behaviour of the H19 methylation imprint in mutant ES cells deficient in the&#13;\nDNA methyltransferase Dnmt-I was examined, compared to wild—type ES cells and&#13;\n"rescued" ES cells in which Dnmt—I function had been restored. The H19 methylation&#13;\nimprint in mutant ES cells was found to be completely erased, and not reinstated in the&#13;\nrescued ES cells. The area containing the H19 methylation imprint was found to be&#13;\nrelatively resistant to de novo methylation.&#13;\n&#13;\nMethylation at the promoter of the skeletal Ot-actin gene, which exhibits a tissuespecific&#13;\npattern of expression, was analysed in both embryos and differentiated tissues.&#13;\nThe embryonic methylation profile was found to be broadly similar to previous models&#13;\nof tissue-specific genes. In the adult, no consistent correlation was found between&#13;\npromoter demethylation and tissue—specific gene expression.&#13;\n&#13;\nThe implications of these analyses of embryonic DNA methylation are&#13;\ndiscussed, in terms of both the technical aspects of DNA methylation deteCtion&#13;\ntechniques and for the regulation of gene expression in the cell.

Topic: Epigenetics and DNA Methylation
Subfield: Molecular Biology
Field: Biochemistry, Genetics and Molecular Biology
Domain: Life Sciences