Diseases of genomic imprinting

Mammals possess an epigenetic system thought to be important for fetus-mother nutrient transfer and normal development, termed genomic imprinting (Constancia et al., 2002; Ferguson-Smith et al., 2003b; Ferguson-Smith et al., 2004; Reik et al., 2003; Walter and Paulsen, 2003). Epigenetic guidelines are imprints laid down in germ cells (in most cases by DNA methyltransferases) governing how genes are expressed depending on their parental origin, be it maternal or paternal (Bjornsson et al., 2004; Brannan and Bartolomei, 1999) (Figure 2.2). Usually the silent gamete is methylated but there are exceptions. In addition, there are DNA methylation-independent imprinting mechanisms, which are operative in extra-embryonic tissues (Lewis et al., 2004; Umlauf et al., 2004). The molecular mechanisms of genomic imprinting are complex, involving multiple imprinted loci, some of which exhibit developmental-specific imprints. Over 60 imprinted genes, which tend to cluster together, thus facilitating coordinate control of imprinted gene expression, have been found in mammals (Beechey, 2004). Three main steps can be visualized during mammalian imprint development; erasure, re-establishment and maintenance (molecular recognition). The first step occurs during primordial germ cell development, the second during gamete maturation and the last step in the developing embryo. The mechanism of imprint erasure is unclear but may involve active DNA demethylation and subsequent histone modifications (Lee etal., 2002). Imposition of DNA methyla-tion at imprinted regions is dependent on Dnmts,

Figure 2.2 Model for genomic imprinting by DNA methylation during development. The imprints are erased during primordial germ cell (PGC) development in males and females, which allows for potential biallelic expression. During gamete formation, the imprints are re-imposed and this process is dependent on DNA methyltransferase activity (notably Dnmt3a) and the co-factor Dnmt3l. After fertilization, the parental specific imprints are maintained by DNA (Dnmtl) and histone modifying activities, resulting in allelespecific expression.

Figure 2.2 Model for genomic imprinting by DNA methylation during development. The imprints are erased during primordial germ cell (PGC) development in males and females, which allows for potential biallelic expression. During gamete formation, the imprints are re-imposed and this process is dependent on DNA methyltransferase activity (notably Dnmt3a) and the co-factor Dnmt3l. After fertilization, the parental specific imprints are maintained by DNA (Dnmtl) and histone modifying activities, resulting in allelespecific expression.

especially Dnmt3a and the cofactor Dnmt3L (Kaneda et al., 2004; Hata et al., 2002). The last step involves read-out of the imprinted regions and can involve selective silencing of one allele due to DNA methylation at regulatory regions (sometimes promoter regions). In many cases, mono-allelic expression is associated with antisense transcripts that are integral to silencing at multiple loci in imprinted regions (Horike et al., 2000).

The association between genomic imprinting and brain disorders with influences on complex behavior is exemplified by the Prader-Willi and Angelman syndromes (PWS and AS), two neurobehavioral disorders with distinct clinical manifestations (Ferguson-Smith et al., 2004; Nicholls and Knepper, 200l). Patients with PWS present with hypotonia at birth, obesity, short stature, mental retardation, hypogonadism and a characteristic facial appearance. AS is characterized by microbrachycephaly, large mouth with tongue protrusion and prognathism; mental retardation is severe with absence of speech. Both diseases are associated with deficiencies in the same region of human chromosome 15q11-q13 due to an unequal crossing over between low copy repeats. These deletions are of paternal origin in PWS

patients and of maternal origin in the case of AS. In PWS, the deletion occurs in an imprinting centre (IC) within the promoter of the imprinted gene SNURF-SNRPN, while in Angelman syndrome deletions are found a few thousand basepairs upstream of the IC (Imprinting Centre), which is active in germline and/or early postzygotic development (Mann and Bartolomei, 1999; Nicholls and Knepper, 2001; Yang et al., 1998). The presence of the IC is integral to the establishment of allele-specific differences in DNA methylation, chromatin organization, histone modification and expression (Nicholls and Knepper, 2001). In PWS, otherwise paternally expressed genes are silenced and methylated, in contrast to AS where genes that are otherwise repressed are now demethylated and switched on. A mouse line harbouring a deletion in the putative PWS-IC region of the SNRPN promoter displays many human PWS-like symptoms and aberrantly silences a number ofimprinted genes in this region. (Yang et al., 1998). A mouse model of AS was generated by deletion of the UBE3A gene (Miura et al., 2002). Once again, the mice displayed striking phenotypic and behavioral similarity to their human counterparts. Microarray analysis has identified candidate genes that are mis-expressed in PWS patients including UBE3A and ATP10C (Bittel et al., 2003). However, PWS (unlike AS) does not appear to be due to single gene mutations. A comprehensive analysis of methyla-tion patterns in PWS/AS families and the mouse models are needed to clarify the molecular mechanisms of these complex diseases and how these methylation patterns are interpreted by nuclear factors (see section on Rett syndrome).

Another disorder of imprinting, the overgrowth disorder Beckwith-Wiedemann syndrome (BWS) on human chromosome 11p15, has been associated with two non-adjacent chromosomal regions; a telomeric domain coincident with the IGF2 and H19 loci and a centromeric domain. (Ferguson-Smith et al., 2004; Walter and Paulsen, 2003; Weksberg et al., 2003). It has been suggested that incorrect imprinting in the telomeric region yields overgrowth and tumors, whereas those in centromeric regions involve the characteristic BWS congenital malformations (Bjornsson et al., 2004; Cui et al., 2002; 2003).

Two basic questions are the timing of the epigenotype spreading and the mechanism of spreading. Is spreading propagated during game-togenesis or is it post-zygotic, or both? In addition, is spreading transcription-mediated by virtue of promoters and anti-sense transcripts, or are ICs spatially arranged to come into contact with distant ICs, perhaps by matrix-attachment sites? An emerging theme is that non-coding RNAs play a role in genome modification in animals as well as plants and fungi (Aufsatz et al., 2002; Volpe et al.,

2003). Antisense RNA transcripts are implicated in the regulation of the autosomally imprinted Igf2r-Air domain and in X-chromosome inactivation in female mammalian cells (Plath et al., 2002; Sleutels et al., 2002). The presence of multiple micro RNA genes at imprinted regions in mammals may be functionally relevant (Beechey, 2004; Seitz et al.,

2004). Recently it has been demonstrated that small interfering or siRNAs targeted to CpG islands of an E-cadherin promoter induced significant DNA methylation and histone H3 lysine 9 methylation in both MCF-7 and normal mammary epithelial cells, leading to gene silencing (Kawasaki and Taira, 2004).

As assisted reproductive technologies (ARTs) are increasingly used to overcome infertility, there is concern that the children conceived may be susceptible to imprinting disorders involving chromatin modification (Maher et al., 2003). As yet, the evidence that ART may be associated with genetic imprinting disorders is inconclusive (Schieve et al., 2004). Parenthetically, Dnmtl overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality (Biniszkiewicz et al., 2002). In addition, somatic cell nuclear transfer often results in multiple imprinting errors (Young and Beaujean, 2004). Loss of imprinting mechanisms can also be observed in many cancers involving reactivation of the normally silent allele of a growth-promoting gene such as Igf2, and/or silencing of the normally active allele of a tumour suppressor gene.

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