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This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and education use, including for instruction at the author’s institution, sharing with colleagues and providing to institution administration. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Review TRENDS in Genetics Vol.23 No.9 Construction and evolution of imprinted loci in mammals Timothy A. Hore*, Robert W. Rapkins* and Jennifer A. Marshall Graves Research School of Biological Sciences, The Australian National University, Canberra, ACT 2601, Australia Genomic imprinting first evolved in mammals around the time that humans last shared a common ancestor with marsupials and monotremes (180–210 million years ago). Recent comparisons of large imprinted domains in these divergent mammalian groups have shown that imprinting evolved haphazardly at various times in different lineages, perhaps driven by different selective forces. Surprisingly, some imprinted domains were formed relatively recently, using non-imprinted components acquired from unexpected genomic regions. Rearrangement and the insertion of retrogenes, small nucleolar RNAs, microRNAs, differential CpG methylation and control by non-coding RNA often accompanied the acquisition of imprinting. Here, we use comparisons between different mammalian groups to chart the course of evolution of two related epigenetic regulatory systems in mammals: genomic imprinting and Xchromosome inactivation. Introduction Diploid organisms have two copies (alleles) of autosomal genes, one from each parent. This redundancy probably evolved by selection for a ‘backup’ copy of each gene, in case one copy is mutated. Genes subject to genomic imprinting (!100 in humans and mice [1]) forgo this safety-net because they inactivate either the maternal or paternal allele, causing attenuation or complete silencing in certain tissues and developmental stages. The functional haploidy caused by silencing one allele means that imprinted genes are often associated with disease [2]. Significantly, imprinted genes tend to be clustered in domains that often include both paternally and maternally silenced genes (Table 1). Imprinted alleles distinguish their parent of origin by parent-specific epigenetic marks, imparted during gametogenesis. These marks often consist of a single differentially marked cis element, known as an imprint control region (ICR). The primary differential epigenetic mark of the ICR (thought to be methylation of CpG nucleotides) is then ‘read’ and propagated in an allele-specific manner throughout an imprinted gene cluster. This occurs in several ways, including antisense transcription, histone modifications, further (sometimes post-zygotic) DNA methylation at auxiliary regions, RNA interference-mediated processes and blocking of enhancers or repressors by insulator proteins (reviewed in * Corresponding author: Graves, J.A.M. ([email protected]). These authors contributed equally to this work. Available online 1 August 2007. www.sciencedirect.com Ref. [3]). This complex process can achieve imprinted expression of multiple maternal and paternal specific genes, sometimes over large distances (>2 Mb) from the ICR. One consequence of genomic imprinting is that viable embryos must receive two haploid genome complements, coming from parents of opposite sex. Thus, natural parthenogenesis (in which an unfertilized egg develops into a new individual) is theoretically impossible in animals with imprinting of essential genes, because the egg would have twice the expression of maternally expressed genes and a complete absence of paternally expressed genes. Indeed, it was the initial failure to produce a parthenogenetic mouse embryo that first demonstrated the non-equivalence of autosomal genetic material between mammalian parents [4,5]. There are several examples of parthenogenetic amphibians and fish, and even a few reptiles and birds (reviewed in Ref. [6]), implying that within vertebrates, genomic imprinting is mammal-specific. The question then becomes, how did genomic imprinting arise in mammals, and why was it selected for, given the apparent costs involved? Here, we review recent discoveries about the transition of mammalian imprinted gene domains from their non-imprinted ancestors, focusing upon novel studies undertaken on the most ancient mammalian clades – the marsupials and monotremes. Given that model species from these clades have recently been sequenced (Table 2), the power and volume of this research is considerable and worthy of retrospection. We show that various imprinted loci, including the locus that controls X chromosome inactivation, are constructed in a surprisingly similar fashion, triggered by unexpected genomic events. We also review how these new data from marsupials and monotremes affects current theories attempting to identify the selective forces that drove the evolution of imprinting. Imprinting in marsupials and monotremes The reptile lineage that gave rise to mammals diverged from other reptiles !310 million years ago (MYA). Monotremes diverged 210 MYA from the therian mammals (marsupials and eutherians), which diverged from each other 180 MYA [7]. These mammal groups all pursue different reproductive strategies. Marsupials (such as kangaroos and opossums) give birth to tiny and underdeveloped (altricial) young that complete development attached to a teat, often protected within a pouch. The placenta is short-lived and less developed than the complex hormoneproducing placenta that supports the extended gestation of eutherians (often referred to as ‘placental mammals’). The 0168-9525/$ – see front matter ! 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2007.07.003 Author's personal copy Review TRENDS in Genetics Vol.23 No.9 441 Table 1. Imprinted domains discussed in this review and the relevant genes they contain Locus name Location a Gene name a Description Prader-Willi and Angelman syndrome 15q11–13/7B3 Frat3 Frequently rearranged in advanced T-cell lymphomas 3 Makorin, ring finger protein MAGE-like protein Nectin SNRPN upstream reading frame d Small nuclear ribonucleoprotein d Cluster of !80 small nucleolar RNAs d UBE3A antisense d Ubiquitin protein ligase ATPase Delta-like homolog Gene trap locus 2 Retrotransposon-like RTL1 antisense Callipyge PEG10 locus 14q32/12F1 7q21–22/6qA1 MKRN3/Mkrn3 MAGEL2/Magel2 NDN/Ndn SNURF/Snurf SNRPN/Snrpn snoRNAs/snoRNAs UBE3A-as/Ube3a-as UBE3A/Ube3a ATP10A/Atp10a DLK1/Dlk1 GTL2/Gtl2 (MEG3) RTL1/Rtl1 (PEG11) RTL1-as/Rtl1-as (PEG11-as) miRNAs/miRNAs snoRNAs/snoRNAs miRNAs/miRNAs DIO3/Dio3 SGCE/Sgce PEG10/Peg10 miRNA-like repeats PPP1R9A/Ppp1R9A (Neurabin) PON1/Pon1 PON3/Pon3 PON2/Pon2 ASB4/Asb4 11q15.5/7qF5 Beckwith-Wiedemann syndrome IGF2R locus 6q25.3/17qA1 X-inactivation centre Xq13.2/XqD IGF2/Igf2 H19/H19 IGF2R/Igf2r Air Xite TSIX/Tsix XIST/Xist JPX/Jpx FTX/Ftx miRNAs ZCCHC13/Cnbp2 Imprint statusa,b,c NO/P P/P P/P P/P P/P P/P P/P P/P M/M M/M P/P M/M NT/P NT/M Cluster of !10 micro RNAs liberated by RTL1as Cluster of !40 small nucleolar RNAs Cluster of !40 micro RNAs Deiodinase, iodothyronine type III Sarcoglycan epsilon Paternally expressed 10 miRNA-like hairpin structures within PEG10 intron Protein phosphatase NT/M Paroxonase 1 Paroxonase 3 Paroxonase 2 Ankyrin repeat and socs box-containing protein Insulin-like growth factor 2 Large non-coding RNA Insulin-like growth factor 2 receptor Igf2r antisense X-intergenic transcription element XIST antisense X-inactive specific transcript Large non-coding RNA Large non-coding RNA Cluster of 3–4 micro RNAs situated within FTX Zinc finger, CCHC domain-containing NT/B NT/M NT/M NT/M NT/M NT/M NT/P P/P P/P NT/NT M/M P/P M/M M/M NO/P NO/M B/M B/P NT/NT NT/NT NT/NT NT/NT a Gene locations, names and imprint status are given as Human/mouse; alternative gene names are in brackets. b Abbreviations: P, paternal expression; M, maternal expression; B biallelic expression; NT, currently not tested for imprint status; NO, no orthologue present. c References and additional imprinted genes can be found at http://igc.otago.ac.nz. d Transcriptionally linked. extraordinary monotremes (such as the platypus) lay eggs as reptiles do and, in the absence of teats, the hatchlings suck milk from the mother’s abdomen. Thus, lactation evolved before the three mammalian clades diverged 210 MYA, viviparity arose 180–210 MYA in therians, and the complex eutherian placenta evolved before the eutherian radiation 105 MYA [7] (Figure 1). Is there imprinting in marsupials and monotremes? The archetypal example of an imprinted gene, insulinlike growth factor 2 (IGF2), is imprinted in humans and mice but not in chicken [8]. The IGF2 orthologue was cloned in the platypus and found to be expressed from both alleles [9], so this locus, at least, and its receptor IGF2R [10,11] are not imprinted in platypus. By contrast, marsupials have imprinted expression of both IGF2 [8,12] and IGF2R [10]. Thus, we can date the emergence of IGF2 and IGF2R imprinting to between 210 and 180 MYA (Figure 1). These results supported a somewhat simplistic notion that genomic imprinting evolved in one fell swoop, along with viviparity. However, recent comparisons of two Table 2. Update on sequencing projects Species Monodelphis domestica Macropus eugenii Ornithorhynchus anatinus a See: http://www.genome.gov/12512299. See: http://www.genome.gov/12512287. b www.sciencedirect.com Common name Gray short-tailed opossum Tammar wallaby Duck-billed platypus Mammalian group Marsupial Marsupial Monotreme Sequence coverage 6.8X 2.1 !6X Refs [84] In progress a In progress b Author's personal copy 442 Review TRENDS in Genetics Vol.23 No.9 Figure 1. Reproductive strategy and imprinting status differs between various vertebrate groups. The evolution of lactation, viviparity and complex placentation can be dated against a phylogeny (asterisks, left) and correlates with changes in the nature of genomic imprinting and X-chromosome inactivation (right). large imprinted gene clusters in marsupials and monotremes – the Prader-Willi–Angelman syndrome domain (PWS–AS) and the Callipyge locus (CLPG) – now provide crucial insight into the evolution of imprinted domains and lead to an alternative model of a gradual and rather haphazard evolution of imprinting in mammals. Evolution of the Prader-Willi–Angelman syndrome imprinted domain Two phenotypically distinct neurological disorders, Prader-Willi and Angelman syndromes (PWS and AS; reviewed in Ref. [13]), are associated with deletions in the same region of human chromosome 15q11–13. The observation that indistinguishable defects caused AS when maternally inherited, but PWS when paternally inherited, suggested that imprinted genes were involved in these diseases. Further characterization of the !2.3 Mb PWS–AS domain divided it into two parts, each responsible for one disorder. The smaller AS subdomain contains two imprinted genes, UBE3A (which has a crucial role in AS) and ATP10A. The genes are both maternally expressed (paternally silenced) in neuronal tissue [14,15]. Proximal to the AS subdomain is the larger PWS subdomain, comprising five protein-coding genes, three of which (MKRN3, MAGEL2 and NDN) are intronless (reviewed in Ref. [13]). The other two protein coding genes (SNURF and SNRPN) are located adjacent to each other and form a bicistronic transcript [16]. All five genes are paternally expressed www.sciencedirect.com (maternally silenced). The PWS subdomain also includes two clusters of small nucleolar RNAs (snoRNAs), also paternally expressed. These are of the C/D box family of snoRNAs, whose members mostly function to guide 2-Omethylation and pseudouridylation of rRNA, but at the PWS–AS loci, snoRNA HBII-52 (which is present as !50 tandem repeats) regulates alternative splicing of the serotonin receptor, possibly contributing to the PWS phenotype [17]. Regulation of the PWS–AS domain has been explored in detail. A differentially methylated ICR located upstream of SNURF and SNRPN controls imprinted expression throughout the entire domain (reviewed in Ref. [18]). Through interaction with the ICR, a second differentially methylated cis element is set up closer to SNRPN. When unmethylated (as it is on the paternally derived allele), this cis element is thought to function as a bidirectional enhancer, activating expression of MKRN3, MAGEL2, NDN and SNURF–SNRPN. A large alternatively spliced non-coding transcript, initiating upstream of SNURF– SNRPN, is responsible for expression of the downstream snoRNAs [19] and extends into the AS sub-domain to produce a transcript antisense to UBE3A (UBE3A-as) [19,20]. Although the mechanism is currently debated, the expression of this antisense transcript is thought to provide the regulatory link between the ICR and UBE3A [21–23]. The expression and location of orthologues of genes within the PWS–AS locus were explored in marsupials Author's personal copy Review TRENDS in Genetics Vol.23 No.9 and monotremes. Surprisingly, only UBE3A could be found in the tammar wallaby genome. Other genes from this region were apparently missing: repeated attempts to clone a marsupial MKRN3 resulted only in the identification of its paralogous source gene (MKRN1) [24], and attempts to clone the orthologue of SNRPN in the wallaby resulted in the isolation of its non-imprinted human chromosome 20 homologue SNRPB [25]. Many attempts to isolate MAGEL2, NDN and SNURF failed to produce any orthologous sequences. Similarity searches for orthologues of these genes in the opossum and platypus genomes also failed to detect any matches. However, searches of the opossum sequence eventually revealed a copy of SNRPN located in tandem with SNRPB. To compound the confusion, SNRPN and UBE3A were not even on the same chromosome in marsupials, let alone adjacent to each other as they are in humans and mice. UBE3A was identified in the platypus, but not SNRPN; SNRPB is present as a single copy in the platypus genome. 443 These confusing observations were explained [26] when a platypus bacterial artificial chromosome (BAC) containing the UBE3A orthologue was isolated and fully sequenced. Surprisingly, a completely different gene (CNGA3, located on human chromosome 2) lay, in place of SNRPN, head to head with UBE3A, and a mere 7 kb away. The same arrangement was found in marsupials, although the distance between the genes was much greater (!60 kb). This UBE3A–CNGA3 arrangement was then discovered in chicken and fish genomes, so it must represent the ancestral arrangement (Figure 2a). A search of the BACs for the marsupial homologues of PWS–AS control sequences did not reveal any similarity between the human ICR upstream of SNRPN and the corresponding region in opossum. Imprinted coregulation of these two genes (at least as it occurs in human and mice) would therefore be impossible in marsupials. Not surprisingly, then, wallaby SNRPN and UBE3A were found to show biallelic expression, as was platypus Figure 2. Evolution of imprinted loci. (a) The Prader-Willi–Angelman Syndrome locus, (b) the Callipyge locus, (c) the PEG10 locus and (d) the X-inactivation centre acquired multiple new genes (retroposed genes, dark blue; other new genes, light blue) and clusters of miRNAs and snoRNAs (thin lines) early in mammalian evolution 210–105 MYA. Some genes from ancestral loci were retained in eutherian mammals (red), but many were lost by translocation (black) or pseudogenization (black with red crosses). Genes acquired imprinted expression (direction of imprinting shown above and below the line) at the same time, or soon after these complex genomic events. The structure of these loci are generalized from multiple eutherian species (human, mouse and dog); minor species-specific changes have occurred, but are not indicated for brevity. www.sciencedirect.com Author's personal copy 444 Review TRENDS in Genetics Vol.23 No.9 UBE3A. This suggested that imprinted expression of the PWS–AS region was accompanied by (or perhaps caused by) the fusion of SNRPN and UBE3A and the evolution of the ICR. Concurrently, SNURF and the snoRNAs were acquired, and MKRN3, MAGEL2 and NDN were retroposed into this domain. This occurred after the divergence of eutherians and marsupials 180 MYA, but before the eutherian radiation 105 MYA. Thus, the PWS–AS domain was constructed relatively recently from non-imprinted components acquired from all over the genome, including protein-coding genes that were translocated or retroposed, snoRNAs and elements that acquired a regulatory function. The locus became imprinted at some point during or after these multiple rearrangements. Evolution of the Callipyge locus The Callipyge locus (CLPG) on human chromosome 14q32 is named after the muscle hypertrophy phenotype with which it is associated in sheep (‘Callipyge’ means ‘beautiful bottom’) [27]. Many similarities have been identified between the PWS–AS and CLPG loci. For example, the CLPG locus also contains multiple paternally expressed genes, including DLK1 (overexpression of which causes the CPLG phenotype [28,29]), RTL1 [30] and DIO3 [31]. This locus also contains a series of non-coding RNAs, including GTL2, RTL1 antisense transcript (RTL1-as), a cluster of C/D box snoRNAs and multiple microRNAs (miRNAs), most of which are found within one large cluster. All these noncoding RNAs are maternally expressed and lie in the same orientation and, as such, are thought to all be a part of the same transcriptional unit, akin to the large UBE3A-as transcript from the PWS–AS domain. Rtl1-as contains a small cluster of miRNAs recently shown to repress Rtl1 by the RNA interference pathway [32]. Situated upstream of GTL2 is a differentially methylated region thought to be an ICR. Deletion of this element from the maternally inherited chromosome in mouse causes bidirectional loss of imprinting throughout the entire CLPG domain [33]. Similarly to UBE3A and SNRPN, DLK1 is biallelically expressed in all marsupial tissues tested to date [34]. No orthologue of GTL2 could be identified outside the eutherian mammals, implying that it must have arisen at the same time as DLK1 imprinting, early in the eutherian radiation. The intronless (retroposed) RTL1 gene is confined to eutherian mammals, as no orthologues could be found in other organisms [32], and unpublished observations suggest that imprinted small RNAs are also eutherian-specific (H. Seitz in Ref. [35]). Taken together, these studies suggest that the CLPG locus underwent similar late construction, in similar manner to the PWS–AS locus (Figure 2b). Therefore, it seems that imprinting was acquired at different times at different loci. Indeed, there are now several examples of the recent acquisition of imprinting – and also the loss of imprinting – in the mouse and human lineages. For example, the paternally expressed and intronless Frat3 lies upstream of the Mkrn3 gene of the PWS–AS region only in the rodent lineage, and is estimated to have retroposed only 18–26 MYA [36]. It appears to have acquired the imprint status of surrounding genes. Conversely, imprinting of IGF2R [37] and many other www.sciencedirect.com genes has been relaxed in humans relative to mice [38]. Examples such as these, in which genes show imprinted expression in some eutherians but not others, suggest that imprinted gene evolution is ongoing and dynamic [1,38,39]. Evolution of the PEG10 locus The evolutionary history of another imprinted region was investigated recently using sequence and expression data from marsupials and monotremes. This locus contains PEG10 (paternally expressed gene 10), which is essential for placental development in mice [40]. PEG10 belongs to the same ‘sushi-ichi’ family of retrotransposon-derived genes as RTL1 of the CPLG locus [41] and similarly hosts several uncharacterized miRNA-like genes [35]. In mice, a region containing the first exons of Peg10 and neighbouring gene Sgce is methylated only on the maternally inherited chromosome [42], accounting for their paternal-specific expression [42–44]. Although not yet tested by deletion studies, this differentially methylated region is probably an ICR, as it is established during gametogenesis [42]. Downstream of PEG10 is a family of paraxonases (PON1, PON2 and PON3) and PPP1R9A and ASB4. All these genes show some level of maternal-specific expression, except for PON1 [42,45], which is a mammal-specific duplicate of the more ancient PON2 or PON3 [46]. Recently, Suzuki and colleagues [47] demonstrated that PEG10 is paternally expressed in the tammar wallaby, but that SGCE, PPP1R9A and ASB4 are not imprinted (the PON genes were not analysed). The first exon of wallaby PEG10 was found to have maternal-specific methylation, just as in mouse. Cell culture assays using the methylation inhibitor 5-aza-20 -deoxycytidine increased PEG10 expression (by derepressing the maternal copy), providing a link between this differentially methylated region and imprinted expression of PEG10. The first exon of wallaby SGCE was not differentially methylated as it is in mouse, presumably accounting for its biallelic expression. Prior to this demonstration, differentially methylated regions in gene promoters were undiscovered in marsupials and often considered unimportant for the regulation of genomic imprinting and X inactivation [10,12,48–52]. Clarifying the role of differential methylation of CpG nucleotides in marsupials is an important task for future research (Box 1). Searches for PEG10 failed in platypus and other nontherians [40,44,47], implying that PEG10 arose in the ancestor of therians after their divergence from monotremes 210 MYA. Thus, the retrotransposition of PEG10 was a prerequisite to the introduction of imprinted gene expression to this region. This further highlights the way in which imprinted domains are often seeded by unique genomic events early in mammalian evolution (Figure 2c). Following PEG10 retroposition, insertion of miRNAs and the spread of imprinting to neighbouring genes seem to have occurred only within the eutherian lineage. Evolution of the X-inactivation centre X-chromosome inactivation is intimately linked with genomic imprinting. It equalizes the dosage of sex-linked genes between mammal females (XX) and males (XY) [53] by transcriptionally silencing one female X using nearly all Author's personal copy Review TRENDS in Genetics Vol.23 No.9 445 Box 1. How do marsupials and monotremes do it? Questions for future research Is DNA methylation involved in marsupial imprinting and X inactivation? Marsupial X inactivation and autosomal imprinting has been thought to lack differential DNA methylation of CpG nucleotides, suggesting that it evolved in eutherians to ‘lock-in’ pre-existing silencing mechanisms [50,51]. However, a recent study shows that DNA methylation is involved in the regulation of the imprinted PEG10 expression in wallaby [47]. Will we find more examples of differential DNA methylation in the marsupial genome? How do marsupials complete X inactivation without XIST? Eutherians (or at least humans and mice) require the XIC and XIST for X inactivation [58], but marsupials have no XIST [66,72–74]. How are modified and variant histones recruited without coating with XIST RNA? Is meiotic sex chromosome inactivation retained in the embryo? Marsupials have meiotic sex chromosome inactivation (MSCI) [85], a process that silences the sex chromosomes during spermatogenesis [86]. Is this initial inactivation retained in the zygote to cause silencing of the paternal X? Are non-coding RNAs required for marsupial X inactivation or imprinting? So far, no non-coding RNAs that regulate imprinting have been identified in marsupials. Marsupials inactivate the X without XIST. They seem to regulate imprinted expression of IGF2R without AIR [49] which has been shown by knockout studies to be required for Igf2r imprinting in mouse [87]. No homologue of H19, the wellstudied non-coding RNA associated with IGF2 [88], has yet been found in a marsupial, nor have any of the imprinted miRNAs and snoRNAs [35]. Does imprinted gene regulation in marsupials not require non-coding RNAs? Or have diverged homologues escaped detection? Are there imprinted genes specific to marsupials and monotremes? The reproductive lifestyles of marsupials and monotremes require fewer maternal resources for gestation than for lactation, and the altered opportunities for parental conflict might have resulted in the acquisition of imprinting by different sets of genes. A global search for monoallelically expressed genes might therefore reveal marsupial- and monotreme-specific imprinted genes. Is there X inactivation or dosage compensation in the monotreme X(s)? Do monotremes inactivate any or all of their five X chromosomes in females or is there some other mode of dosage compensation? Is monotreme X inactivation imprinted as it is in marsupials? of the same repressive epigenetic mechanisms as imprinting [54]. Moreover, the first example of imprinting in mammals was the discovery, in 1971, that inactivation in marsupials silenced only the paternal X [54]. By contrast, all adult eutherians tested show random X inactivation, producing a mosaic of cells with either the paternal X or the maternal X inactivated. However, in extraembryonic tissues of rodent and cow (but evidently not human), the paternally derived X is preferentially inactivated [55–57], a process that is reversed in the epiblast and replaced by random inactivation in the embryo (Figure 3). In humans and mice, X inactivation depends on the X-inactive specific transcript (XIST), a key non-coding RNA, which coats the inactive X in cis, initiating heterochromatic silencing [58]. The enigmatic XIST locus is surrounded by non-coding RNAs, which collectively form the X-inactivation centre (XIC) [59,60]. XIST has www.sciencedirect.com Figure 3. X inactivation in eutherian and marsupial mammals. In both, the sex chromosomes are inactivated (white) at male meiosis (MSCI) but remain active in female meiosis (red). In eutherian embryos, the paternal X is reactivated (yellow) at some time after fertilization and there is a 2X-active stage. Paternal inactivation occurs (or is retained from MSCI) in the extraembryonic cells (EE) and random inactivation occurs in the embryo proper (inner cell mass or ICM) so that female eutherian mammals are mosaics of cells with maternal-specific (red) and paternalspecific expression of the X (yellow). In marsupials, no 2X-active developmental stage has been demonstrated, and it is possible that the paternal X remains inactive after fertilization, then is partially reactivated in some tissues (orange patch). an antisense transcript called TSIX. In mice, Tsix works in conjunction with a neighbouring non-coding RNA (Xite) to control the ‘counting’ of Xs within the cell [61], and ‘choice’ of which one to inactivate [62,63]. No orthologue of Xite has yet been discovered in humans, and it is debated whether or not TSIX in human functions to repress XIST as it does in mice, especially considering that human XIST lacks imprinted expression [63,64]. The XICs of human and mouse contain at least two other large non-coding RNAs (JPX and FTX) upstream of XIST [60]. This region has a strikingly high concentration of histone H3 lysine-9 methylation, and has been proposed to act as a ‘nucleation’ centre from which XIST-mediated X inactivation spreads [65]. Interestingly, the FTX gene contains a small cluster of miRNAs [66], which might mediate chromatin remodelling of this locus during initiation of X inactivation, as occurs in other systems (reviewed in Refs [67,68]). XIST is paternally expressed in the extraembryonic tissues of mouse and cow [69,70], accounting for the imprinted X inactivation in these tissues. Thus, the XIC can be considered a bona fide imprinted locus. Is marsupial X inactivation also regulated by an XIST gene within an X-inactivation centre? Searches for a marsupial or monotreme XIST over many years failed to Author's personal copy 446 Review TRENDS in Genetics Vol.23 No.9 identify any orthologue [71]. The availability of opossum, tammar wallaby and platypus genome sequence enabled a detailed search of whole genomic sequence and confirmed the absence of any XIST orthologue in non-eutherians [66,72–74]. Furthermore, no sequence similarity to TSIX, Xite, FTX, JPX or the miRNAs nestled within FTX was found in marsupials or monotremes. Unexpectedly, BACs containing XIC flanking markers were localized to regions far apart on the opossum X chromosome [66,73,74] – and far apart on a platypus autosome [66]. Evidently, then, the region that formed the XIC in eutherians was disrupted independently in monotremes and eutherians, suggesting that, as with the PWS–AS locus, an influx of retroposons, repeats or other foreign sequences rendered it susceptible to rearrangement. Surprisingly, in view of its disruption in marsupials and monotremes, the region that became the eutherian XIC is represented by a single region on chicken chromosome 4p, and a single (unmapped) scaffold in frog, and this must therefore represent the ancestral arrangement. Between the same set of flanking markers lies a set of protein-coding genes in chicken and frog that are absent from the human and mouse X, and one of which, LNX3, is present in the opossum genome [72]. Analysis of near-background exonic similarity between LNX3 and parts of XIST exons showed that LNX3 was the ancestor of at least a part of the eutherian XIST. As LNX3 is common to all amniotes except eutherians and XIST is found only in eutherians, it was proposed that LNX3 gave rise to XIST by pseudogenization during early evolution of eutherian mammals. However, as the overlapping regions do not seem to be structurally similar and do not include the essential stemloop ‘A-repeats’ of XIST, perhaps LNX3 supplied only transcriptional capability, rather than functional motifs [66]. Thus, it seems that the entire eutherian XIC and all of its non-coding RNAs evolved after the divergence of marsupials and eutherians. Around the same time, the XIC also received the retroposed gene ZCCHC13 [60]. Therefore, the XIC shows an evolutionary trajectory remarkably similar to that of the autosomal PWS–AS, CLPG and PEG10 imprinted loci (Figure 2d). Why was imprinting selected? There are many hypotheses to explain why the advantages of diploidy were abandoned by the 100 or so imprinted loci. Perhaps the most widely debated is that imprinting was selected by intra-genomic conflicts within offspring over resources supplied to them by their mother; a theory known as the kinship hypothesis (Box 2; reviewed in Refs [75,76]). For instance, conflict between parental genomes could explain why IGF2 (a growth-enhancing gene) and IGF2R (a repressor of growth) first became imprinted in the ancestor of marsupials and eutherians. It is in the interests of the paternal genome to increase foetal growth by increasing IGF2 expression at the expense of the mother; but it is in the interests of the maternal genome (and its future descendants) to limit this growth by expressing IGF2R. However, the relative advantages of the CLPG and PWS–AS genes to the maternal and paternal genomes www.sciencedirect.com Box 2. The kinship hypothesis " Kin are related individuals who share some of their genes by recent common descent. ‘Symmetrical kin’ share the same paternal and maternal lineage (such as full siblings), and ‘asymmetrical kin’ do not (such as half-siblings). " The kinship hypothesis proposes that genomic imprinting arose out of conflicts between maternally and paternally derived genes over interactions among asymmetrical kin [75,76]. " In many animals (including mammals), the most significant asymmetrical interaction is between mothers and their offspring. Often, mothers administer a significant amount of nourishment and care to developing offspring. Thus, in offspring of polygamous species, a theoretical conflict arises between maternally and paternally derived genes over access to maternal resources. " It is in the best interests of fathers to ensure their offspring receive maximum maternal resources, even if this reduces the mother’s capacity to rear further offspring (which may, after all, not be his). " Conversely, mothers maximize their fitness by rationing their resources equally over all their offspring, regardless of who the father is. are not striking, although CLPG has an overgrowth phenotype [27], PWS (and AS in mice) phenotypes include abnormal feeding behaviour [77,78], and AS affects behaviour in ways that suggest a trigger for maternal care [79]. This parental conflict is expected to play itself out in different ways in the egg-laying non-mammals and monotremes, the marsupials, and the eutherians (Figure 1). In an egg, the paternal genome has no opportunity to manipulate the maternal resources, which are laid down at the mother’s discretion. Consistent with this is the finding that no imprinted genes have been discovered in egg-laying animals. The altricial young of marsupials have a limited opportunity to exploit the uterine environment because birth occurs very early compared with the prolonged gestation in eutherians. Despite this, genes such as IGF2, IGF2R, PEG1/MEST and PEG10 are imprinted in marsupials [8,10,12,47] and probably affect marsupial placental growth and development, just as in eutherians [40,80]. Theoretically, the evolution of lactation provides opportunities, even for monotreme hatchlings, to demand more maternal resources at the expense of the mother and her reproductive future [81]. The advent of whole-genome analysis technologies that can exploit marsupial and monotreme sequence data might even reveal imprinted genes specific to marsupials and monotremes (Box 1). A second wave of evolution of imprinting might account for recently evolved eutherian-specific imprinting at loci such as PWS and CLPG, driven by an increased potential for conflict [34]. Access to maternal resources would have become greater as the placenta became more invasive and more crucial to the prolonged intra-uterine development of eutherian offspring. However, not all imprinted genes need to have been selected for imprinting due to parental conflict. Many (e.g. Frat3 and other genes retroposed into imprinted regions) seem to have been ‘innocent bystanders’ that were caught up in domain-wide repressive chromatin changes [36]. It could also be that some genes became imprinted for entirely different reasons, such as the need to equalize gene dosage following duplication [39], to protect against spontaneous development of unfertilized eggs [82] or because of intra-locus sexual conflict [83]. Author's personal copy Review TRENDS in Genetics Vol.23 No.9 Concluding remarks Comparative analysis of large imprinted loci across the divergent mammalian groups discussed here shows that construction of imprinted domains can occur in a strikingly similar fashion. 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