Genomic imprinting: antagonistic mechanisms in the germ line and early embryo.

tions of both a maternal and paternal genome (Barton et al. 1984; McGrath and Solter 1984). This unique constraint is due to a subset of genes whose monoallelic expression is dependent upon the parent of origin. Therefore, despite identical genetic information, alleles of such imprinted genes can be discriminated from one another by transcriptional activators or repressors in the nuclei of all somatic cells. Also implied by this phenomenon are mechanisms in each gamete that must establish a transcriptional state for an allele, one that is capable of being interpreted and stably maintained throughout the lifetime of the organism. Correct establishment and maintenance of these states is paramount, as misregulation of imprinted genes is associated with many developmental defects as well as with numerous cancers (Lalande 1996; Tycko 1999). Recent data have uncovered cis-elements and trans-factors that control the regulation of various imprinted genes in the soma. However, the mechanisms that govern establishment of imprints during gametogenesis remain incompletely understood. The organization of imprinted genes within the genome provides several clues about their regulation. Approximately 70 imprinted genes have been identified in mammals (http://www.mgu.har.mrc.ac.uk/research/imprinted/). Not only is their imprinted expression well conserved between mouse and man, but their genomic organization is as well, as imprinted genes are often associated in clusters throughout the genome (Verona et al. 2003). The high level of conservation between imprinted expression patterns and clustered organization suggests a functional link between the two. Chromosomal rearrangements or deletions within these clusters often cause misregulation of linked genes, providing further evidence of a functional connection between their organization and regulation (Reik and Maher 1997). Central to their allele-specific expression, differential DNA methylation has been associated with almost all imprinted loci (Brannan and Bartolomei 1999). Targeted mutagenesis has shown that these differentially methylated domains (DMDs; also differentially methylated regions, DMRs) are critical to the regulation of neighboring imprinted genes (Thorvaldsen et al. 1998; Fitzpatrick et al. 2002; Lin et al. 2003). Because it is stable and heritable through cell division, DNA methylation is the leading candidate for the imprinting “mark,” the epigenetic coding that distinguishes one allele from another. The critical importance of DNA methylation to imprinted gene regulation has been demonstrated in mice that carry mutations in several members of the DNA methyltransferase (Dnmt) family, the enzymes responsible for methylating DNA in mammals. Hypomorphic mutations of the first such enzyme identified, Dnmt1, have demonstrated that this protein is primarily involved in maintaining methylation at both imprinted and nonimprinted loci (Li et al. 1993). An oocyte-specific, maternally loaded alternative splice form of Dnmt1, Dnmt1o, functions to maintain methylation during a brief window of preimplantation development (Howell et al. 2001). The identification and characterization of three Dnmt3 genes has linked these related proteins to establishment of imprints, as these genes are highly expressed during gametogenesis in both parents (La Salle et al. 2004). Compound mutations of Dnmt3a and Dnmt3b genes have uncovered a vital, yet possibly redundant, role in establishing methylation imprints (Okano et al. 1999). More recently, conditional null alleles of Dnmt3a have demonstrated an essential role for this de novo methyltransferase in the acquisition of many, but not all, methylation imprints in both parental germ lines (Kaneda et al. 2004). Alternative forms of Dnmt3a have also been identified, potentially adding greater complexity to the establishment of methylation imprints (Chen et al. 2002). Interestingly, the only member of this family that does not have DNA methyltransferase activity in vitro, Dnmt3l, is required for establishment of methylation at imprinted loci at least in oocytes (Bourc’his et al. 2001). Biochemical data suggest that Dnmt3l may exert its activity by interacting with Dnmt3a and 3b (Hata et al. 2002). Therefore, Dnmt3l may provide sequence specificity to the other Dnmt3 proteins. Differentially methylated domains can be divided into those that are maternally or paternally hypermethylated. The majority of known DMDs, such as those for the Snurf-snrpn, Igf2r, Kcnq1ot1, and Peg3 loci, are maternally hypermethylated, while the differentially methylated regions of H19 and Rasgrf1 are paternally hypermethylated (Stöger et al. 1993; Plass et al. 1996; Shemer et al. 1997; Tremblay et al. 1997; Li et al. 2000; Fitzpatrick et al. 2002). Recent work has focused on the paternally hypermethylated H19 DMD as a paradigm not only for the regulation of imprinting, but for its establishment as well. H19 and Insulin-like growth factor 2 (Igf2) are two closely linked, yet oppositely imprinted genes (BarGenomic Imprinting: Antagonistic Mechanisms in the Germ Line and Early Embryo