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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
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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
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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
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(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
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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.
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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.
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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
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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
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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
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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
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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
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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. An eclectic set of components, including
intronless genes, large non-coding RNAs and small noncoding RNAs (snoRNAs and miRNAs), arrived at the Prader-Willi–Angelman syndrome, Callipyge and PEG10 loci
by translocation, retroposition or mutation from existing
components. The X-inactivation centre of eutherians was
generated in similar fashion, further reinforcing the links
between X inactivation and genomic imprinting. Despite
these recent discoveries, there is much to learn about the
evolution of imprinting and X-inactivation in marsupials
and monotremes (Box 1). It is therefore important to
intensify our study of divergent mammalian models of
imprinting and X inactivation. This will better define
the extent to which these enigmatic epigenetic phenomena
exist, how they evolved and why they persist.
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