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Regulatory Mechanisms

Cytogenet Genome Res 113:81–89 (2006) DOI: 10.1159/000090818

How imprinting centres work A. Lewis W. Reik Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge (UK)

As one might expect, genes within an imprinting cluster share many regulatory elements and often have similar devel-opmental and tissue-specifi c patterns of expression. The im-printing of genes in each cluster is regulated coordinately by an element called an imprinting centre (IC). Deletion or dis-ruption of an IC causes loss of imprinting at many surround-ing genes usually with major phenotypic effects. Knockouts of ICs in mice often result in embryonic lethality or severe growth defects and mutations in human ICs lead to imprint-ing disorders such as Prader-Willi (PWS) and Beckwith-Wiedemann syndromes (BWS). Here we review recent ad-vances in our understanding of the mechanisms by which an IC can control parental allele-specifi c expression and epige-netic marks throughout an imprinting cluster. We discuss how these mechanisms may be used and combined within different clusters and also examine the hierarchy of epigenetic events that occur at and around ICs in the early embryo.

Features of imprinting centres

An IC is defi ned as one or more discrete DNA elements which regulate imprinted gene expression and epigenotype throughout an imprinting cluster. Thus genetic manipulation

Abstract. Imprinted genes tend to be clustered in the ge-nome. Most of these clusters have been found to be under the control of discrete DNA elements called imprinting centres (ICs) which are normally differentially methylated in the germline. ICs can regulate imprinted expression and epige-netic marks at many genes in the region, even those which lie several megabases away. Some of the molecular and cellular mechanisms by which ICs control other genes and regulatory regions in the cluster are becoming clear. One involves the insulation of genes on one side of the IC from enhancers on the other, mediated by the insulator protein CTCF and high-er-order chromatin interactions. Another mechanism may in-volve non-coding RNAs that originate from the IC, targeting

Request reprints from Annabelle LewisLaboratory of Developmental Genetics and ImprintingThe Babraham Institute, Babraham Research CampusCambridge CB2 4AT (UK)telephone: +44 1223 496356; fax: +44 1223 496022e-mail: annabelle.lewis@bbsrc.ac.uk

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histone modifi cations to the surrounding genes. Given that several imprinting clusters contain CTCF dependent insula-tors and/or non-coding RNAs, it is likely that one or both of these two mechanisms regulate imprinting at many loci. Both mechanisms involve a variety of epigenetic marks including DNA methylation and histone modifi cations but the hierar-chy of and interactions between these modifi cations are not yet understood. The challenge now is to establish a chain of developmental events beginning with differential methyla-tion of an IC in the germline and ending with imprinting of many genes, often in a lineage dependent manner.

Copyright © 2006 S. Karger AG, Basel

Over 20 years ago nuclear transplantation experiments in mice were used to show that both parental genomes are nec-essary for complete embryogenesis indicating that genes can function differently depending on whether they are inherited maternally or paternally (McGrath and Solter, 1984; Surani et al., 1984). It soon became clear, however, that this parental effect was limited to specifi c chromosomal regions (Cattanach and Kirk, 1985). We now know that these regions contain clusters of imprinted genes which are differentially marked in the maternal and paternal germlines. Nearly 80 imprinted genes have been characterised in the mouse to date and the large majority of them do indeed exist in clusters in the chro-mosomal regions defi ned by Cattanach and colleagues rather than spread throughout the genome (http://www.mgu.har.mrc.ac.uk/research/imprinting/).

Supported by MCR BBSCR and EU.

Manuscript received 7 June 2005; accepted in revised form for publication by F. Ishino, 15 September 2005.

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of an IC defi nes the extent of a cluster; it is important to note that some large imprinting clusters can be further subdivided into domains containing separate ICs whose functions are limited to the domains. The fi rst IC to be genetically charac-terised was the differentially methylated region (DMR) up-stream of H19 (also called DMD or ICR) ( Fig. 1 ). Deletion of this region in mice causes loss of imprinting at both H19 and Igf2 (Thorvaldsen et al., 1998) and mutations in the homolo-gous region in humans have been found in patients with the overgrowth disorder Beckwith-Wiedemann Syndrome (BWS) (Sparago et al., 2004; Prawitt et al., 2005). The DMR at the

H19 IC is established in the germline resulting in high levels of DNA methylation in sperm and no methylation in oocytes. This paternal-specifi c methylation is then maintained throughout development, escaping the reprogramming events that occur in the early embryo (Morgan et al., 2005). All ICs contain such germline DMRs. In addition to germline DMRs there are also post-zygotic DMRs that are established after fertilization during the genome wide wave of demethylation and de novo methylation. The establishment of such post-zy-gotic DMRs (which do not act as ICs), is in fact dependent on the IC within the cluster in which they reside. DNA meth-ylation in both types of DMR is erased in primordial germ cells before the new parental-specifi c methylation is estab-lished.

Another paternally methylated germline DMR, the IG-DMR in the Dlk1/Gtl2 locus ( Fig. 1 ), has also been shown to act as an IC by regulating imprinting of all the genes in the cluster, although its effects are limited to the maternal chro-mosome (Lin et al., 2003). The paternal germline DMR with-in the Rasgrf1 locus is known to control imprinting of the Rasgrf1 gene although it is not yet known how it affects other nearby genes (Yoon et al., 2002) .

Similarly several maternally methylated germline DMRs act as ICs ( Fig. 1 ). Examples include intronic DMRs within the Igf2r/Air cluster (Wutz et al., 1997) and the IC2/ Kcnq1 cluster (Fitzpatrick et al., 2002), and more recently two dis-tinct germline DMRs within the Gnas cluster have been pro-posed to act as ICs (Liu et al., 2000; Coombes et al., 2003). The IC in the PWS locus was originally defi ned in humans using small deletions in PWS and Angelman syndrome (AS) patients (Buiting et al., 1999; Ohta et al., 1999; Bielinska et al., 2000). This IC is bipartite with deletions of a DMR (PWS-IC) in the promoter region of SNRPN leading to PWS when paternally inherited and deletions in a region 35 kb upstream (AS-IC) leading to AS when maternally inherited. The PWS-IC is maternally methylated in the mouse germline (Shemer et al., 1997) although there are confl icting reports about the status of the human IC (El-Maarri et al., 2001; Geuns et al., 2003).

In addition to differential DNA methylation, ICs also show allelic differences in chromatin structure, namelyDNase I hypersensitivity and covalent modifi cations of his-tones tails. Allele-specifi c DNase I hypersensitive sites have been found at the Igf2/H19 IC (Khosla et al., 1999), the IC within the IC2/ Kcnq1 cluster (IC2 or KvDMR1) (Kanduri et al., 2002; Mancini-DiNardo et al., 2003) and at the PWS-IC (Schweizer et al., 1999). One of the putative ICs in the Gnas cluster also lies in the same region as previously described al-lele-specifi c hypersensitive sites (Coombes et al., 2003). The availability of antibodies to many of the known histone tail modifi cations involved in epigenetic gene regulation has led to several studies of allele-specifi c modifi cations in both the mouse and human at imprinted genes. Chromatin immuno-precipitation has been used to look at histone modifi cations at the PWS/AS locus, the Igf2/H19 region, the IC2 cluster, Igf2r , U2af1-rs1 and part of the Gnas cluster (Pedone et al., 1999; Grandjean et al., 2001; Gregory et al., 2001; Fournier et al., 2002; Yang et al., 2003; Lewis et al., 2004; Sakamoto

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Fig. 1. Schematic diagrams of imprinting clusters (adapted from O’Neill, 2005). Maternally expressed genes are shown as red rectangles and paternally expressed genes are shown as blue. Non-imprinted genes are represented by unfi lled rectangles. Tissue-specifi c imprinted genes are represented by stripes. Triangles inside a gene indicate the transcript is processed into microRNAs. Small boxes indicate snoRNAs. Genes tran-scribed from the sense and antisense strands are shown above and below the line respectively. Maternally methylated germline DMRs are repre-sented by red lollipops and paternally methylated DMRs are represented by blue ones. Imprinting centres are represented by black boxes.

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et al., 2004; Umlauf et al., 2004). Some of these fi ndings will be discussed in detail later but generally it has been found that repressive histone modifi cations such as methylation at his-tone H3 lysine 9 and lysine 27 are found at ICs and other DMRs on the methylated allele, whereas activating histone modifi cations such as H3 and H4 acetylation and H3 lysine 4 are found on the unmethylated allele. The allele-specifi c histone modifi cations discovered so far are generally restrict-ed to the vicinity of DMRs but recent studies show that this is not always the case. In extraembryonic tissues the whole of the IC2 cluster is marked by allele-specifi c differences in his-tone modifi cations in promoter, exonic and intergenic regions whereas in embryonic tissues only the DMRs are marked (Lewis et al., 2004; Umlauf et al., 2004). Until now most analyses at other imprinted loci have been limited to DMRs so it will be interesting to carry out more studies in non-dif-ferentially methylated regions to see if allelic markings occur across other clusters and whether the phenomenon is limited to the placenta or is present in other lineages.

Another common feature of ICs is their ability to act as a chromatin insulator. An insulator is defi ned as a sequence of DNA that blocks enhancers from interacting with gene pro-moters when positioned between the two and/or acts as a bar-rier to the spread of transcriptionally repressive condensed chromatin. The CCCTC binding factor (CTCF) is known to bind to insulators and mediate their enhancer blocking activ-ity. A CTCF dependent insulator exists within the Igf2/H19 IC. On the unmethylated maternal allele, the insulator pro-tein, CTCF, binds to target sites in the IC preventing the en-hancers downstream of H19 from accessing the Igf2 promot-ers (Bell and Felsenfeld, 2000; Hark et al., 2000; Kanduri et al., 2000; Szabo et al., 2000). On the paternal allele, DNA methylation blocks CTCF binding. This abolishes insulator activity and allows the enhancers to access Igf2 promoters resulting in Igf2 transcription. Since this discovery, CTCF has been found to bind to IC2 (M. Higgins, pers. comm.), Rasgrf1 (P. Soloway, pers. comm.) and putative binding sites have been found in the Gtl2 and Grb10 loci (Takada et al., 2002; Hikichi et al., 2003). Due to the wide variation in the se-quences for CTCF binding sites it is likely that there are more CTCF sites at imprinted loci that have not yet been discov-ered. There are also non-CTCF dependent insulators at im-printing loci. A DMR (and putative IC) in the Peg3 locus contains binding sites for the protein YY1 and functions as a methylation sensitive insulator (Kim et al., 2003). It is pos-sible that YY1 or unknown proteins may bind to other ICs to give them insulator activity in a similar manner to CTCF.

Non-coding RNAs have been found in great numbers in all species and have many and diverse roles including tran-scriptional regulation, RNA processing and protein transport (Storz, 2002). It is therefore not surprising that non-coding RNAs have been found at imprinting clusters. There are many different types of imprinted RNAs, from those that are processed into 21–25-nt microRNAs to 1 100 kb long un-spliced RNAs. A recent review (O’Neill, 2005) summarises our current understanding of these RNAs and the essential roles they play in imprinting but for the purposes of this ar-ticle we will focus on non-coding RNAs that have promoters

within or near ICs. The best characterised of these RNAs is the Air RNA which is a non-coding RNA transcribed in an-tisense to Igf2r . The promoter for Air lies within the IC and the 108-kb Air transcript overlaps the Igf2r promoter (Lyle et al., 2000). Other similar antisense RNAs have promoters at the IC2 ( Kcnq1ot1 ) (Mitsuya et al., 1999), and also near the PWS-IC ( Ube3a antisense) although the precise position of the promoter is not known (Rougeulle et al., 1998). In the Gnas locus the Nesp and a non-coding antisense transcript Nespas are reciprocally expressed but it is not yet known how important the Nespas transcript is for imprinting. Hence the defi ning properties of ICs (alone or in combination) current-ly appear to be (a) that they regulate other imprinted genes within the same cluster in cis , (b) differential DNA methyla-tion established in the germlines and maintained in somatic tissues of the offspring, (c) differential histone modifi cations, (d) that they act as chromatin insulators, (e) that they contain promoters for non-coding RNAs.

Mechanisms of regional imprinting control

ICs are able to regulate genes which lie megabases away. The mechanisms by which they do this are now being unrav-elled although it appears that each IC may use different vari-ants or combinations of mechanisms. The fi rst model pro-posed for coordination of epigenotype across an imprinting cluster was linear spreading of DNA methylation (Turker and Bestor, 1997). After examining regional control of DNA methylation in the Igf2/H19 region we proposed an alterna-tive model: long range chromatin interactions or ‘looping’ (Lopes et al., 2003). It now appears that both of these models contain some truth.

ICs and looping In the Igf2/H19 region interactions between DMRs create

parent-specifi c chromatin loops (Murrell et al., 2004). The CTCF dependent insulator is positioned in the IC between Igf2 and H19 with the enhancers lying downstream of H19. On the unmethylated maternal chromosome the IC interacts with the DMR1 region of Igf2 and a downstream matrix at-tachment region (MAR3) in a CTCF dependent manner (Ku-rukuti et al., unpublished) . This creates an inactive chromatin domain surrounding Igf2 which is specifi c to the unmethyl-ated maternal allele. On the methylated paternal chromo-some CTCF does not bind to the IC and the IC interacts with Igf2 DMR2 instead of DMR1. It is not known which proteins are involved in the interaction on the paternal chromosome. There is also new evidence that the downstream enhancers are directly interacting with Igf2 promoter regions (Kurukuti et al., unpublished). This interaction occurs on the paternal allele but is prevented on the maternal allele by CTCF bind-ing to the IC and the resultant inactive chromatin loop. Mu-tation of the CTCF binding site on the maternal IC disrupts the chromatin loop and allows the enhancers to interact with the maternal Igf2 promoters. Interestingly, mutation of this CTCF binding site also causes an increase in DNA methyla-tion in the maternal DMR1 showing that CTCF can coordi-

Cytogenet Genome Res 113:81–89 (2006) 84

nate the epigenotype across the whole Igf2/H19 region (Ku-rukuti et al., unpublished).

Chromatin looping to form an inactive domain has also been observed at the imprinted Dlx5 locus (Horike et al., 2005). In this case the looping is mediated by the methyl bind-ing and Rett syndrome associated protein MeCP2. In the brains of MeCP2 null mice the inactive loop is absent and there is loss of imprinting of Dlx5 . Although it is not known whether the loop is allele-specifi c, the methylation sensitive binding of MeCP2 to a DMR to form an inactive chromatin structure on the paternal chromosome and an active structure on the maternal allele is an attractive explanation for the ma-ternal-specifi c expression of Dlx5 . However there is no dif-ferential DNA methylation in the vicinity of the Dlx5 locus. Thus the MeCP2 mediated looping likely allows Dlx5 to be regulated in an allele-specifi c manner by a distant differen-tially methylated element, i.e. an IC which is currently unde-fi ned.

The examples above are just two possible mechanisms by which chromatin looping could be involved in the epigenetic regulation of imprinting clusters. It is likely that such mecha-nisms occur at many of the known imprinting clusters. Dis-ruption of imprinting in humans in the GNAS cluster has been linked with deletions in the distant (non-imprinted) STX16 region in patients with pseudohypoparathyroidism (Linglart et al., 2005). Since STX16 is over 200 kb upstream of GNAS this must surely involve some higher-order chromatin struc-tures but the interactions involved are unknown. The preva-lence of CTCF binding sites at ICs suggests that this particu-lar mechanism could be used in other imprinted loci. Loss of MeCP2 does not affect the expression of many imprinted genes (Balmer et al., 2002) but it is possible that other meth-yl binding domain proteins (MBDs) may play similar roles or that there is redundancy between MBDs at imprinted loci. Indeed, the unknown protein(s) involved in interactions be-tween the methylated DMRs in the paternal specifi c chroma-tin loop in the Igf2/H19 locus (Murrell et al., 2004) is prob-ably an MBD.

Another method of forming discrete domains of active or silent chromatin, perhaps on a larger scale, could involve teth-ering of imprinted regions to fi xed structures in the nucleus. Two of the matrix attachment regions (MARs) in the Igf2/H19 region are restricted to the paternal allele (Weber et al., 2003). If the maternal, unmethylated IC (which binds CTCF) is deleted the maternal MARs also become attached. Thus the chromatin structure of the paternal allele, in the absence of CTCF binding, seems to depend on MARs while higher-order structure of the maternal allele depends on CTCF binding. In addition, CTCF is known to associate with a nuclear matrix protein, called nucleophosmin (Yusufzai and Felsenfeld, 2004) so it is likely that the nuclear matrix is also instrumen-tal in the organisation of chromatin on the maternal allele. MARs on both parental alleles have also been found in large numbers between the two imprinting clusters on mouse distal chromosome 7 ( Igf2/H19 and IC2; Purbowasito et al., 2004). Thus MARs may also act as boundary elements to block the spreading of chromatin modifi cations or to form separate chromatin domains or loops. On an even larger scale, studies

now suggest that ICs are required for the movement of the two parental alleles to different subnuclear compartments (Gribnau et al., 2003; M. Higgins, pers. comm.). The com-partments differ in their replication timing and also in their heterochromatic and euchromatic characteristics.

Labrador and Corces (2002) proposed a model in which boundary or insulator elements throughout the genome estab-lish chromatin domains, organising the chromatin fi bres into local compartments of condensed (inactive) and decondensed (active) chromatin. This model can easily be extended to in-clude imprinted genes: an unmethylated insulator may bind to a nuclear structure dividing the imprinting cluster into two and dividing promoters and enhancers into separate compart-ments. The same insulator, inactivated by methylation, will not be able to form two separate domains and therefore en-hancers and promoters will interact. This gives us a picture of the genome, organised into many different regulatory domains of higher order chromatin structure interspersed with a few imprinted regions in which the chromatin organisation is de-pendent on the methylation state of an insulator element.

Non-coding RNAs and chromatin spreading The best characterised mammalian non-coding RNA is

Xist , the driving force behind the process of X chromosome inactivation. The Xist RNA coats the future inactive X and triggers the events which lead to gene silencing along the length of the X chromosome (reviewed in Heard, 2004). This silencing begins in regions directly fl anking the Xist locus and spreads outwards (Huynh and Lee, 2003). As discussed above, non-coding RNAs are also prevalent at imprinting clusters. In two well characterised clusters (IC2 and Igf2r/Air ) these RNAs are expressed from the paternal allele while the sur-rounding genes are paternally silenced. This situation is very similar to X chromosome inactivation in mouse extra embry-onic lineages where it is imprinted with Xist being expressed specifi cally from the paternal allele and preferential inactiva-tion of the paternal X. The parallels between the two pro-cesses have led to proposals that they may have coevolved (Huynh and Lee, 2005; Reik and Lewis, 2005).

Evidence for the functional role of autosomal imprinted non-coding RNAs comes from a truncation of the Air tran-script which results in loss of imprinting of Igf2r and also of two placental-specifi c imprinted genes, Slc22a2 and Slc22a3 , located upstream of the Air promoter (Sleutels et al., 2002). Thus the critical element within the Igf2r/Air IC is the Air transcript. It is not known, however, how the Air RNA medi-ates silencing of the surrounding genes. Possible models in-clude an RNAi based mechanism, transcription through spe-cifi c DNA elements or a chromosome coating mechanism as in X chromosome inactivation.

Further insights into the role of non-coding RNAs come from studies of the IC2 locus. The Kcnq1ot1 RNA has many similar features to Air and Xist . Its transcription is initiated from the unmethylated paternal IC in the IC2 cluster and all the fl anking genes are paternally silenced. Many of the genes have no associated DMRs, are imprinted only in the placen-ta and, unusually, retain imprinted expression in the absence of maintenance DNA methylation (Lewis et al., 2004). This

Cytogenet Genome Res 113:81–89 (2006) 85

independence from DNA methylation could be explained by the presence of paternal allele-specifi c histone methylation at H3 lysine 9 and lysine 27 across the entire IC2 region (Umlauf et al., 2004). These modifi cations only occur in extraembry-onic lineages and suggest spreading of a repressive chromatin organisation outwards from the IC. It is likely that the histone methylation on the paternal allele is mediated by the PRC2 polycomb complex since members of PRC2, Eed and the his-tone methyltransferase Ezh2, associate specifi cally with the paternal allele of the IC2 cluster (Umlauf et al., 2004). Im-printed X chromosome inactivation is also independent of maintenance DNA methylation, and histone methylation and the PRC2 complex is associated specifi cally with the inactive X (Sado et al., 2000; Heard, 2004). Indeed, both X inactiva-tion and imprinting of some genes in the IC2 cluster are dis-rupted in the absence of the PCR2 complex member, Eed (Wang et al., 2001; Mager et al., 2003). The above evidence suggests that imprinting at Igf2r/Air and IC2 may involve an X inactivation like mechanism, most probably in the placen-ta only. The kinetics of X chromosome inactivation and its regulation by the X inactivation centre are much better un-derstood than the mechanisms used at imprinting centres. Using the pathway of X chromosome inactivation as a mod-el, the next important task is to investigate the order of events and the enzymes involved in the silencing pathway in early preimplantation development.

A recent publication reveals that the UBE3A antisense RNA plays a role regulating the imprinted expression of UBE3A , the gene which is disrupted in AS patients (Make-donski et al., 2005). The UBE3A antisense gene is controlled by MeCP2 binding at the PWS-IC and therefore both UBE3A and its antisense are also disrupted in Rett syndrome patients. Unlike other genes in the PWS/AS cluster, imprinting of UBE3A is restricted to the brain, has no differential DNA methylation and the gene lies on the telomeric side of the im-printing centre (Soejima and Wagstaff, 2005). It is tempting to speculate that the paternally expressed non-coding anti-sense RNA may regulate the maternally expressed sense gene using the same mechanism as Xist , Air and Kcnq1ot1 . This would involve the establishment and spreading of a repressive chromatin structure specifi cally in the brain, but there are arguments against this. Although both the human UBE3A antisense and the mouse Ube3a antisense transcripts are sev-eral hundred kb long, it has been shown in the mouse that there are alternatively spliced transcripts some of which are processed into C/D snoRNAs (Landers et al., 2004). This makes RNAi based mechanisms or the effects of transcription itself equally as likely as a coating mechanism.

ICs can play a dual role The IC2 cluster and the Igf2r / Air cluster contain placental-

specifi c imprinted genes on one side of the IC and genes im-printed in both embryonic and extraembryonic lineages on the other. In the mouse IC2 cluster these two sets of genes are clearly regulated in different ways. The genes on the telomer-ic side of the IC are regulated by DNA methylation and have monoallelic expression and histone methylation at H3 lysine 27 in ES cells which are equivalent to the blastocyst stage

(Lewis et al., 2004; Umlauf et al., 2004). The centromeric, placentally imprinted genes are regulated by a DNA meth-ylation independent mechanism and their histone modifi ca-tions and imprinted expression are only partly established in ES cells (Lewis et al., 2004; Umlauf et al., 2004; Green and Lewis, unpublished). In addition, the imprinting of most of these genes is not as tightly controlled as those regulated by DNA methylation; the expression is biased towards the ma-ternal alleles but there are still low levels of expression from the paternal alleles. Removing the IC disrupts imprinting of the whole locus in all lineages (Fitzpatrick et al., 2002), but an 800-kb YAC transgene containing most of the locus only exhibits imprinting of the ‘telomeric’ genes (Cerrato et al., 2003).

We suggest that the IC regulates imprinting in the IC2 cluster in two distinct ways ( Fig. 2 ). One involves CTCF bind-ing to the insulator on the unmethylated paternal IC and us-ing higher-order chromatin structures to restrict enhancer ac-cess. On the maternal chromosome CTCF binding is blocked by DNA methylation. This mechanism is active in all lin-eages and results in stable imprinting of genes on the telo-meric side of the locus including Cdkn1c/p57 Kip2 and Phlda2/Ipl . The other mechanism occurs only in the extraembryonic lineage but may begin in the preimplantation embryo. This involves the Kcnq1ot1 RNA, possibly coating the chromo-some, and recruiting repressive histone modifi cations to the paternal allele. The histone modifi cations are recruited to genes on both sides of the IC but are probably irrelevant on the telomeric side since the stronger, looping-based mecha-nism takes precedence. The RNA/histone methylation mech-anism is much less stable, and is not maintained through the later stages of gestation.

The Air RNA is important for imprinting in both embry-onic and extraembryonic lineages (Sleutels et al., 2002) but there are clear differences between the placental-specifi c im-printed genes which have no associated DMRs, and Igf2r which exhibits methylation dependent imprinting in many tissues. There are obvious parallels between the placental-spe-cifi c imprinting of Slc22a2 and Slc22a3 and the centromeric genes of the IC2 cluster, so an Xist-style coating mechanism is again an appealing model, although it must be noted that Air and Kcnq1ot1 are transcribed in opposite directions rela-tive to the placental-specifi c imprinted genes. Furthermore, unlike imprinted X inactivation, imprinted expression of Igf2r is dependent on DNA methylation as well as the Air RNA (Li et al., 1993) making it less likely that Igf2r imprint-ing is regulated by an Xist-style coating mechanism. RNAi cannot be ruled out although it has been shown that the over-lap between the two transcripts is not necessary for imprinting (Sleutels et al., 2003). No CTCF sites have been reported in the Igf2r/Air IC but an insulator model is still a possibility perhaps with Air transcription regulating the insulator activ-ity in an allele-specifi c manner. RNA FISH experiments in fi broblasts have revealed that the Air RNA forms unusual clusters or aggregates away from chromosomes (Braidotti et al., 2004). These aggregates do not occur in ES cells which do not exhibit Igf2r imprinting and perhaps indicate a new, un-characterised mechanism.

Cytogenet Genome Res 113:81–89 (2006) 86

Other clusters may also employ more than one mechanism to regulate imprinting. The PWS/AS cluster IC is described as bipartite. Establishment of imprinting on the paternal chromosome requires the PWS-IC in cis whereas establish-ment of imprinting on the maternal chromosome requires the AS-IC. In addition the tissue-specifi c maternal expression of Ube3a requires the Ube3a antisense RNA. Thus there may be as many as three separate mechanisms regulating imprinting in this cluster. In the Dlk1/Gtl2 cluster the IG-DMR acts as an IC for the maternal allele only and its removal has no ef-fect on imprinting when inherited paternally (Lin et al., 2003). Either the epigenetic marks and expression patterns on the paternal chromosomes represent the ‘default’ state or there may yet be another, unidentifi ed element of this IC. The Igf2/H19 IC contains a methylation sensitive insulator which pre-vents expression of Igf2 from the maternal chromosome, but it also contains a separate silencer element which represses transcription from H19 on the paternal allele (Drewell et al., 2000). Thus in general, ICs need to be viewed as multipartite and multifunctional genetic elements, and the outcomes of genetic analyses depend on which part of an IC has been de-leted.

Epigenetic modifi cations and their interactions

It is now clear that there is no single all-encompassing mechanism of imprinting and that imprinting clusters behave in many different ways. Most of the mechanisms discussed

here are not unique to imprinted loci but act throughout the genome to regulate gene expression. It is the allele-specifi c epigenetic marks which actually regulate imprinting by steer-ing the ICs down different pathways on different parental chromosomes. We know that most ICs have germline DMRs but we do not yet understand the relationship between this primary DNA methylation, methylation in DMRs estab-lished post-zygotically and also the various histone modifi ca-tions that occur at the ICs, post-zygotic DMRs and through-out the IC2 locus in the placenta (and perhaps other tis-sues).

In plants and fungi it has been shown that CpG methyla-tion is dependent on H3 lysine 9 methylation (Tamaru and Selker, 2001; Jackson et al., 2002). This same relationship has been shown in mouse ES cells (Lehnertz et al., 2003) with ES cells homozygously mutated for the heterochromatin-specifi c lysine 9 methylases, Suv39h1 and Suv39h2 showing reduced DNA methylation levels in major satellite repeats in pericen-tromeric heterochromatin. Perhaps more relevant is the ob-servation that DNA methylation is reduced at the imprinted locus Snrpn in ES cells mutant for the histone methylase G9a (Xin et al., 2003). (G9a methylates histone H3 at lysine 9 pri-marily in euchromatic regions.) However this difference is not observed in day 9.5 embryos (E9.5).

Other studies indicate that the relationship between his-tone methylation and DNA methylation might not be entire-ly unidirectional. In Arabidopsis , erasure of CpG methylation caused a dramatic loss of H3 lysine 9 methylation in hetero-chromatin (Tariq et al., 2003). In vitro assays in mammalian

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CTCF

PatPlacenta

Fig. 2. A model showing mechanisms of imprinting at the IC2 locus. The top panel shows the IC2 cluster with maternally ex-pressed genes in red and paternally expressed genes in blue. The pictures below show poten-tial higher order chromatin structures com-bined with silencing due to non-coding RNA and histone methylation on the maternal and paternal alleles. On the maternal chromosome the IC2 cluster is in an accessible conformation in both embryo and placenta. The enhancers (yellow circles) are able to interact with the pro-moters on the telomeric side promoting expres-sion in most tissues. The same or different en-hancers may also access the centromeric genes, some of which are expressed only in the pla-centa. On the paternal chromosome, the un-methylated IC binds to the insulator protein CTCF (large blue circle) in embryonic and pla-cental tissues. This interaction either establish-es or localises the telomeric genes to a repres-sive chromatin domain (grey background), pos-sibly by interacting with an upstream DNA element (hatched rectangle) where histone methylation occurs (dark red hexagons). The enhancers are prevented from accessing the promoters. In addition, in placental tissues the Kcnq1ot1 RNA (wavy lines) may coat the chro-mosome, targeting histone methylation to both sides of the IC as a second mechanism of repres-sion.

Cytogenet Genome Res 113:81–89 (2006) 87

cell lines have shown that MeCP2 facilitates H3 lysine 9 methylation of the Igf2/H19 IC (Fuks et al., 2002), thus pro-viding potentially a mechanistic link between DNA methyla-tion and histone methylation. However, DNA methylase de-fi cient ES cells (both Dnmt1 defi cient and Dnmt3a and Dnmt3b double defi cient) showed no change in centromeric lysine 9 methylation, questioning whether such a mechanistic link does exist in mammals. Unfortunately, due to technical limitations it is very diffi cult to study the kinetics of histone methylation in mammals at early developmental stages in vivo so at the moment the best model systems are mouse em-bryonic (ES) and trophoblast (TS) stem cells which are rough-ly equivalent to the ICM and TE lineages in blastocysts at embryonic day 3.5 (E3.5), the earliest precursors to the em-bryonic and extraembryonic lineages, respectively. These can then be compared to DNA methylation studies which can be carried out in vivo. In ES cells mutant for the maintenance DNA methylase Dnmt1 we have observed that the IC2 loses the maternal-specifi c lysine 9 and lysine 27 methylation seen in wildtype ES cells (Green and Lewis, unpublished). This same effect is seen in some BWS patients where patients with a loss of methylation at the IC also have a lack of histone methylation (Higashimoto et al., 2003). In wildtype ES cells there is some paternal-specifi c lysine 27 methylation up-stream of the Cdkn1c promoter whereas in Dnmt1 –/– ES cells

there are no allelic differences in this region. The paternal-specifi c methylation at lysine 27 is more obvious in E9.5 em-bryos and placentae and there is also some lysine 9 methyla-tion present. We know that this region is not differentially methylated in the germline but becomes paternally DNA methylated at about E7.5 to E8.5, i.e. after H3 lysine 27 meth-ylation appears (Bhogal et al., 2004). Interestingly, DNA methylation at Cdkn1c depends on the Swi/Snf nucleosome remodeller Lsh (Fan et al., 2005) suggesting perhaps that the reading of histone marks by the DNA methylation machinery requires nucleosome remodelling.

Thus we can piece together a model of how maternal germ-line DNA methylation can lead to a paternal postzygotic methylation mark ( Fig. 3 ): a property of the unmethylated paternal IC such as the Kcnq1ot1 RNA, an insulator medi-ated higher-order chromatin structure, or perhaps intranucle-ar localisation leads to polycomb protein association and H3 lysine 27 methylation at preimplantation stages. After im-plantation the lysine 27 methylation recruits lysine 9 meth-ylation and DNA methylation probably in that order. Once again we can see similarities with the pathway of X chromo-some inactivation. In the Igf2/H19 locus the paternal germ-line methylation is necessary to create other paternal, postzy-gotic DMRs, which on the maternal allele are prevented from becoming methylated by higher order contacts brought about

Germline DNAmethylation at IC

gametes

Fertilization

Blastocyst

implantation

5

PGCs

Paternal Allele(unmethylated IC)

Maternal Allele(methylated IC)

Expression of non-coding RNA

H3 K27 methylation

H3 K9 methylation

De novo DNA methylation

of post-zygotic DMR

IC2/Aircluster

methylation at IC

gametes

Fertilization

Blastocyst

implantation

5

PGCs

Paternal Allele(unmethylated IC)

Maternal Allele(methylated IC)

Gametes

Fertilization

Blastocyst

Implantation

E 9.5

PGCs

Paternal allele(unmethylated IC)

Maternal allele(methylated IC)

Germline DNAmethylation at IC

Gametes

Fertilization

Blastocyst

Implantation

E 9.5

PGCs

Maternal allele(unmethylated IC)

Paternal allele(methylated IC)

CTCF binding to unmethylatedI IC

CTCF dependent higher order

chromatin structure

Protection of all DMRs from de novo

DNA methylation

Igf2/H19 cluster

Epigenetic steps leading to methylationof post-zygotic DMRs

e.g. histonemodifications

De novo DNA methylationof post-zygotic DMRs

Fig. 3. The predicted order of establishment of epigenetic modifi ca-tions at imprinting clusters in early embryonic development. Hatched arrows indicate progressions for which there is no experimental evidence. Light grey text indicates predicted steps which have not been demon-strated. The left panel shows the predicted developmental sequence in the IC2 and Igf2/Air clusters which both contain a maternally methyl-ated IC, non-coding RNAs and postzygotic paternally methylated DMRs. The germline DMR leads to allele-specifi c expression of a non-coding RNA. This RNA may be responsible for recruiting histone H3 K27 meth-ylation which occurs in early embryos followed by H3 K9 methylation. (The prevalence of these modifi cations is dependent on lineage.) The histone methylation then targets DNA methylation of a postzygotic

DMR. The right panel shows the predicted developmental sequence of allele-specifi c epigenetic modifi cations in the Igf2/H19 cluster. This be-gins in the germline with a paternally methylated IC. On the paternal chromosome, methylation of this IC leads to the establishment of postzy-gotic paternally methylated DMRs, and although little is known about histone modifi cations in this region, it is assumed that they must play a role in establishing the epigenotype of the region (progression shown in grey). On the maternal chromosome, CTCF binds to the unmethylated IC at an unknown time in early development. This leads to the establish-ment of allele-specifi c higher-order chromatin structures which then pro-tect the IC and the other DMRs from de novo DNA methylation.

Cytogenet Genome Res 113:81–89 (2006) 88

by CTCF. We do not know the epigenetic stages involved in the establishment of the postzygotic DMRs but a possible pathway involves histone methylation. Stem cell lines are use-ful but to truly understand these epigenetic cascades we need to develop techniques to look at locus-specifi c histone modi-fi cations in the early embryo. Knocking out enzymes involved in these crucial processes inevitably results in embryonic le-thality so we also need to improve conditional knockout and RNAi technology to allow depletion of the enzymes at critical stages and in a lineage-specifi c manner.

We must also defi ne and investigate what really occurs in the so called spreading and looping mechanisms. We know that at the early stages of imprinted X inactivation silencing spreads outwards from an X inactivation centre – but how? Heterochromatin spreading occurs due to recruitment of the Suvar methylases by HP1, methylation of adjacent nucleo-somes followed by HP1 binding and so on (Hall et al., 2002). HP1 is not involved in X inactivation, so do the polycomb complexes play a similar role? X chromosome/autosome translocations show that Xist mediated repression can also

spread into autosomes but not as effi ciently (Sharp et al., 2002). This implies that specifi c sequences on the X are need-ed to promote spreading. Some genes on the X chromosome and some genes in the imprinted IC2 cluster escape repres-sion, so do specifi c sequences also protect against silencing? These escaped genes are likely to loop out, so is there insula-tor dependent looping on the X, too (Filippova et al., 2005)? When we refer to looping mechanisms that create domains of active or repressive chromatin, there must be some spreading of chromatin structure within the domain. How does this oc-cur? Although we currently treat looping and spreading as mutually exclusive mechanisms, perhaps we should investi-gate whether they sometimes work in tandem.

Acknowledgements

We would like to thank M. Higgins, J. Peters, K. Green and G. Smits for valuable comments on the manuscript.

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