DNA methylation in early mammalian development

Abstract

In mammalian genomes, the base cytosine is frequently modified to S-methylcytosine by the action of DNA methyltransferases. DNA methylation may affect gene expression within the cell by acting to repress transcription, and has been suggested to be involved in numerous cellular processes including tissue-specific gene control, repression of transposon activity, X-chromosome inactivation, genomic imprinting and tumourigenesis. While the pattern of mammalian DNA methylation is typically stable in the adult, widespread changes occur to genomic methylation levels during embryonic development, with a generalised demethylation of the genome at implantation followed by extensive de novo methylation in the postimplantation embryo. Embryonic changes to genomic methylation levels are important in determining the adult methylation state, however limitations in the methods used to detect DNA methylation have prevented a detailed study of DNA methylation in the embryo.

Bisulfite sequencing is a technique for the detection of 5—methylcytosine that offers considerable advantages over previously developed methods, and may be used to characterise DNA methylation in detail. However, bisulfite sequencing has not previously been used for the detection of DNA methylation in embryonic samples. In this project, the bisulfite sequencing technique is adapted for the detection of DNA methylation from mouse embryos, and the methylation state of single—copy genes determined throughout embryonic development. The various parameters affecting DNA methylation analysis in mouse embryos are examined.

The CpG island within the promoter of the Rb gene was found to be completely unmethylated through all stages of development, supporting a model in which the CpG island is protected from de novo methylation.

The methylation state of an allele-specific methylation imprint located 5‘ to the imprinted H19 gene was determined, and found to be methylated only at the paternal allele for all developmental stages examined. However, unlike the Rb CpG island, the control of allele—specific methylation was not absolute, with a low level of demethylation at the paternal allele and a low level of methylation at the maternal allele observed within the area bounded by the methylation imprint. The methylation of a boundary region for the H19 methylation imprint was also characterised.

The behaviour of the H19 methylation imprint in mutant ES cells deficient in the DNA methyltransferase Dnmt-I was examined, compared to wild—type ES cells and "rescued" ES cells in which Dnmt—I function had been restored. The H19 methylation imprint in mutant ES cells was found to be completely erased, and not reinstated in the rescued ES cells. The area containing the H19 methylation imprint was found to be relatively resistant to de novo methylation.

Methylation at the promoter of the skeletal Ot-actin gene, which exhibits a tissuespecific pattern of expression, was analysed in both embryos and differentiated tissues. The embryonic methylation profile was found to be broadly similar to previous models of tissue-specific genes. In the adult, no consistent correlation was found between promoter demethylation and tissue—specific gene expression.

The implications of these analyses of embryonic DNA methylation are discussed, in terms of both the technical aspects of DNA methylation deteCtion techniques and for the regulation of gene expression in the cell.