ANRV386-GG10-11 ARI 18 May 2009 18:45

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Evolution of GenomicImprinting: Insights fromMarsupials and MonotremesMarilyn B. Renfree,1,2 Timothy A. Hore,1,3

Geoffrey Shaw,1,2 Jennifer A. Marshall Graves,1,3,∗

and Andrew J. Pask1,2,4,∗

1ARC Center of Excellence for Kangaroo Genomics; email: m.renfree@unimelb.edu.au2Department of Zoology, The University of Melbourne, Melbourne, Victoria 3010, Australia3Research School of Biological Sciences, The Australian National University, Canberra,ACT 0200, Australia4Department of Molecular and Cellular Biology, University of Connecticut,Connecticut 06269

Annu. Rev. Genomics Hum. Genet. 2009.10:11.1–11.22

The Annual Review of Genomics and Human Geneticsis online at genom.annualreviews.org

This article’s doi:10.1146/annurev-genom-082908-150026

Copyright c© 2009 by Annual Reviews.All rights reserved

1527-8204/09/0922-0001$20.00

∗Co-corresponding authors.

Key Words

marsupials, monotremes, placenta, brain, retrotransposons,comparative genomics

AbstractParent-of-origin gene expression (genomic imprinting) is widespreadamong eutherian mammals and also occurs in marsupials. Most im-printed genes are expressed in the placenta, but the brain is also a fa-vored site. Although imprinting evolved in therian mammals before themarsupial-eutherian split, the mechanisms have continued to evolve ineach lineage to produce differences between the two groups in terms ofthe number and regulation of imprinted genes. As yet there is no evi-dence for genomic imprinting in the egg-laying monotreme mammals,although these mammals also form a placenta (albeit short-lived) andtransfer nutrients from mother to embryo. Therefore, imprinting wasnot essential for the evolution of the placenta and its importance in nu-trient transfer but the elaboration of imprinted genes in marsupials andeutherians is associated with viviparity. Here we review the recent anal-yses of imprinted gene clusters in marsupials and monotremes, whichhave served to shed light on the origin and evolution of imprintingmechanisms in mammals.

11.1

Review in Advance first posted online on July 16, 2009. (Minor changes may still occur before final publication online and in print.)

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Genomic imprinting:parent-of-origin–specificexpression

INTRODUCTIONParent-of-origin silencing of specific genes,better known as genomic imprinting, is a cis-acting mechanism that leads to the silencing ofgenes in the parental germlines that renderseither the maternally or paternally inheritedcopy silenced while the other copy is active inthe newly formed embryo (and placenta). Thisepigenetic silencing raises many fundamentalquestions about the mechanism, as well as theevolutionary forces that might have selected forsuch an apparently contrary system. Marsupialsand monotremes, Australia’s iconic mammaliangroups, can provide some clues to how, and evenwhy, genomic imprinting evolved.

To date, about 80 mammalian genes(mapped to 10 chromosomes in the mouse)show parent-specific gene expression (7, 67).An epigenetic mark, or “imprint” distinguishesparental alleles from each other. This epige-netic mark is established at an imprint controlregion (ICR) during gametogenesis and is inter-preted and propagated postfertilization, givingrise to parent-specific expression of genes in theembryo. ICRs are methylated in the germline,and while maternal ICRs are usually promot-ers for antisense transcripts, paternal ones areintergenic (22, 95). Most imprinted genes areclustered together in a handful of domains scat-tered throughout the genome, each typicallycontaining several protein-coding genes. Thefunctions of 26 of the 78 imprinted genes inthe mouse have been investigated, largely fromknockout studies. Around 50% of imprintedgenes are involved as embryonic or neonatalgrowth regulators, about 20% are involved inneurological processes, and the remainder haveno obvious biological function identified as yet(7). Recently, these protein-coding imprintedgenes have been divided into three distinct cat-egories: genes that act only in the placenta, im-printed genes with no associated fetal growthaffects that act in early postnatal stages, and anetwork of genes that are expressed in the em-bryo and placenta that act prenatally, many ofwhich are also involved in postnatal metabolicregulation and many of which are expressed inthe brain (13).

As a consequence of parent-specific ex-pression, species possessing genomic imprint-ing require the presence of both parentalgenomes for normal development of the fetusto term. The failure to make viable diploidswith both chromosome complements from thefemale germline (a gynogenote) or the malegermline (an androgenote) provided the firstevidence that the gametic genomes are notfunctionally equivalent (61, 62, 94). Further-more, this experiment explained why partheno-genetic species, which are found in all othervertebrate taxa, are not found in mammals.Although this was not the first discovery ofimprinting in mammals [paternal-specific ex-pression of X-linked genes in kangaroos wasdiscovered a decade earlier (89)], these exper-iments initiated intensive research into parent-specific gene expression.

Genomic imprinting shares many regula-tory mechanisms with other types of epige-netic gene control. DNA methylation is usedthroughout development to regulate gene tran-scription from pluripotent cells into differenti-ated tissues of the developing embryo (81, 95).De novo DNA methylation is established in aparent-specific manner at the ICR of imprinteddomains during gametogenesis, using the samemembers of the DNA methyltransferase 3 fam-ily as are used to silence genes in development(47, 49). This epigenetic mark is thought to bethe major way in which the cell distinguishesbetween parental alleles. Mechanisms involvedin propagating this initial mark also appear tobe mediated by general factors that have beenhijacked from other epigenetic processes forthe purpose of establishing imprinting. For in-stance, the regulation of imprinting at the in-tensively studied insulin-like growth factor 2(IGF2) locus is largely dependent upon the in-sulator protein CTCF (Figure 1), which bindsto more than 20,000 different sites in the humangenome (8, 54), only a small fraction of whichare predicted to be imprinted. The ICR of theIGF2 locus contains a CTCF binding site thatis methylated only on the paternally derivedchromosome (99). CTCF binds the unmethy-lated maternally derived ICR, and in doing so

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ANRV386-GG10-11 ARI 18 May 2009 18:45

mediates the formation of unique chromosomeloops (68) that block (or insulate) IGF2 fromdownstream enhancers, effectively switchingoff its expression (9, 37). In contrast, the pa-ternally derived IGF2 promoter has uninhib-ited access to these enhancer sequences becauseCTCF cannot bind the paternally methylatedICR, allowing IGF2 expression.

Nonprotein-encoding RNAs are a promi-nent feature of imprinted loci, and 8 of the12 known clusters contain at least one non-coding RNA (71). Small hairpin RNA such asmicroRNAs are used widely during develop-ment to silence genes and in at least some casesare used to impart parent-specific silencing ofimprinted genes (19). Large noncoding RNAsalso appear at many imprinted loci, such as thematernally expressed H19 transcript that orig-inates immediately adjacent to the IGF2 ICR.Some of these large noncoding RNAs may actto silence surrounding loci within an imprintedregion. This would be similar in function to thenoncoding RNA transcriptXIST, which is es-sential for X-chromosome inactivation (silenc-ing) in female humans and mice, and coats theinactive X and recruits it to a compartmentwithin the nucleus where gene expression is re-pressed (40).

Why genomic imprinting has evolved,and been selected for, presents a conundrum.Monoallelic expression caused by genomicimprinting increases the risk of genetic disease,since organisms will only have a single activeallele of any imprinted gene. All mutations inthe single active copy are therefore dominant(107). Even without gene mutations, correctexpression of imprinted genes can be easilyupset. If an individual receives two sister chro-mosomes from one parent through uniparentaldisomy, imprinted genes will have either twicethe amount of “wild-type” gene expression orno expression at all (25). Furthermore, loss ofimprinting can occur at some imprinted geneswhereby the silenced allele is reactivated. Suchan epi-mutation is considered to be a majorcause of familial and sporadic cancer, and wasfirst discovered for the imprinted insulin-likegrowth factor 2 (IGF2) in the early 1990s

ICR Enhancers

IGF2 H19

CTCF

Maternalchromosome

IGF2 H19Paternalchromosome

Boundary

IGF2 H19Maternalchromosome

IGF2 H19Paternalchromosome

DMD

Boundaryelement

DMR1 silencer

Silencer Enhancers

Slc22a3Maternalchromosome

Paternalchromosome

Slc22a2 Slc22a1 Igf2r Mas 1ICE

Slc22a3 Slc22a2 Slc22a1 Igf2r Mas 1ICE

AIR-NC

ncRNA

Figure 1Regulation/mechanisms of imprinting. In the IGF2-H19 cluster, H19 appearsto be mainly regulated by DNA methylation. Although H19 is a noncodingRNA, it does not function as a silencer of IGF2. IGF2 is paternally expressed( pale blue) and H19 maternally expressed ( pale pink). The two genes share anenhancer region. The ICR is unmethylated on the maternal allele, permittingan insulator, CCCTC binding factor (CTCF), to bind and prevent interactionswith a downstream enhancer acting as a boundary controller (9, 37). Absence ofICR methylation allows maternal H19 expression. When the ICR is methylated(as on the paternal allele), H19 expression is prevented and IGF2 hasunencumbered access to its enhancers, promoting expression. In the IGF2Rcluster the methylated ICE contains the AIR ncRNA promoter that is directlysilenced by the DNA methylation imprint (7). Red lines/orange arrows showblocking or activation of the DMR silencer; orange arrows indicatetranscription; methylation shown by closed circles; open circles unmethylated;active silencers shown by black boxes; ellipses with cross hatching areenhancers; site of action of silencers or enhancers by blue arrows/lines in cis.CTCF site in blue hexagon. ICE:imprinting control element.

www.annualreviews.org • Imprinting in Mammals 11.3

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Eutherians:worldwide mammaliangroup that is typicallycharacterized byextended pregnancy;includes humans,mice, and commondomestic mammals

Marsupials: mammalgroup from Australasiaand South Americathat give birth to tinyaltricial young thatlargely depend uponlactation for earlydevelopment

Monotremes:Australasian mammalsthat lay eggs andsuckle young with milk

(74, 78). Indeed, the cost of imprinting is re-flected in its association with many human dis-eases or syndromes, including various cancers,such as Prader-Willi, Angelman, Beckwith-Wiedemann, Retts, and Silver-Russell syn-dromes. However, the persistence of genomicimprinting for over 125 million years ofmammalian evolution would suggest thatdespite the association with disease, imprintingmust confer some evolutionary advantages.Comparative genomics provides a novel way toexamine the origins of genomic imprinting inmammals and the selective forces responsiblefor its prevalence.

THEORIES OF THE ORIGINOF GENOMIC IMPRINTING:THE HOW AND THE WHY

How: Host Defense Mechanismsor de novo Imprinting?

Before we can discuss selection forces actingon imprinting, it is important to discuss howit arose. The host defense hypothesis proposesthat imprinting evolved as a consequence ofDNA methylation driven by machinery de-signed to silence foreign DNA elements that in-sert into the genome (6). The insertion or rapidexpansion of foreign DNA elements withinthe cell attracts DNA methylation and histonemodifications (7). This hypothesis is supportedby the finding that the same enzyme requiredfor genomic imprinting, de novo methyltrans-ferase 3L (Dnmt3L), also plays a major rolein the methylation and silencing of retrotrans-posons in the male germline (6, 7, 11). The hostdefense hypothesis can be tested by compara-tive genomics, as highlighted later.

Why: Parental Conflictor Coadaptation?

Many theories have been advanced to explainwhy genomic imprinting evolved, i.e., whatthe selective advantages are. Here we discussthe two major theories whose predictions areclosely entwined with the organism’s mode of

reproduction, and could therefore be tested bycomparisons of imprinting between eutheri-ans, marsupials, and monotremes. The parentalconflict (also known as genetic conflict andmore recently as the kinship hypothesis) the-ory is perhaps the most widely accepted, buthas recently been challenged by the coadapta-tion theory.

The genetic conflict hypothesis states thatthere will be selection for paternal genes thatmaximize transfer of nutrients to the fetus, evento the detriment of the mother (34, 66). Themother may protect herself against the demandsof the fetus by suppressing the growth inducedby the paternal genes. Thus, optimal fitness forthe male is achieved when the female carryinghis young invests the maximum amount of nu-trition into his fetus(es), regardless of the fit-ness consequences on the mother. The mother,on the other hand, achieves optimal fitness bybalancing the nutrient provision to the currentfetus(es) so that she has resources to supportsubsequent pregnancies. Two corollaries of theconflict hypothesis are that imprinting wouldbe expected in any polygamous animal in whichthere is a contribution of maternal resources tothe embryo (usually via the placenta) and thatimprinted genes affect fetal nutrient delivery ordemand (72).

During development, the fetus can belikened to a parasite of the mother that de-pends on the placenta to transfer nutrients andgases. As the placenta is of fetal origin, pater-nally derived genes in the offspring have ampleopportunity to sequester maternal resources.For example, the trophoblast produces severalhormones that regulate maternal physiologyfor fetal benefit. In some eutherian speciesincluding humans and mice, fetal trophoblastcells invade and remodel the maternal vascularsystem so that the mother is unable to influencethe nutrient volume of blood reaching theplacenta or to divert it to other tissues. It istherefore easy to envisage how paternally ex-pressed genes might pillage maternal resourcesduring the extended pregnancy of eutherianmammals. Indeed, many imprinted genes affectboth the demand and supply of nutrients across

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the placenta (5, 80). Of particular relevance tothe parental conflict hypothesis is the observa-tion that paternally expressed genes promotegrowth of the placenta and the fetus, whereasmany maternally expressed genes (MEG)appear to repress fetal growth. For instance,the paternally expressed growth factor IGF2promotes growth of the placenta and fetus(14), whereas the maternally expressed IGF2receptor (IGF2R) functions antagonistically toIGF2 by binding to it and transporting it to thelysozymes for degradation (55).

This hypothesis can also explain why somegenes are imprinted in the brain to influencefeeding behavior in the young. For example,the XLalphaS protein from the GNAS complexis paternally expressed in neonatal mice (76)and humans (39). When knocked out, this genecauses a striking failure of the mother to suckleher young (28, 77). Although the parental con-flict or kinship hypothesis does fit many ofthe observations associated with genomic im-printing, it does not fit all. For instance, pa-ternally inherited deletions of human chromo-some 15q11-13 cause the neurological disorderPrader-Willi syndrome that is characterized byovereating and obesity postweaning (70), theopposite of what might be expected from theparental conflict hypothesis. Although explana-tions as to how this might still conform to con-flict theory have been provided (35, 102), thisand other inconsistencies remain major chal-lenges for the parental conflict hypothesis (33).

Coadaptation

Recently, Keverne and colleagues have made astrong case for the idea of coadaptation (15, 16,50, 97). They propose that genomic imprintingcoadaptively regulates mammalian embryonicdevelopment and reproductive behavior (50).Germline reprogramming of imprints occursin mammals to restore totipotency to thegametes. They argue that the genetic conflicttheory may be at odds with the mechanismsthat underpin genomic imprinting at themolecular level, since control over genes thatare paternally expressed is often achieved in the

MEG: maternallyexpressed gene

female germline where the imprint is formed,which leads to silencing of the maternal allele.It is difficult to explain how an effect on thefetal-placental unit that is disadvantageous tomothers would be selected.

The coadaptation theory has particularlyinteresting implications for the maternal-offspring relationship. Since a subset of genesthat are expressed in the placenta are also ex-pressed in the hypothalamus, and most of thelatter are paternally expressed, these genes maybe developmentally coordinated (Figure 2).Imprinted genes have a role in brain function-ing with effects not only during postnatal de-velopment, but also on social behavior in theadult (44). The placenta and the maternal hy-pothalamus must function as one unit, despitethe fact that they are derived cells with differentgenotypes. Because the placenta can hormon-ally regulate the maternal hypothalamus, as wellas the developing fetal hypothalamus, and be-cause these events overlap in time, it is difficultto reconcile with the parental conflict hypoth-esis. The placental and fetal expression of onegene that has been most studied, Peg3, resultsfrom imprinting of the same maternal allele, butthis gene in the maternal hypothalamus was im-printed in the germline of the mother’s mother(50). Thus control at the level of the maternalgenome is most likely to be a result of selectionfor coexpression of genes in the placenta andhypothalamus, i.e., for parent-infant coadapta-tion with expression primarily through matri-lineal control.

Peg3 is a key example of a gene that is im-printed in all three tissues: the fetal placenta, thefetal hypothalamus, and the maternal hypotha-lamus. Peg3 is paternally expressed in the brainand in the placenta, and is essential for normalmaternal and pup sucking behavior (16). A mu-tation in Peg3 causes a failure of milk ejection(57), and it appears to act in a similar way toGnasxl in regulating suckling behavior (15, 44).Intrauterine development of knockout mice isslower than normal, and postnatally the pupsdo not suck while Peg3 knockout mothers failto rear their pups due to a lack of motheringbehavior (15, 16). Male sexual behavior is also

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Placenta and fetal hypothalamus regulate growth andsignal to maternal hypothalamus to activate maternalcare and milk production

Maternal hypothalamus regulates utero-placentalnutrient transfer

Maternal hypothalamus regulates milk let down andnursing behavior

Pup hypothalamus regulates sucking, thermoregulation,and growth

Maternallyimprinted gene

Maternally imprinted gene

G1

G1

G2

G2

Placenta

Figure 2Coadaptive evolution. The maternal hypothalamusregulates transfer of nutrients to the fetus across theplacenta. The placenta and the fetal hypothalamusregulate growth and signal to the mother’s hypotha-lamus to activate maternal care and milk production.The mother’s hypothalamus regulates milk let-downand maternal behavior. After birth, the pup’shypothalamus regulates their sucking activity, ther-moregulation and growth. Genomic imprinting at anallele that is maternally expressed in the placenta (e.g.Peg3) is also the allele expressed at the same time inthe brain (hypothalamus) of the fetuses and mothers.Placental expression is functionally constrainedby its interaction with the maternal hypothalamus.Co-adapting these functions provides theselection pressures for development of the maternalhypothalamus, which in the female offspring in thenext generation again interacts with the placenta (50).

controlled by Peg3, since mutant mice fail toshow gender-specific sexual behavior and havea lack of aggression to other male mice (97). Im-printing in this case could not be explained un-der the evolutionary selective forces that wouldgovern parental conflict.

Keverne & Curley (50) argue that the si-lencing of maternal alleles could confer anotherpossible advantage in exposing novel recessivealleles of imprinted genes to rapid selection.Since mammals are diploid, the incidence ofbeneficial mutations is a rate-limiting step inadaptation. Haploid expression, as occurs whenone allele is silenced, will rapidly fix a trait in apopulation, so beneficial mutations will be se-lected for and spread more quickly. However,genomic imprinting produces haploid domi-nance for lethal, as well as beneficial, allelesand deleterious mutations (by far the most com-mon) in imprinted genes would be expected tooffset any such advantage. Keverne & Curley(50) conclude that “transgenerational interac-tions between hypothalamus, placenta and fetalhypothalamus provide a template for mother-infant coadaptive evolution through herita-ble traits regulated by chromosomal epigeneticmarks in the maternal germline.”

The occurrence of parthenogenesis amongall nonmammal vertebrate taxa implies that ver-tebrate genomic imprinting evolved only inmammals. It is therefore instructive to exam-ine the evolutionary changes that differentiatedmammals from their reptile ancestors, and askwhether these correlate with imprinting, andwhether informative differences may be foundin different groups of mammals. These consid-erations may offer some means of distinguish-ing the major hypotheses put forward to explainhow and why imprinting evolved in mammals.

IMPRINTING IN MAMMALS

The three groups of living mammals dif-fer greatly in their modes of reproduction(Figure 3). Thus, it may be possible to use dif-ferences in imprinting between these groups todistinguish hypotheses that genetic conflicts inutero or coadaptation between the fetus and

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Reptiles Monotremes Marsupials Eutherians

Reptilian ancestors

BirdsNow

100 mya

200 mya

300 mya

400 mya

Imprinting INS, IGF2r, MEST bymethylation; PEG1 DMRImprinting of CDKN1C, DLK- DIO3and SNRPN-UBE3A

Degradationof BORIS

Imprinting INS, IGF2r,MEST by histone mods

Methylation controls IGF2-H19 domain evolutionof DNMT3LRestriction of BORIS expression to germlineInsertion of PEG10 by retrotransposon imprintingof IGF2R

Gene duplication of CTCF to form BORIS

Sauropsids Synapsids

Therapsids

Therian mammals

Prototherian mammals

Figure 3Evolution of imprinting in mammals. This figure illustrates the major events in the evolution of genomicimprinting in mammals as the amniote vertebrates evolved and diverged. Blue boxes show the evolution ofthe insulator elements CTCF/BORIS; yellow shows the evolution of methylation mediated imprinting andgreen shows 3 genes regulated by histone-mediated imprinting in marsupials that are regulated by DNAmethylation in eutherians.

its mother are drivers of genomic imprinting.Despite a paucity of experimental data inmarsupials and monotremes, commitment toviviparity and complex placentation in the three

mammalian groups correlate to their level ofgenomic imprinting. This strong connectionwith the placenta, both functionally and phy-logenetically, has meant that most theories

www.annualreviews.org • Imprinting in Mammals 11.7

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Therian: marsupialand eutherianmammals

attempting to explain why imprinting evolveddeal specifically with viviparity and early de-velopment (48). Since imprinting is widespreadamong eutherian mammals, and is present inmarsupial mammals but not monotreme mam-mals or birds that lay eggs, one assumption hasbeen that it must be associated with the evolu-tion of viviparity. Yet, although there are manyviviparous animals, from seahorses, scorpions,and selachian sharks as well as fish, amphibians,and reptiles (4), genomic imprinting has not sofar been identified in any of these. Thus a cru-cial test of the conflict hypothesis was whethergenomic imprinting exists in viviparous taxaother than in the Eutheria (72). While pla-centation in teleost killi-fish was not associatedwith imprinting per se, it was associated withpositive selection in the IGF2 gene, indicativeof parental conflict (73). Perhaps without themeans to establish epigenetic imprinting, sucha mechanism was unable to evolve.

Neonatal

Maternal

0.08%

Monotreme

0.12%

Marsupial

15%

Eutherian

Figure 4Maternal investment between mammalian groups. The mass of marsupial andmonotreme neonates relative to their mother (0.12% and 0.08%, respectively)is much smaller than that of eutherian neonates (15%) (38). Despite this, thetotal mass of monotreme litters at weaning relative to the mothers mass isaround 50% [calculated from echidna data reported in (32)]. This proportion issimilar for marsupial (55%) and eutherian mammals (59%) of comparable size(38). Thus, the maternal investment of marsupials and monotremes is greaterduring lactation than placentation but the total is very similar between all threeextant mammalian groups.

Mammal Relationships and Modesof Reproduction

Early mammals evolved from a reptile-like an-cestor about 310MYA (Figure 3). Monotrememammals (platypus and several species ofechidna) diverged from therian mammals about166 Mya, and the therians split into euthe-rian mammals and marsupials between 125 and148 Mya (10, 60).

The three groups of mammals differprincipally in their mode of reproduction.Eutherians nurture their young in utero foran extended period of time and most deliverhighly developed young at birth. Marsupialshave a brief intrauterine life and are born tinyand altricial, but have developed an elaborateand sophisticated lactation that supports theyoung for the remainder of their development.The relatively short-lived marsupial placentais fully functional, produces hormones, andis essential for fetal development (26, 27,82–84, 88, 101). Furthermore, there is amaternal recognition of pregnancy, and despiteits altricial development at birth, the fetusprovides endocrine signals to the mother thatare responsible for the initiation of parturition(43, 86, 88, 90). Monotremes lay eggs after abrief period of intrauterine growth supportedby the uterine secretions transferred acrossthe yolk sac, and suckle altricial hatchlingswith milk that, like marsupial milk, changesin composition during the lactation period.Thus there are significant differences in theproportion of maternal investment allocated togestation and lactation (Figure 4). Marsupialsand monotremes have elaborated lactation,whereas eutherians have elaborated placenta-tion: Marsupials have traded the umbilical cordfor the teat (84). Nevertheless, many eutherianmammals also deliver highly altricial young,such as, for example, bears, rodents, and theinsectivorous shrews that all give birth to highlyaltricial young. This point is often forgotten incomparisions across the three groups of mam-mals. Conversely, the guinea pig is probablythe most precocial mammal at birth, since new-born young can survive without that essential

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mammalian character, mother’s milk. Humansare considered secondarily altricial, since theyneed to deliver offspring that can pass throughthe birth canal and yet allow for the consider-able growth of the brain that occurs postnatally.However, the common ancestral mammal waslikely to have delivered tiny altricial young(85) so the imprinting that we observe incurrent-day mammals must have resulted fromselection for these mechanisms in animals withmore limited placental dependence than mostcurrent day eutherian mammals.

Imprinting in Eutherian, Marsupial,and Monotreme Genomes

Imprinting is widespread among eutherianssuch as humans, mice, and common domes-ticated mammals. Many of these genes areknown to be very important or essential for fe-tal growth and placental development in euthe-rians (67, 100). Imprinting has also been ob-served in several of the same gene clusters inmarsupials (87), but some genes have differentexpression patterns in eutherians and marsu-pials. No imprinted genes have been found inmonotremes (42, 46, 52), although investiga-tions have been limited by availability of tis-sues, and no developing young or fetuses havebeen available for examination. Below we dis-cuss the similarities and differences in eachof the major domains of imprinted genes andhave grouped these under three different waysthat the imprinted domains may have evolved,namely stepwise accumulation of imprinted fea-tures, genesis of a new genomic region by retro-transposition, and generation of a new genomicregion by genome reorganization.

Stepwise Accumulation of ImprintedFeatures: IGF2–H19 ImprintedCluster

IGF2 and H19, the best-characterized im-printed loci, reside in an imprinted domain thatalso includes the imprinted genes INS, KCNQ1,KCNQ1OT1, CDKN1C, TSSC5, and TSSC3.IGF2 is a key fetal and postnatal growth factor

that is highly conserved in vertebrates. Deletionof the paternally expressed IGF2 gene resultsin intrauterine growth restriction (20), whereasdeletion of the maternally expressed H19 geneor overexpression of the IGF2 gene results in fe-tal overgrowth (93, 103, 104) (Figure 2). SinceIGF2 is so intimately involved in growth ofthe fetus in eutherian mammals and has beenimplicated in several aspects of placental de-velopment, including blood vessel formation(36), trophoblast invasion (30, 63), and nutri-ent transfer (14), it and its receptors and an-tagonists are logical genes on which to basea test of the genetic conflict hypothesis. Theentire region has been extensively investigatedin marsupials and partially investigated in theplatypus. Despite the short-lived attachment ofthe marsupial placenta and relatively minor in-trauterine growth, IGF2 is paternally imprinted(as it is in eutherian mammals), in at least threemarsupial species (the tammar wallaby, Macro-pus eugenii, South American short-tailed grayopossum, Monodelphis domestica, and the NorthAmerican opossum, Didelphis virginiana) (51–53, 72, 91, 96). Functional analyses in the tam-mar wallaby placenta have also demonstratedan equivalent biological function for IGF2 pro-moting vascular endothelial growth factor pro-duction and yolk sac vasculogenesis as seen ineutherian placentas (1).

The oppositely imprinted H19 gene encodesa noncoding RNA molecule that is thought tofunction as a microRNA (miRNA) precursorinvolved in the posttranscriptional downregu-lation of specific mRNA targets. H19 was ini-tially thought to be absent from the marsupialgenome as a homologous sequence could notbe detected in the genome with conventionalalignment tools. However, recently devel-oped and more sophisticated alignment toolsthat also examine DNA structure have detectedan actively transcribed paternally imprinted or-thologue in the marsupial genome contiguouswith marsupial IGF2 and paternally imprinted(91). Marsupial H19 orthologues (from boththe tammar wallaby and opossum) show conser-vation of the microRNA precursor structure,miR-675, and exon structure conservation,

www.annualreviews.org • Imprinting in Mammals 11.9

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DMR: differentiallymethylated region

suggesting functional selection on both fea-tures. Methylation of the paternal regionupstream of H19 encompasses a CTCF insu-lator binding site as in eutherians, and spreadssomatically into the H19 gene (91). Theconservation in all therians of the mechanismcontrolling imprinting of the IGF2-H19 locussuggests a critically regulated role for thesegenes in mammalian development (91). It alsosuggests that the complexity of imprintingmechanisms is conserved across disparatemammalian species and evolved in response toevolutionary pressures in the common therianancestor.

Recently, a DMR (differentially methylatedregion) has been identified in the IGF2 region ofthe opossum genome; however, demethylationof this region caused activation of the mater-nal opossum allele while demethylation in micepromotes repression of the paternal IGF2 allele(56). So while the same mechanism regulatesgene expression, its effects on gene silencinghave evolved differently in different lineages.

Another eutherian imprinted gene withinthe same cluster that appears to act antago-nistically with IGF2 in placental developmentis CDKN1C ( p57KIP2). Placental dysplasiacaused by a lack of p57KIP2 can be suppressedby reducing the levels of Igf2 (12). p57KIP2

is downregulated in mice with high serumlevels of IGF2 and its expression is reduced inIGF2 treatment of embryonic fibroblasts (29).CDKN1C ( p57KIP2) is syntenic to IGF2 andH19 in marsupials but, interestingly, it is notimprinted (96). Marsupials provided an inter-esting system in which to examine the evolutionof this antagonistic relationship since CDKN1C( p57KIP2) is not imprinted in the marsupialgenome whereas IGF2 is. Despite the absenceof imprinting, CDKN1C protein is produced atthe same time and in the same cell types as IGF2in the tammar wallaby placenta (1, 3). There-fore, imprinting of the two genes could haveevolved concurrently to balance their maternaland paternal influences on the growth of theplacenta. CDKN1C imprinting in eutheriansis regulated by the antisense RNA productionfrom the adjacent KCNQ1 gene, producing the

KCNQ1OT1 transcript. Marsupials also have anadjacent copy of the KCNQ1 gene and produceand antisense KCNQ1OT1 transcript, despitethe absence of CDKN1C imprinting, suggest-ing that antisense transcription at this locuspreceded imprinting of this domain (2). Thesefindings demonstrate the differential stepwiseaccumulation of control mechanisms withinimprinted domains in different mammalianlineages (Figure 5), and show that CDKN1Cplacental expression and interaction with IGF2preceded its acquisition of imprinting.

INS (insulin) is another maternally im-printed gene found within the same region andthat directly regulates the expression of IGF2.Mice have two different insulin genes, believedto be due to gene duplication (Ins1 and Ins2),whereas humans and marsupials have only one(INS). Ins2 and its human orthologue (INS) areexclusively paternally expressed in the yolk sac(21, 65). Similarly, INS is maternally imprintedand paternally expressed in the marsupial yolksac [the primary placenta in most marsupials (1,87)]. In all species, INS imprinting is restrictedexclusively to the yolk sac, suggesting that im-printing at this locus evolved as the result ofselection within this tissue in the therian ances-tor of marsupials and eutherians, in direct sup-port of the kinship hypothesis. This also sug-gests that imprinting evolved in the placentasindependently of other tissues.

Together, the complete analysis of this re-gion (IGF2/H19) illustrates the differential ac-quisition and maintenance of imprinting at dif-ferent loci in marsupials and eutherians. Thesame regulatory mechanisms appear to existwithin the region in both lineages, but im-printing is more widespread in eutherians. Thismight possibly be due to the increased selec-tive pressure of parental conflict during the ex-tended eutherian gestation. However, supportfor this proposal requires more detailed func-tional investigations to determine links betweenthe genes in marsupials compared to those ineutherians. Such studies will help to shed fur-ther light on the selective pressures that mayhave led to the extended imprinting seen ineutherians.

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IFG2 Receptor

The IGF2 receptor (IGF2R) is another genethat is imprinted in mice and humans. Althoughit is not on the same chromosome as IGF2, itplays a critical role in its signaling. IGF2R an-tagonistically regulates IGF2 by binding it atthe cell surface, and the ligand-receptor com-plex is internalized, targeted to lysosomes, anddegraded (107). In mice, Igf2r imprinting is reg-ulated by methylation of a CpG island in intron2 of the gene, and silenced by the production ofthe antisense Igf2r RNA transcript (Air). IGF2R

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 5Different mechanisms governing the evolution ofimprinted domains by (a) stepwise accumulation ofimprinted genes, (b) genesis of a new imprintedregion by retrotransposition or (c) genomereorganization or (d ) divergent evolution. (a) Thetop diagram shows the CDKN1C region in themarsupial (top) and the mouse (bottom). Imprintinghas spread in this region to encompass neighbouringloci, and the formation of another DMR has led toimprinted expression of the antisense RNAKCNQOT1, which is biallelic in marsupials. (b) ThePeg10 region in monotremes (top) marsupials(middle) and eutherians (bottom). Imprinting wasattracted to the region along with the insertion ofPeg10 in the therian ancestor. This imprint has thenspread to neighboring loci in eutherians to form anew imprinted gene cluster. (c) The top diagramshows the marsupial and monotreme organization ofUBE3A and SNRPN on separate chromosomes. Ineutherians (bottom diagram) these regions have beenbought together through genome reorganisationalong with the insertion of several genes and theexpansion of a SnoRNA cluster. This has led to theacquisition of imprinting to this region only in theeutherian lineage. (d ) The top diagram shows theorganization of the Dio3 cluster in the therianancestor. Different evolutionary forces have driventhe divergent evolution of this region in eutherians(bottom left diagram) and marsupials (bottom right). Inmarsupials there was a loss of Rtl1 and an expansionof repeats but no imprinting. In eutherians there isretention of Rtl1, an antisense transcript from thisgene, a DMR within the locus, and imprinting ofthe region. Red boxes indicate maternally expressedgenes; blue boxes indicate paternally expressedgenes; gray boxes indicate genes with unknownimprint status; black boxes indicate non-imprintedgenes. Blue lollipops show paternal methylation; redlollipops show maternal methylation.

is also imprinted and maternally expressedin marsupials, despite the absence of severalkey features that regulate its imprinting inmice, including differential methylation, a CpG

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www.annualreviews.org • Imprinting in Mammals 11.11

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PEG: paternallyexpressed gene

island within intron 2 and antisense IGF2RRNA production (51, 53, 106). Imprinting atthis locus therefore occurred in the therian an-cestor, but is maintained by different imprintingmarks and mechanisms in the different mam-malian lineages.

Genesis of a New Imprinted Regionby Retrotransposition: PEG10and DLK/DIO3/RTL1

One of the most consistent genomic changes,coincident with the evolution of imprintingat many loci, is repeat invasion or expansion.Retrotransposons are widespread in the mam-malian genome, and several are associated withthe acquisition of imprinting.

Analyses of PEG10 a retrotransposon-derived imprinted gene, across the three ex-tant mammal groups, provided the first ex-ample of the genesis of an imprinted domainderived from the insertion of a retrotransposon-derived gene (Figure 5). The absence of thisgene from monotremes implies that PEG10 wasinserted into the genome of mammals after themonotreme–therian mammal split. The inser-tion of this foreign DNA element is coincidentwith the advent of imprinting to this regionin the therian ancestor. In eutherians the im-print has spread from this gene to encompassneighboring loci where, as in marsupials, it isrestricted to the PEG10 locus (96). This gene isalso differentially methylated in both marsupi-als and eutherians and shows complete silencingfrom the methylated allele. This was the first ex-ample of differential methylation in marsupialsand provided the first evidence that this mech-anism of imprinting regulation clearly evolvedin the therian ancestor. PEG10 also provides di-rect evidence in support of the hypothesis thatimprinting arose from a host defense mecha-nism, to show that foreign DNA insertion candrive the acquisition of genomic imprinting.

The DLK1-DIO3 cluster is a developmen-tally important region in the mouse thatcontains the protein-coding genes Delta-likehomologue 1(Dlk1), retrotransposon-like gene 1(Rtl1/Mart1), and type3 diodinase (Dio3) that are

expressed from the paternal allele (17, 58). Italso contains multiple long and short noncod-ing RNAs including miRNAs and small nucle-olar RNA (snoRNA) genes expressed from thematernal chromosome. There is an intergenicDMR in mouse and human that is methylatedduring spermatogenesis (98). It is expressed inthe fetal and extraembryonic tissues, as well aspostnatally in the brain (18). In seven verte-brates examined, there are evolutionary con-served regions common to particular subgroupsand to all vertebrates (23). A detailed and ex-tensive analysis of this cluster across eutheri-ans, marsupials, and a monotreme shows thatimprinting at this domain must have evolvedafter the marsupial-eutherian split (23, 24). Inmarsupials, the region has accumulated a largenumber of LINE repeats, such that the mar-supial locus is 1.6 megabases, double that ofeutherians (Figure 5). Thus there seems tohave been region-specific resistance to expan-sion by repetitive elements in eutherians to-gether with addition of noncoding miRNAsand C/D snoRNAs (Figure 5) (23). This re-gion highlights the different trajectories the im-printed regions can follow in different mam-malian lineages. Still unknown is whether theoriginal insertion of the RTL1 gene after thedivergence of monotremes from therian mam-mals drove the evolution of imprinting thatwas subsequently lost in marsupials or whetherimprinting only arose at the region after themarsupial–eutherian divergence and coincidentwith the evolution of sno- and miRNAs anddegradation of the gene in marsupials.

Genesis of a New Imprinted Regionby Genome Reorganization andInsertion: SNRPN and UBE3A

Even more fundamental differences betweenthe imprint status in eutherians and marsupialswere described for genes within the Prader-Willi–Angelman syndrome (AS) region (79).Unexpectedly, UBE3A, the gene thought tobe responsible for AS, is not located nextto SNRPN and other genes of human chro-mosome 15q11-13 in platypus, chicken, and

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zebrafish. Instead, UBE3A is next to CNGA3,a gene located on chromosome 2 in humans.The UBE3A-CNGA3 conformation provedto be shared with the wallaby and opossumgenomes, implying that it was the ancestralvertebrate arrangement. A rearrangement thatbrought UBE3A and SNRPN together musthave occurred in a eutherian ancestor aftertheir divergence from marsupials. The onlynon-eutherian group possessing a SNRPN or-thologue was isolated from marsupials, whereSNRPN was found situated adjacent to SNRPB.The position of SNRPN in marsupials andits absence from the genomes of monotremesor nonmammal vertebrates together implythat SNRPN arose from SNRPB by tandemduplication.

There are four other imprinted protein-coding genes upstream of SNRPN in the euthe-rian PWS-AS domain of humans and mice. Allappeared to be missing from the genomes ofmarsupials, monotremes, and nonmammalianvertebrates. The intronless MKRN3, MAGEL2,and NDN genes all have closely related introncontaining genes in the noneutherian species,so they were likely to have been added to thePWS-AS domain by retrotransposition afterthe divergence of marsupials from eutherians(31, 79). The fourth gene, SNURF, is of par-ticular importance because it is implicated inthe regulation of expression at the PWS-AS do-main in humans and mice. No marsupial pro-genitor of the SNURF protein-encoding gene,or the upstream regulatory elements, includ-ing the ICR, which controls imprinting of thisentire region, could be found. Thus, it was per-haps not surprising that SNRPN in wallaby andUBE3A in wallaby and platypus were found tobe nonimprinted (79). Other interesting fea-tures of the PWS-AS region also appear tohave a recent origin. For instance, at least oneof the extensive snoRNA arrays of this locusin eutherian mammals seems to have expandedfrom a single snoRNA copy that is shared withmarsupials (69).

Thus, the PWS-AS region is unique in thesense that it was assembled from a range ofcomponents found in disparate regions of the

genome, and acquired imprinting only in theeutherian lineage (42).

Conclusions from Analysisof Individual Gene Clusters

We conclude from these comparisons that, ingeneral, fewer genes are imprinted in marsu-pial clusters than in eutherians: only 6 of the 13genes so far investigated in detail in marsupialsare imprinted (87). This could suggest that se-lection for imprinting has been more stringentin the eutherian lineage. Alternatively, theremay be as-yet undiscovered marsupial-specificimprinted genes. Imprinting at the IGF2 andIGF2R [but not CDKN1C ( p57KIP2)] loci aswell PEG10 predated the marsupial-eutheriandivergence. However, imprinting at the DLKDIO3 RTL1 domain probably occurred in theeutherian lineage after the two groups di-verged, as did assembly and imprinting at theSNRPN-UBE3A domain. Thus, although theevolution of viviparity and a complex pla-centa in an ancestral therian mammal seemsto have set up the conditions under which im-printing was selected, imprinting has been ac-quired and regulated differently in the differentgroups.

Comparisons of elements within imprinteddomains in eutherians and marsupials providessome clues to the molecular changes accompa-nying the acquisition of imprinting. An increasein repeats was coincident with the acquistion ofimprinting at the CDKN1C locus. Marsupialswith an unimprinted CDKN1C gene also havesignificantly fewer LTR and DNA elements inthe region and in intron 9 of KCNQ1. In ad-dition, there are fewer LINEs in the regionin the tammar wallaby compared with humanand mouse. Furthermore, the CpG island inintron 10 of KCNQ1 and promoter elementscould not be detected. Accumulation of repeatelements within this locus in eutherians wascoincident with the acquisition of imprintingin this locus. Another feature is the accumu-lation of retrotransposed intronless genes intoimprinted domains, some of which also acquireimprinting.

www.annualreviews.org • Imprinting in Mammals 11.13

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EVOLUTION OF GENES THATCONTROL GENOMICIMPRINTING

DNA methylation and histone deacetylationplay an important role for establishment andmaintenance of genomic imprinting “memo-ries” in humans and mice. During normal de-velopment, gene expression is regulated by asequence of methylation and demethylationin both somatic and germline cells. We nowknow that this is controlled by demethylase en-zymes (95), and there is selective erasure and re-establishment of these epigenetic marks by the

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Figure 6Lifecycle of imprinted genes: germline methylation and demethylation. Theepigenetic marks controlling imprinted expression in somatic cells aredependent upon the sex of the parent from which they were inherited. In germcells these marks are erased and new sex-specific marks are established duringoogenesis and spermatogenesis.

de novo methylases DNMT3a and -3b in thedeveloping germ cells (Figure 6). This com-plex establishment and erasure of these epi-genetic marks is now well understood at leastfor mouse and human. The methylation ismaintained by the actions of another methy-lase, DNMT1. Almost all methylation is erasedsoon after fertilization, and the basic patternis re-established around implantation in eu-therian mammals (Figure 6). This demethyla-tion is nonspecific. Later in development, spe-cific demethylation of tissue-specific genes canoccur.

Only one study has been conducted on theglobal methylation of marsupials. The patternof methylation of the marsupial genome issimilar to that of the mouse (92), with threelevels of methylation: hypermethylated in theembryonic DNA, intermediate in the vascularyolk sac, and hypomethylated in the avascularyolk sac. Germline establishment of differentialmethylation has been confirmed in marsupialsfor the H19 gene in male sperm (91), suggest-ing the same mechanisms regulate methylationin all therian mammals.

Brother of regulator of imprinted sites(BORIS) is a germline-specific zinc-finger pro-tein closely related to CTCF that has beenassociated with a function of establishing im-printing, and to have an evolutionary historythat correlates with the evolution of imprint-ing. It shares with CTCF an almost identi-cal DNA binding domain and binds to almostidentical sites (59), but differs in the N- andC- terminal regions, indicating that they per-form differing functions. Like members of theDNMT3 family and a histone modifier, pro-tein arginine methyltransferase 7 (PRMT7),BORIS was found to be essential for establish-ing methylation at an Igf2/H19 ICR transgeneinserted into Xenopus oocytes (45). As CTCFand BORIS can both bind the Igf2/H19 ICR,but BORIS is expressed in the germline andCTCF is expressed everywhere else, it was pro-posed that BORIS establishes and CTCF inter-prets, the imprint mark of the IGF2/H19 ICR.The apparent absence of BORIS sequence inthe chicken genome suggests that the BORIS

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gene is a mammal-specific duplicate of CTCFand that its evolution was intimately linked withthe onset of genomic imprinting (59). How-ever, a recent analysis shows that BORIS is amuch more ancient duplicate of CTCF, as tracesof its sequence can be identified in birds andfully intact sequence exists in at least two rep-tiles as well as marsupials and monotremes (41)(Figure 3). In nonimprinted species (platypusand the lizard bearded dragon), BORIS is ex-pressed in multiple somatic tissues, as well as thegermline, whereas in the imprinted marsupialand eutherians mammals, it is strictly germlinespecific. Thus, rather than the genesis of BORISbeing correlated with the evolution of imprint-ing, it appears that restriction of BORIS expres-sion to the germline was the major event thatsignals its recruitment to the regulation of ge-nomic imprinting.

The phylogenetic distribution of DNMT3Lis also consistent with a major role in the es-tablishment of genomic imprinting. Eutherianand marsupial mammals possess an orthologueof DNMT3L, but the genomes of nonimprintedspecies such as birds and fish do not (108).The genesis of DNMT3L by duplication fromone of the other DNMT3 family members maywell have been a critical event in the evolu-tion of genomic imprinting. No DNMT3L or-thologue has yet been discovered in the platy-pus genome, despite in-depth sequencing ofits genome (105). This is consistent with theproposition that monotremes do not possessimprinting (see below). Thus, genome compar-isons between monotremes and therian mam-mals provide the perfect system in which toexamine changes in the genome that were co-incident with the onset of genomic imprinting.

EVOLUTION OF MAMMALIANIMPRINTING

Recently sequenced genomes of a marsupial(the opossum) (64) and a monotreme (the platy-pus) (105) have become available, enabling thefirst whole-genome comparisons to be madeacross all three mammalian lineages. It is nowpossible to examine the changes that occurred

Imprinted cluster:collection ofneighboring geneswhose imprintedexpression iscontrolled from asingle imprint controlregion (ICR)

in the mammalian genome that coincided withthe advent of genomic imprinting. Orthologuesof every imprinted gene isolated in marsupi-als and eutherian mammals were examined inthe platypus genome (105) (75). In the platy-pus almost all imprinted gene cluster ortho-logues mapped to similar clusters with similargene arrangements, consistent with studiesin birds. Comparisons of the complete se-quence of imprinted clusters between eutheri-ans and marsupials (with confirmed imprinting)and monotremes (without confirmed imprint-ing and no identified DNMTL3 orthologue)showed a significant accumulation of repeats ofspecific classes (LTRs and DNA elements) co-incident with the acquisition of genomic im-printing (Figure 7). It appears that the adventof imprinting arose coincident with repeat in-vasion and expansion in the eutherian genome(75). This finding is in direct support of the hostdefense hypothesis (6) that imprinting evolvedfrom mechanisms within the cell that act to si-lence foreign invading DNA sequences. Thissuggests that host defense–mediated silencingcould have occurred all over the genome tostem the rapid expansion of repeat elementsin therians, but where this silencing led to aselective advantage (in genes that would bene-fit from parental-specific imprinting) this was

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a bEutherianMarsupialMonotremeBird

Figure 7Comparative analysis of DNA and LTR repeats within orthologous regions ofthe platypus genome to those imprinted in marsupials and/or eutherians. Thehistogram shows the mean percent of sequence covered by LTRs (a) and DNAelements (b) across eutherians, a marsupial, a monotreme and a bird. Therewere significantly fewer LTR and DNA elements across the platypusorthologous imprinted regions (asterisks) compared to all other mammalianspecies. Other repeat classes did not differ significantly between mammaliangroups (75). The significant increase in these two classes of repeats iscoincident with the acquisition of genomic imprinting in mammals.

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selected for and retained. Host defense–initiated imprinting was confirmed with thePEG10 locus in mammals in which its inser-tion into the genome following the therian-monotreme split coincided with its acquisitionof imprinting.

Comparisons of the many imprinted loci be-tween the different mammalian groups havealso shown that different mechanisms may haveacted to drive and maintain imprinting at dif-ferent loci. Hence, there may be many differ-ent mechanisms that all result in imprinting,rather than one common mechanism that droveimprinting at all sites in mammals. Early com-parative observations suggested that imprintingwas less stringent and less complex in marsupialmammals, and only become stringent and elab-orated in eutherians. However, more recent de-tailed imprinted analyses have shown that mar-supial imprinted regions are just as complex aseutherian domains.

CONCLUSIONS

A strong connection between imprinting andthe placenta, both functionally and phyloge-netically, has meant that most theories put for-ward to explain the genomic imprinting dealspecifically with viviparity and early develop-ment. Thus, studies of imprinting in marsupialsand monotremes, with their different placentalstructures and maternal investments, are highlyrelevant to assessing the two major hypothesesthat attempt to explain the evolution of imprint-ing through the maternal-fetal relationships—namely, the parental conflict and coadaptationtheories. At present, both hypotheses have goodexplanatory power for particular examples. Forinstance, the parental conflict hypothesis ac-counts well for most of the observations of thedirection of imprinting of growth genes andtheir regulators, whereas the coadaptation hy-pothesis makes more sense of the imprintingof genes involved in placental function, feed-ing, and maternal behavior. The two hypothe-ses may not be mutually exclusive. It is easy tosee how parental conflict might initially act toselect for differential parental control of fetal

growth, but that these genes and their neigh-bors and regulators might then become subjectto selection for coadaptation of placenta and hy-pothalamus. Alternatively, imprinting may beselected for and maintained due to parental con-flict at some loci, but due to coadaptation at oth-ers. The imprinting we observe in current-daymammals is the result of evolutionary pressuresthat drove the selection of this mechanism ina common ancestor, the reproductive details ofwhich we can only guess.

We cannot deduce from the fossil recordwhat the reproductive strategy of the stemmammals or stem therian mammals would havebeen. The therian ancestors were almost cer-tainly small, nocturnal, polyovular animals, andfetal development would have included a periodof placental attachment (27), but how long thislasted, and how invasive the placenta was, areunknown. The common ancestor to all mam-mals, the mammal-like reptiles, presumably laideggs, like monotremes, and presumably lackedany genomic imprinting. To date, investigationsin marsupials and monotremes have been lim-ited to examining regions orthologous to euthe-rian imprinted domains, but it is equally likelythat each lineage will have evolved its own spe-cific imprinted domains as a result of the spe-cific selective pressure imposed by its particularmode of reproduction and mating scheme (42).Further studies on these interesting and uniquemammals are awaited with interest.

Current comparative investigations of im-printed genes in mammals are revealing sev-eral ways in which imprinted regions can beestablished. These correlate with the insertionof retrotransposons and accumulation of re-peat elements within the genome as a whole.These findings have strongly supported thehost-defense hypothesis to explain how im-printing arose.

Why imprinting has been maintained ismore complex to address. The retention of ge-nomic imprinting and its complex regulatorymechanisms in marsupials suggests that selec-tion for parental conflict or coadaptation isstill strong enough in marsupials to have main-tained this state. It would also suggest that these

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selective pressures are not strong enough inegg-laying monotremes to have driven imprint-ing, or that in the absence of DNMT3L theydo not have a mechanism to establish it in thefirst instance. Continued analysis of imprinting

in all three extant mammalian lineages willbe essential to our deeper understanding ofthis seemingly perverse regulation that is socritical for normal development in therianmammals.

SUMMARY POINTS

1. Insertion of transposons can drive the evolution of imprinting (PEG10; DLK/DIO3/Rtl1).

2. Accumulation of repeat elements may have sparked the evolution of mammalian imprint-ing, or at least provided a mechanism for differential parental control to be applied to anancient monoallelic regulatory system.

3. Complex imprinted gene regulatory elements evolved in a step-wise manner.

4. Regions have become progressively more complex in the eutherian lineage.

5. Imprinting can spread to affect neighboring loci, leading to the evolution of compleximprinted regions.

6. In general, imprinted regions in marsupials contain fewer imprinted genes than in eu-therians.

7. The parental conflict theory provides a good explanation of imprinted growth genes andtheir regulators, but the co-adaptation theory accounts better for the imprinting of genesthat govern fetal and maternal behavior.

8. Genes that regulate imprinting have an evolutionary trajectory that correlates with theevolution of imprinting: for instance, the BORIS gene was evidently an ancient duplicationof CTCF, but has recently become germline-specific in therian mammals.

FUTURE ISSUES

1. Determination of the true function and phylogenetic extent of imprinted genes withinmammals and other vertebrates:

(a) Discoveries will become more rapid in the age of high-throughput sequencing thatcan be applied at the transcript level;

(b) Species of particular interest will be those associated with viviparity, placentation,and opportunities for parental conflict or mother-offspring coadaptation;

(c) Review of this information should offer more clues regarding how and why genomicimprinting evolved.

2. Identification of key factors that have evolved to direct general epigenetic silencing mech-anisms in a parent-specific manner:

(a) Understanding the true innovative power of epigenetic mechanisms such as ge-nomic imprinting will allow insight into how large evolutionary changes can occur with-out major changes in DNA sequence;

(b) Understanding the interacting functions of epigenetically controlled genes.

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DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.

ACKNOWLEDGMENTS

We thank our many collaborators, without whom much of the work reported here would not havebeen possible. In particular, we thank Eleanor Ager, Rob Rapkins, Fumi Ishino, Shunsuke Suzuki,Tomo Kaneko Ishino, Anne Ferguson Smith, Carol Edwards, Wolf Reik, Guillaume Smits, andGavin Kelsey.

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RELATED RESOURCES

Imprinted gene catalogueshttp://www.har.mrc.ac.uk/research//genomic imprinting/http://igc.otago.ac.nz/home.htmlVertebrate genomes including platypus and opossumhttp://genome.ucsc.edu.auhttp://www.ensembl.orghttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=search&term=Tammar wallaby trace archiveshttp://www.ncbi.nlm.nih.gov/Traces/trace.cgi?

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