DNA Methylation and Genomic Imprinting

DNA methylation was implicated in several developmental processes such as X-chromosome inactivation and genomic imprinting. DNA methylation in X-inactivation is discussed in Chapter 4 (this Vol.). Genomic imprinting is perhaps the best studied developmental process in which DNA methylation plays a pivotal role. The fact that the parental genomes contribute unequally to the development of a mammalian fetus was first demonstrated by nuclear transfer studies (McGrath and Solter 1984; Surani et al. 1984). While androgenetic embryos that contain two paternal copies of the genome show very poor embryonic development with normally developed extraembryonic tissues, parthenogenetic embryos, which contain two maternal genomes, show normal embryo development and underdeveloped extraembryonic tissues. These observations suggested that both sets of parental genomes are required for proper development. This conclusion gained further support by genetic experiments in which mice containing uniparental duplications of subchro-mosomal regions showed phenotypes that were either embryonic lethal or developmentally retarded (Cattanach and Kirk 1985).

This phenomenon suggested that expression of a subset of genes depends on their parental origin and that such differential expression of genes must be controlled by epigenetic modifications that take place during gametogenesis when the parental alleles are in separate compartments. The epigenetic mark could then serve as a signal to discriminate between the two parental alleles post fertilization and help to maintain this discrimination during embryonic development and adult life. The mark should then be erased and created anew during gametogenesis according to the gender of the offspring.

What epigenetic modification could best fulfill these requirements? DNA methylation is a good candidate since it can be erased by demethylation, established through de novo methylation and propagated by the maintenance methyltransferase (see Fig. 1). In fact, differentially methylated regions (DMRs) were found in all imprinted genes (Razin and Cedar 1994). Interestingly, DMRs are usually located in CpG islands. This is in contrast to the general rule that characterizes CpG islands being invariably unmethylated. The monoallelic methylation of DMRs is established in the gametes by Dnmt3a, Dnmt3b and Dnmt3L. Dnmt3L which in itself lacks methyltransferase activity is believed to recruit Dnmt3a and Dnmt3b to methylate the maternal allele in imprinted genes (Bourc'his et al. 2001; Hata et al. 2002). The differential methylation that is established in the gametes or early in embryo development is maintained throughout development (Fig. 4). Therefore methylation of the DMRs must escape the genome-wide demethylation that takes place in the early embryo and also the global de novo methylation that takes place post implantation (Kafri et al. 1992; see Fig. 2). However, the imprint must be erased by an as yet unknown demethylase that should work at the time period when the primordial germ cells migrate to the gonads (Fig. 4).

In fact, several imprinted genes (Igf2r, H19, Snrpn and Xist) among others have been shown to obey these rules (Brandeis et al. 1993; Stoger et al. 1993; Ariel et al. 1995; Tremblay et al. 1995; Shemer et al. 1997). DNA methylation may therefore play a dual role in the imprinting process. The differential methylation may mark the parental alleles, allowing the transcriptional machinery of the cells to distinguish between the parental alleles. In addition, DNA methylation can directly affect promoter activity of the imprinted genes.

Fig. 4. DNA methylation changes of imprinted genes during gametogenesis and embryo development

Alternatively, DNA methylation can indirectly affect monoallelic expression of a gene by silencing a promoter of an antisense gene, thus allowing production of antisense RNA only from the allele on which the imprinted gene is repressed (Rougeulle et al. 1998; Lyle et al. 2000).

Loss of function of DMRs, or defects in their methylation, frequently cause loss of imprinting associated with neurobehavioral disorders. Such epigenetic defects within a 2-Mb domain on human chromosome 15q11-q13 cause two different syndromes. Prader-Willi syndrome is caused by loss of function of a large number of paternally expressed genes (Buiting et al. 1995), while silencing of the maternally expressed genes within the domain causes Angelman's syndrome (Reis et al. 1994). The imprinting of this entire domain is regulated by an imprinting center that constitutes a DMR within the 5' region of the imprinted SNRPN gene. Individuals with deletions of this region on the paternal allele have Prader-Willi while another sequence located 35 kb upstream of SNRPN confers methylation of the SNRPN DMR on the maternal allele, thereby inactivating the paternally expressed genes on the maternal allele. When this upstream region is deleted on the maternal allele, the SNRPN DMR does not become methylated (Perk et al. 2002). Consequently, the entire domain on the maternal allele remains unmethylated and all paternally expressed genes on the maternal allele are activated while maternally expressed genes are silenced, thus causing Angelman's syndrome. A model was proposed suggesting that the upstream sequence together with the SNRPN DMR constitute a complex imprinting box responsible for both the establishment and maintenance of the imprinting state at PWS/AS domain on both alleles (Shemer et al. 2000).

Altered allelic methylation and expression patterns of the imprinted gene IGF2 have been found in Beckwith-Wiedemann syndrome (BWS) patients. BWS is a pre- and post-natal growth syndrome associated with predisposition for childhood tumors. Translocation breakpoints in a number of BWS patients map to the imprinted gene KCNQ1 which is located in the center of the 800-kb BWS region on human chromosome 11p15.5. The translocations in BWS are associated with loss of imprinting of IGF2 but not H19 (Brown et al. 1996). It appears that this impairment in imprinting involves the differentially methylated intronic CpG island in KCNQ1. In a small number of BWS patients, hypomethylation of the KCNQ1 CpG island correlated with biallelic expression of IGF2 (Smilinich et al. 1999; Paulsen et al. 2000). Deletion of this CpG island on the paternal chromosome 11 leads to silencing of KCNQ1 antisense transcript and activation of KCNQ1, p57KIP2 and SMS4 which are located downstream on the normally repressed paternal allele. It is therefore possible that this CpG island is at least part of an imprinting center on human chromosome 11p15.5 and its orthologous region on mouse chromosome 7.

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