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1.6.1.1 D evelopm ental reg u latio n of m éth y latio n

5-methylcytosine is found exclusively at cytosine-guanine (CpG) dinucleotides. Shortly after fertihsation most genes are actively demethylated in a process thought to allow erasure of epigenetic information originating in the highly differentiated gametes (Razin and Shemer, 1995). The pluripotent state which results precedes differentiation and the creation of specific cell lineages. Following implantation a wave of de novo méthylation then occurs (Figure 1.7). The majority of CpGs become modified but all CpG islands associated with ‘housekeeping’ genes remain unmethylated (Bird, 1986). During development tissue- specific genes are demethylated in those tissues in which they are expressed. Thus in adult cells there is a clear correlation between undermethylation of those genes that are active and full méthylation of those that are inactive. Primordial germ cells escape the global preimplantation de novo méthylation but undergo a similar de novo méthylation process in the gonads to produce gamete specific méthylation patterns (Razin and Shemer, 1995).

Méthylation is clearly necessary for normal development since mice lacking a functional DNA methyltransferase gene fail to develop properly and die in midgestation (Li

et al., 1992). Further evidence links méthylation directly to control of gene expression. Méthylation of genes in vitro prevents their expression following transfection into fibroblasts (Yisraeli et al., 1988). Conversely, treatment of cells with 5-azacytidine, an inducer of déméthylation, leads to the activation of several endogenous genes (Jones,

Chapter 1. Introduction Méthylation level De novo Déméthylation méthylation Maintenance of méthylation ^ —-—-—■—► Adult Gamete Gamete Zygote Gamete _Gamete PGC Blastocyst

Cleavage Implantation Gastrulation Gametogenesis

Developmental time

F igure 1 .7 . Temporal changes in DNA méthylation levels during development, adapted from Jaenisch (1997). The DNA of the zygote is substantially methylated. During cleavage there is global déméthylation of the genome. After implantation a wave of de novo

méthylation occurs. Throughout subsequent organogenesis and differentiation the overall level of méthylation is maintained. During germ cell development DNA becomes de novo

methylated; however, some sequences are demethylated at later stages of gametogenesis. Sex-specific de novo méthylation or déméthylation of imprinted genes occurs during gametogenesis. PGC: primordial germ cells.

1.6.1.2 D ifferential m éthylation of im p rin te d genes

DNA méthylation is an epigenetic event stable enough to be propagated through many cell generations but also easily altered by de novo méthylation or déméthylation at specific sites during development. These properties make it ideal as a potential means of marking parental alleles in imprinted genes.

The first evidence that parental allele specific expression is associated with differential méthylation came from an unexpected observation in mouse transgenic experiments designed to determine the effect of an activated c-myc oncogene. (Swain et al.,

1987). The transgene used was not derived from an endogenously imprinted gene. However, it was found to be expressed only when inherited from the father. When

inherited from the mother it was completely silent. This parent-specific expression pattern correlated exactly with differential méthylation of the transgene. Silent maternal copies of the transgene were highly methylated whereas active paternal copies were unmethylated. Méthylation of the transgene was acquired by its passage through the female parent and eliminated during gametogenesis in the male. It was clear that a similar mechanism could underlie parent-of-origin-specific expression of imprinted genes.

Allele-specific méthylation of many imprinted genes has since been observed in late embryo and adult somatic cells. The differential méthylation varies considerably in extent from gene to gene. Allelic differences have been observed in promoters, introns, exons and regions distal to the transcription unit. In general, méthylation is associated with the inactive allele, as has been observed for non-imprinted genes. However, there are notable exceptions to this rule. For example, the Igf2r gene is methylated at the start of transcription on the silent paternal allele but also in an intron on the expressed maternal allele (Stoger et a l, 1993).

Where the inactive chromosome is methylated, the mechanisms of transcriptional repression are likely to be similar to those proposed for non-imprinted genes. Méthylation may prevent binding of methylation-sensitive transcription factors to their target sequences. Alternatively, proteins such as MeCPl and MeCP2, which themselves block the actions of transcription factors, may bind specifically to methylated sequences (Boyes and Bird, 1991; Nan et al., 1997). Binding of MeCPs to methylated DNA reduces the accessibility of chromatin to endonucleases such as Dnase I. Some imprinted genes show marked reduction in DNase I hypersensitivity in regions of extensive méthylation (Feil and Kelsey 1997). This observation supports a role for such proteins in allelic silencing. It seems that it is assembly of methylated DNA into a stable chromatin structure which results in loss of transcriptional activity (Kass etal., 1997).

For genes such as Ig flr and Igf2, where the active chromosome is methylated, other mechanisms need to be postulated. In these genes méthylation may be interfering with binding of transcriptional repressors and/or specific transcription factors may be binding preferentially to methylated DNA (Constancia et a l, 1998).

Chapter 1. Introduction

1.6.1.3 Evidence for méthylation as a genomic imprint

To assess the role of DNA méthylation in the imprinting process, Li et al. (1993) examined the expression of three imprinted genes H I 9, Igf2 and Igf2r in DNA methyltransferase mutant mice. Expression of all three genes was affected. In the case of

H19, the normally inactive paternal allele was activated. In contrast, the same mutation resulted in silencing of the normally active paternal allele of Igf2 and maternal allele of

IgfZr. DNA méthylation was therefore required to maintain monoalleleic expression of all three of these imprinted genes during embryonic development. These results provided the first demonstration of a causal link between DNA méthylation and parental allele activity of imprinted genes. Functional studies using transgenic mice support the importance of differentially methylated sites in maintaining the imprint. For example, yeast artificial chromosome (YAC) transgenes of IgfZr can reproduce the imprinted méthylation and expression pattern of the endogenous gene (Wutz et al., 1997). Deletion of a CpG island in the second intron (region 2) results in loss of imprinting and biallelic expression. This differentially methylated region therefore seems to be involved in maintenance of the Igf2r

imprint.

If differential méthylation of imprinted genes does have a role in determining allele- specific expression then it would be predicted to resist the wave of déméthylation which occurs after fertilisation. Evidence for this has been found at loci within several imprinted genes. Methylated sequences within the intron of Igf2r, the upstream region of H I9 and the promoter and 5’ region of Xist all retain their méthylation status in preimplantation embryos (Stoger et al., 1993; Tremblay et al., 1997; Ariel et al., 1995). These differentially methylated residues may function as ‘imprinting signals’ marking the parental origin of alleles in the embryo and adult. However, strong méthylation differences are not maintained at all imprinted loci. For example, close to the promoter of H I 9, both parental alleles are relatively hypomethylated in the blastocyst although highly methylated in sperm. The higher level of méthylation of the paternal allele is established postimplantation (Tremblay gr a/., 1997).