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- The Company of Biologists
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- © 2021 The Company of Biologists. All rights reserved.
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- 0950-1991
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- 0950-1991
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- 10.1242/dev.030403
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Abstract
REVIEW 1771 Development 136, 1771-1783 (2009) doi:10.1242/dev.030403 Martha V. Koerner*, Florian M. Pauler*, Ru Huang and Denise P. Barlow Non-coding RNAs (ncRNAs) that regulate gene expression in cis that equalises X-linked gene expression between the sexes. Xist is or in trans are a shared feature of prokaryotic and eukaryotic expressed from, and localises to, the inactive X chromosome and, genomes. In mammals, cis-acting functions are associated with by an unknown mechanism, targets repressive chromatin macro ncRNAs, which can be several hundred thousand modifications and gene silencing to this chromosome. Tsix overlaps nucleotides long. Imprinted ncRNAs are well-studied macro with the entire Xist gene in an antisense orientation and silences Xist ncRNAs that have cis-regulatory effects on multiple flanking on the active X chromosome (reviewed by Wutz and Gribnau, genes. Recent advances indicate that they employ different 2007). The next-best-studied mammalian macro ncRNAs are those downstream mechanisms to regulate gene expression in involved in genomic imprinting (see Glossary, Box 1; Box 2). To embryonic and placental tissues. A better understanding of date, 90 genes show imprinted expression in the mouse these downstream mechanisms will help to improve our general (http://www.har.mrc.ac.uk/research/genomic_imprinting), and their understanding of the function of ncRNAs throughout the imprinted status is mostly conserved in humans genome. (www.otago.ac.nz/IGC). Imprinted genes mostly occur in clusters that contain 2-12 genes, and in most of these clusters at least one Introduction gene is a macro ncRNA. So far, two of the three tested imprinted In recent years, tiling-array analyses (see Glossary, Box 1) and macro ncRNAs have been shown to be required for the imprinted genome-wide cDNA sequencing have shown not only that most of expression of the whole cluster (Barlow and Bartolomei, 2007). the mammalian genome is transcribed, but also that the majority of Thus, imprinted macro ncRNAs are able to regulate small clusters the mammalian transcriptome consists of non-coding (nc) RNAs of autosomal genes in cis and offer an excellent model system not (Carninci et al., 2005; Engstrom et al., 2006; Kapranov et al., 2002; only to investigate how ncRNAs regulate genes epigenetically, but Katayama et al., 2005; Okazaki et al., 2002). Because a full also to investigate the general biology of ncRNA transcripts. classification system for ncRNAs is still outstanding, they are In this review, we focus on six well-studied mouse imprinted generally described according to their mature length, location and clusters and their associated macro ncRNAs (Fig. 1) and review orientation with respect to the nearest protein-coding gene. For three main areas: first, how imprinted macro ncRNAs are example, a new group called large intervening non-coding (linc) themselves epigenetically regulated by DNA methylation imprints, RNAs comprise ncRNAs that lie outside annotated genes in the and their role in inducing imprinted expression and epigenetic mouse genome (Guttman et al., 2009). When their function is modifications in imprinted clusters; second, what is currently known known, ncRNAs can also be classified by whether they act in cis or about the organisation and the transcriptional biology of imprinted trans (see Glossary, Box 1). Trans-acting functions are associated macro ncRNAs; and third, why developmental and tissue-specific with short ncRNAs, such as short interfering (si) RNAs (21 nt), variation in imprinted expression indicates that multiple micro (mi) RNAs (~22 nt), piwi-interacting RNAs (26-31 nt) and mechanisms might operate downstream of imprinted ncRNAs. short nucleolar (sno) RNAs (60-300 nt). By contrast, cis-acting functions have so far only been associated with macro ncRNAs (see Imprinted macro ncRNAs are epigenetically Glossary, Box 1), which can be up to several hundred thousand regulated nucleotides long. Interestingly, whereas the number of protein- A key feature of imprinted gene clusters is the presence of an imprint coding genes is no indication of an organism’s morphological control element (ICE) (see Glossary, Box 1), which has been complexity, macro ncRNA number increases with complexity, genetically defined by deletion experiments in mice or through the indicating a potential functional role in gene regulation (Amaral and mapping of minimal naturally occurring deletions in humans (Table Mattick, 2008). In support of this hypothesis, many ncRNAs show 1). The ICE is epigenetically modified on only one parental distinct cell-type-specific and developmental-stage-specific chromosome by a DNA methylation ‘imprint’, which is acquired expression profiles (Dinger et al., 2008; Mercer et al., 2008). To date, during maternal or paternal gametogenesis and is maintained on the however, only a few macro ncRNAs have been analysed in detail same parental chromosome in the diploid embryo. As the other and shown to have functional gene-regulatory roles (Yazgan and parental chromosome lacks ICE DNA methylation, this region in a Krebs, 2007; Prasanth and Spector, 2007). diploid cell is also known as a gametic differentially methylated The best-known functional mammalian macro ncRNAs are the region (gDMR). The unmethylated ICE controls the imprinted inactive X-specific transcript (Xist) and X (inactive)-specific expression of the whole cluster; upon its deletion, imprinted genes transcript, antisense (Tsix), which are overlapping transcripts are no longer expressed in a parental-specific pattern (Bielinska et required for X chromosome inactivation in female mammals – an al., 2000; Fitzpatrick et al., 2002; Lin et al., 2003; Thorvaldsen et al., epigenetic dosage-compensation mechanism (see Glossary, Box 1) 1998; Williamson et al., 2006; Wutz et al., 1997). Note that the term ‘imprinted’ refers to the presence of DNA methylation on the ICE and not to gene expression status and that the above-mentioned Research Center for Molecular Medicine of the Austrian Academy of Sciences, Dr deletion experiments show that only the unmethylated ICE is active. Bohr-Gasse 9/4, Vienna Biocenter, A-1030 Vienna, Austria. Four of the six well-studied imprinted clusters in the mouse (Igf2r, Kcnq1, Pws/As, Gnas) are maternally imprinted and thus gain their *These authors contributed equally to this work Author for correspondence (e-mail: [email protected]) ICE DNA methylation imprint during oogenesis. This imprint is DEVELOPMENT 1772 REVIEW Development 136 (11) maintained only on the maternal chromosome in diploid cells (Fig. Box 1. Glossary 1A-D). The remaining two clusters (Igf2, Dlk1) are paternally imprinted and gain their ICE DNA methylation imprint during Cis-acting function spermatogenesis. This imprint is maintained only on the paternal The ability of a DNA sequence or transcript to regulate the chromosome in diploid cells (Fig. 1E,F). expression of one or more genes on the same chromosome. This In the Igf2r, Kcnq1 and Gnas clusters, the ICE contains a contrasts with trans-acting function (see below). promoter for a macro ncRNA [Airn (108 kb), Kcnq1ot1 (91 kb) and CTCF Nespas (~30 kb), respectively] that has an overlap in the antisense A CCCTC-binding factor that is an 11-zinc-finger protein that binds direction with only one gene in each imprinted cluster (for insulator elements. references, see Table 2). In the Pws/As cluster, the provisionally Differentially methylated region (DMR) named Snrpn-long-transcript (Snrpnlt, also known as Lncat) is an A CG-dinucleotide-rich genomic region that, in diploid cells, is unusually long macro ncRNA that could cover 1000 kb of genomic methylated on one parental chromosome and unmethylated on the sequence. The Snrpnlt ncRNA overlaps in antisense orientation with other. Gametic DMRs acquire their parental-specific DNA the Ube3a gene, which is located 720 kb downstream. The size of methylation during gametogenesis, either in the developing haploid the ICE in this cluster has not been precisely determined in the oocyte or sperm, whereas somatic DMRs acquire their parental- mouse, as the smallest available ICE deletion only shows a partial specific DNA methylation in somatic diploid cells. or mosaic imprinting defect (Bressler et al., 2001). In the paternally DNA/RNA FISH imprinted Igf2 and Dlk1 clusters, the ICE is found 5-14 kb upstream A fluorescence in situ hybridisation technique that uses a of the H19 macro ncRNA (2.2 kb) and the provisionally named complementary DNA or RNA strand to determine the localisation of Gtl2-long-transcript (Gtl2lt; length unknown), respectively. H19 DNA sequences or RNA transcripts in cell nuclei. lacks any known transcriptional overlap with the other genes in the Dosage compensation cluster, whereas Gtl2lt overlaps with Rtl1. In short, although the An epigenetic regulatory mechanism present in mammals, flies and organisation of these six well-studied imprinted clusters appears to worms that equalises the expression of genes on the X chromosome be complex, they generally follow two simple rules: (1) an between XY/X0 males and XX females. unmethylated ICE is required for macro ncRNA expression; and (2) Epigenetics most imprinted mRNA genes are not expressed from the Modifications of DNA or chromatin proteins that alter the ability of chromosome from which the macro ncRNA is expressed. DNA to respond to external signals. Imprinted macro ncRNAs can host short ncRNAs Gene regulation in cis The length of the macroRNAs of the imprinted clusters, i.e. Airn, See cis-acting function. Kcnq1ot1, Nespas, Snrpnlt, H19 and Gtl2lt, ranges from 2.2-1000 Gene regulation in trans kb (Table 2). Intriguingly, as shown in Fig. 1, the majority of these See trans-acting function. imprinted macroRNAs, with the possible exception of Kcnq1ot, also Genomic imprinting serve as host transcripts for trans-acting short RNAs, such as An epigenetic mechanism that induces parental-specific gene siRNAs, which are involved in gene silencing by the RNA expression in diploid mammalian cells (see Box 2). interference pathway (reviewed by Mattick and Makunin, 2006), Imprint control element (ICE) miRNAs, which function as translational gene repressors (reviewed A short DNA sequence [also known as an ICR (imprint control by Cannell et al., 2008), and snoRNAs, which are involved in rRNA region) or IC (imprinting centre)] that controls the imprinted processing (reviewed by Brown et al., 2008). For example, siRNAs expression of multiple genes in cis. All known ICEs are also gametic encoded by the Au76 pseudogene region lie within the Airn ncRNA DMRs; however, their identification requires the in vivo analysis of a in the Igf2r cluster and are found in oocytes (Watanabe et al., 2008). deletion of the DMR. In the Pws/As cluster, two snoRNA clusters (Snord115, Snord116) Insulator element are located within the Snrpnlt macro ncRNA (Cavaille et al., 2000; A genetic boundary element that binds insulator proteins to Huttenhofer et al., 2001). In the Gnas cluster, two miRNAs are separate a promoter on one side of the insulator element from the located within the Nespas macro ncRNA (Holmes et al., 2003). In activating effects of an enhancer located on the other side. the Igf2 cluster, both the H19 ncRNA and the protein-coding Igf2 Macro ncRNAs gene contain a miRNA within their transcriptional unit (Cai and ncRNAs that can be as short as a few hundred nucleotides or as long Cullen, 2007; Landgraf et al., 2007). In the Dlk1 cluster, four as several hundred thousand nucleotides, the function of which separate macro ncRNAs have been described [Gtl2 (Meg3), Rtl1as, does not depend on processing into short or micro RNAs. Rian, Mirg] that might be contained within the Gtl2lt ncRNA. Gtl2, Rtl1as and Mirg contain multiple miRNAs, whereas Rian contains Tiling-array analysis multiple snoRNAs (Cavaille et al., 2002; Houbaviy et al., 2003; Commercial chips containing 25-60 nt oligonucleotide probes Huttenhofer et al., 2001; Kim et al., 2004; Lagos-Quintana et al., designed to continuously cover a genomic region that are used to 2002; Seitz et al., 2004; Seitz et al., 2003). Interestingly, miRNAs produce unbiased maps of histone modifications following generated from Rtl1as have been shown to be involved in the trans- chromatin immunoprecipitation (ChIP-chip), or of DNA methylation following methylated DNA immunoprecipitation (MeDIP-chip), or of silencing of Rtl1 through an siRNA-mediated pathway (Davis et al., gene expression following cDNA hybridisation (RNA-chip). 2005). Few of the small ncRNAs in these clusters have been analysed in detail and most probably have trans-acting functions, Trans-acting function which indicates that they are unlikely to be involved in regulating The ability of a DNA sequence or transcript to regulate the imprinted gene expression that depends on a cis-acting mechanism. expression of one or more genes on different chromosomes or to However, their presence suggests a functional link between macro regulate mature RNAs in the cytoplasm. ncRNAs and short ncRNAs. DEVELOPMENT Development 136 (11) REVIEW 1773 Imprinted ncRNAs – atypical mammalian Box 2. Genomic imprinting: basic biology, history and transcripts? clinical implications An unusual feature of many imprinted ncRNAs is that they are Mammals are diploid organisms whose cells contain two matched unspliced or spliced with a low intron/exon ratio, in contrast to the sets of chromosomes, one inherited from the mother and one from majority of mammalian mRNA-encoding genes, which are intron the father. Thus, mammals have two copies of every gene with the rich. Notably, the export of ncRNAs to the cytoplasm correlates with same potential to be expressed in any cell. Genomic imprinting is an splicing (Table 2). The H19 ncRNA is fully spliced and exported to epigenetic mechanism that affects ~1% of genes and restricts their the cytoplasm (Brannan et al., 1990; Pachnis et al., 1984), whereas expression early in development to one of the two parental the Kcnq1ot1 ncRNA is unspliced and retained in the nucleus chromosomes. Genes that show parental-specific expression were (Pandey et al., 2008). The Airn ncRNA produces unspliced and hypothesised to exist in mammals following a series of landmark observations that began to accumulate thirty years ago. These spliced transcripts at a ratio of 19:1, and only the spliced transcripts included the failure of embryos to develop by parthenogenesis in the are exported to the cytoplasm (Seidl et al., 2006). In the Gnas cluster, absence of fertilisation, the phenotype of embryos that had inherited both Exon1A and Nespas ncRNAs are also found as spliced and two copies of one parental chromosome in the absence of the other unspliced forms, but their cellular localisation is unknown (Holmes parental copy, and the inability to generate viable embryos that et al., 2003; Li et al., 1993; Liu et al., 2000; Williamson et al., 2002). contained two maternal or paternal pronuclei through oocyte Both Airn and Kcnq1ot1 were shown by RNA fluorescence in situ nuclear transfer experiments. This hypothesis was corroborated in hybridisation (RNA FISH; see Glossary, Box 1) to form RNA 1991 by the discovery of three imprinted genes: the maternally ‘clouds’ at their site of transcription (Braidotti et al., 2004; Nagano expressed Igf2r gene, the paternally expressed Igf2 gene and the et al., 2008; Terranova et al., 2008). We do not yet know whether maternally expressed H19 ncRNA. Whereas genomic imprinting now these ncRNA ‘clouds’ explain their ability to repress flanking genes offers one of the best models in which to investigate epigenetic gene regulation in mammals, it also has considerable implications for or whether this ‘cloud-like’ appearance is a consequence of a lack of modern molecular medicine in the management of genetic diseases splicing, as mRNA genes mutated to inhibit splicing also show that map to autosomes but are only inherited from one parent, and nuclear retention and intranuclear RNA focus formation (Custodio in the efforts to apply assisted reproductive or cloning technologies et al., 1999; Ryu and Mertz, 1989). to human reproduction. Although not all imprinted macro ncRNAs have been studied in sufficient detail, at least some have been shown to have unusual transcriptional features that are not generally associated with mammalian mRNA genes, such as reduced splicing potential or low 1999). Instead, a methylation-sensitive insulator element (see intron/exon ratio, nuclear retention and accumulation at the site of Glossary, Box 1) contained in the ICE regulates the ability of transcription. Investigations into the control of these unusual enhancers that lie downstream of H19 to interact physically with the transcriptional features, and into their role in the functional upstream H19 and Igf2 promoters (Fig. 1E). On the unmethylated properties of imprinted ncRNAs, are only just beginning. An maternal allele, CTCF (see Glossary, Box 1) binds the ICE and obvious genetic element that might account for unusual restricts the access of enhancers to the H19 promoter. On the transcriptional features is the promoter. It was recently shown in methylated paternal allele, CTCF cannot bind, and the enhancers fission yeast that splicing regulation is promoter driven (Moldon et interact preferentially with the Igf2 promoter, facilitating its al., 2008). It was therefore surprising to find that the endogenous transcription (Bell and Felsenfeld, 2000; Hark et al., 2000). An Airn promoter does not control the low splicing capacity of the Airn additional mechanism to induce imprinted expression is present in ncRNA (Stricker et al., 2008), and further work is required to the newly described H13 imprinted cluster on mouse chromosome determine how these unusual macro ncRNA features arise. 2 (Wood et al., 2007) (not shown in Fig. 1). The H13 (histocompatibility 13 antigen) (Graff et al., 1978) gene contains an Macro ncRNAs are functional in imprinted intronic, maternally methylated gDMR. The transcription of full- expression length functional H13 from the maternal chromosome depends on The presence of macro ncRNAs in imprinted clusters raises the the methylation of this gDMR. On the paternal allele, the question of whether they play a functional role in imprinting. In the unmethylated gDMR acts as a promoter for the Mcts2 retrogene, and case of the two paternally expressed imprinted macro ncRNAs Airn Mcts2 expression correlates with the premature polyadenylation of and Kcnq1ot1, experiments in which the ncRNA was truncated by H13 (Wood et al., 2008). To date, it is unknown whether the Mcts2 the homologous insertion of a polyadenylation cassette have retrogene is coding or non-coding, nor whether Mcts2 expression or demonstrated that these ncRNAs are indeed necessary for imprinted the unmethylated gDMR is required to block the production of full- expression. Paternal chromosomes that carry a truncated Airn or length H13 transcripts. Kcnq1ot1 ncRNA lose the repression of all protein-coding genes in Thus, two out of the three tested imprinted macro ncRNAs act the imprinted cluster in both embryonic and placental tissues, in cis to induce the imprinted expression of flanking genes. These whereas maternal alleles are unaffected (Mancini-Dinardo et al., macro ncRNAs might also possess additional functions. For 2006; Shin et al., 2008; Sleutels et al., 2002). These experiments example, as their promoter is contained in the ICE, they could showed that these macro ncRNAs act by repressing multiple play a role in acquiring the gametic DNA methylation imprint. flanking genes in cis in both embryonic and placental tissues. However, a recent publication indicates that the transcription of Nespas is similar to Airn and Kcnq1ot1 in that it is transcribed from overlapping imprinted protein-coding genes, rather than of a promoter contained within the unmethylated ICE on the paternal ncRNAs, is needed to acquire methylation imprints in the Gnas allele and has an antisense orientation with respect to the imprinted cluster (Chotalia et al., 2009). This work shows that a truncation protein-coding Nesp gene. However, it is not yet known whether of the Nesp mRNA transcript (Fig. 1D) by the insertion of a Nespas has a cis-silencing role similar to Airn and Kcnq1ot1. By polyadenylation cassette, which abolishes transcription through contrast, the maternally expressed H19 ncRNA is known to be the ICE, impairs acquisition of the ICE methylation mark in dispensable for the imprinted expression of Igf2 (Schmidt et al., oocytes. This might represent a common theme for oocyte- DEVELOPMENT 1774 REVIEW Development 136 (11) Fig. 1. Six well-studied imprinted A Igf2r chr. 17, 490 kb clusters. (A-F) The genomic Plg Slc22a2 Slc22a1 Igf2r Slc22a3 Mas organisation of six well-studied mouse imprinted clusters. The maternal Me chromosome is shown as a pink bar, the paternal chromosome as a blue Au76 Airn siRNAs bar. Protein-coding genes are shown as boxes: solid red box, maternally expressed gene on the expressed allele; transparent red box, maternally B Kcnq1 chr. 7, 780 kb expressed gene on the repressed Cd81 Cdkn1c Phlda2 Cars Tnfrsf22 Osbpl5 allele; solid blue box, paternally Ascl2 Trpm5 Slc22a18 Nap1/4 Tnfrsf26 Tspan32 Tssc4 Kcnq1 Tnfrsf23 expressed gene on the expressed allele; transparent blue box, paternally Me expressed gene on the repressed allele. Macro ncRNAs are shown as Kcnq1ot1 wavy lines: red for maternally expressed, blue for paternally expressed. Arrows indicate transcriptional direction: solid arrows, strong transcription; dashed arrows, C Pws/As chr. 7, 3700 kb weak transcription. Note that many of Atp10a Ube3a Ndn Mkrn3 Snurf/Snrpn Magel2 Frat3 the indicated genes show tissue- or temporal-restricted gene expression Me (not indicated). See key for further Snrpnlt (Lncat) details. chr., chromosome; ICE, imprint ? Ipw Snurf U exons Ube3a-ats Pwcr1 control element; miRNA, microRNA; siRNA, short interfering RNA; snoRNA, short nucleolar RNA. snoRNAs Snord115 Snord116 Gnasxl D Gnas chr. 2, 80 kb Nesp Gnas Me miR-296 Nespas Exon1a miR-298 E Igf2 chr. 7, 80 kb miR-675 Igf2 Ins2 H19 miR-483 Igf2as Me F Dlk1 chr. 12, 830 kb Gtl2lt Dio3as Mico1 Dlk1 Rtl1 Dio3 Rian Mirg Gtl2 Mico1os Rtl1as miRNAs C/D snoRNAs miRNAs Me Key: Expression only/ Me Methylated ICE Small ncRNA preferentially from Macro ncRNA Methylation/imprinted paternal allele Unmethylated ICE expression restricted Expression only/ Possible to placenta Somatic DNA methylation preferentially from continuation ? Conflicting data No DNA methylation maternal allele of macro ncRNA Enhancer Gametic DNA methylation * Silent paternal copy outside of ICE Non-imprinted gene Silent maternal copy DEVELOPMENT Development 136 (11) REVIEW 1775 Table 1. Imprint control elements of six well-studied imprinted clusters ICE (a gametic DMR) Associated histone modifications Genomic position (UCSC mm9) Imprinted cluster DNA methylated ICE Unmethylated ICE 1,2 3 3 Igf2r Region 2 Maternal: Paternal: Chr17: 12934307-12935355 H3K9me3, H4K20me3 H3K4me3, H3K4me2, H3K9ac 4 5,6 5,6 Igf2 H19 DMD Paternal: Maternal: Chr7: 149766154-149767791 H3K9me3, H4K20me3 H3K4me2, H3K4me3, H3ac 7 5,8 5,8 Kcnq1 KvDMR1 Maternal: Paternal: Chr7: 150481310-150482463 H3K9me, H3K27me H3K4me, H3K9ac, H3K14ac, H3ac, H4ac 9 5,10 5,10 Pws/As Part may be contained in 4.8 kb , which is Maternal: Paternal: orthologous to the human PWS-IC H3K9me3, H4K20me3, H3K4me2, H3K4me3, H3ac, H4ac Chr7: 67147668-67152741 H3K27me3 (ES only) Gnas Nespas DMR Overlapping H3K4me3/H3K9me3 peaks in ES cells, H3K4me3 Chr2: 174117482-174125568 paternal bias (unmethylated ICE) 13 5,6 5,6 Dlk1 IG-DMR Paternal: Maternal: Chr12: 110765047-110769203 H3K9me, H4K20me3 H3K4me3, H3K4me2, H3ac, H4ac ac, acetylation; Chr, mouse chromosome; DMR, differentially methylated region; H3, histone 3; H4, histone 4; ICE, imprint control element; K, lysine; me2, dimethylation; me3, trimethylation; PWS-IC, Prader-Willi imprint control element; UCSC, University of California Santa Cruz genome browser. 1 2 3 4 5 6 7 References: (Wutz et al., 1997), (Wutz et al., 2001), (Regha et al., 2007), (Thorvaldsen et al., 1998), (Verona et al., 2008), (Delaval et al., 2007), (Fitzpatrick et al., 8 9 10 11 12 13 2002), (Umlauf et al., 2004), (Bressler et al., 2001), (Wu et al., 2006), (Williamson et al., 2006), (Mikkelsen et al., 2007), (Lin et al., 2003). specific DNA methylation imprints, given that an overlapping There are several examples in which differential imprinted mRNA gene has been reported to be transcribed through five expression correlates with differential ncRNA expression. In the other maternal gDMRs in oocytes, but not in sperm. Perhaps mouse brain, the imprinted expression of the Ube3a gene in the surprisingly, ncRNA transcription might play a role in Pws/As cluster is seen in neurons that express the Ube3a-ats maintaining the unmethylated state of the ICE in the early transcript, which might be continuous with Snrpnlt (Fig. 1C), but it embryo. This is indicated by experiments in which the Airn is not seen in glial cells that lack this antisense RNA (Yamasaki et promoter was deleted from the paternal chromosome in al., 2003). Glial cells show imprinted expression of Igf2r and express embryonic stem (ES) cells, which resulted in the methylation of the Airn ncRNA, but neurons lack Airn and show non-imprinted the normally unmethylated paternal ICE (Stricker et al., 2008). Igf2r expression (Yamasaki et al., 2005). Imprinted expression of In summary, macro ncRNAs have been shown to function in Igf2r also correlates with Airn expression in embryonic inducing parental-specific gene expression in the Igf2r and development. Preimplantation embryos lack Airn and express Igf2r Kcnq1ot1 imprinted clusters. In the Igf2 imprinted cluster, the from both parental chromosomes, whereas post-implantation ncRNA is expressed from the parental chromosome, which silences embryos express Airn only from the paternal chromosome and Igf2r the protein-coding genes but does not itself play a functional role in only from the maternal chromosome (Sleutels et al., 2002; Szabo the silencing. The functions of ncRNAs in the other imprinted and Mann, 1995). In terms of in vitro models for studying clusters shown in Fig. 1 are yet to be tested. imprinting, it was shown recently for the Igf2r cluster that ES cell differentiation mimics the onset of imprinted expression and the gain Developmental and tissue-specific imprinted of epigenetic modifications seen in the developing embryo (Latos et expression al., 2009). This work establishes the utility of ES cells to study the How might developmental or tissue-specific imprinted expression imprinted expression that is typical for embryonic tissue. However, arise? In this section, we discuss mechanisms that might as the imprinted expression of the Slc22a2 and Slc22a3 genes in the differentially regulate imprinted expression, and describe recently Igf2r cluster is restricted to the placenta labyrinth layer (Fig. 2A), developed in vitro model systems that provide an excellent tool with this cannot be analysed in ES cells, which arise from a cell lineage which to study these mechanisms (Fig. 2). Genomic imprinting that does not contribute to this tissue. Trophoblast stem (TS) cells consists of a cycle of events that begins when the ICE DNA are an obvious ES cell analogue for the study of genes that show methylation imprint is established on one parental allele during imprinted expression only in placental tissues (Fig. 2B). However, gametogenesis. After fertilisation, when the embryo is diploid, the differentiated TS cells appear to be an unsuitable model for the later ICE methylation imprint is maintained on the same parental allele stages of placental development, as the expression patterns and through the action of the DNA methyltransferase DNMT1 (Li et al., histone modifications detected in vivo are not recapitulated in vitro 1993). In subsequent developmental or tissue-specific regulated (Lewis et al., 2006). steps, imprinted expression can be maintained by additional The placenta provides a good example of tissue-specific epigenetic modifications or lost in the absence of such factors. In the variation in imprinted expression, as the majority of imprinted examples already discussed, temporal- and tissue-specific imprinted genes in the mouse only show imprinted expression in the expression could be achieved by regulating ncRNA expression and placenta (Wagschal and Feil, 2006). This occurs because in many function (for the Igf2r and Kcnq1 clusters) or by regulating insulator imprinted clusters a small number of centrally positioned genes formation (for the Igf2 cluster). To complete the genomic imprinting show ‘ubiquitous’ imprinted expression (i.e. in embryo, placenta life cycle, the ICE methylation imprint and any secondary epigenetic and adult), whereas additional genes in the cluster that extend modifications are erased during early germ cell development to upstream or downstream have imprinted expression only in the allow the parental gametes to acquire a maternal or paternal DNA placenta (Fig. 1). As experiments that involve either ICE deletion methylation imprint ready for the next generation (reviewed by or ncRNA truncation (as described above) show that imprinted Barlow and Bartolomei, 2007). expression in the embryo and the placenta are controlled by the DEVELOPMENT 1776 REVIEW Development 136 (11) Table 2. Non-coding RNAs in imprinted clusters in the mouse genome Imprinted cluster Unspliced or (mouse Macro spliced/subcellular chromosome) ncRNA Published size localisation Cis-silencing function Host for small RNAs 1, 2 4 5 Igf2r Airn * 108 kb 95% unspliced (N) Yes Au76 siRNA (17) 5% spliced to ~1 kb (C) 6 7 7,8 9,‡ 8,10 Kcnq1 Kcnq1ot1 ~90 kb Unspliced (N) Yes (7) † 11-13 Pws/As Snrpnlt Numerous splice Spliced n.d. 11 11-13 14,15 (7) (Lncat) variants Snord64 MBII-13 Might contain all ncRNAs listed below 14,15 Genomic size: Snord115 MBII-52 11 16,17 1000 kb Snord116 MBII-85 Snurf U n.d. n.d. n.d. exons 18 18 18 Pwcr1 2.2 kb Unspliced n.d. 19 19 Ipw 0.5-12 kb Several splice n.d. Genomic size: variants 13,§ 60 kb mainly (N) Ube3a- n.d. Spliced/unspliced n.d. ats n.d. 13,§ mainly (N) 21 25 Gnas Nespas Unspliced at least Several spliced n.d. mmu-mir-296 22 25 (2) 3.35 kb and unspliced mmu-mir-298 22-24 Spliced 1.4-15.8 versions 22,23 kb Genomic size: at least 30 kb Exon1A Unspliced >1.1 Spliced and n.d. 23 23,26 kb unspliced Spliced >1.4 kb Genomic size: 19 kb 27 28 28 29 30 31 Igf2 H19 2.2 kb Spliced (C) no mmu-mir-675 (7) Genomic size: 2.5 kb 32 32 Igf2as 4.8 kb Alternative n.d. Genomic size: promoters, spliced 10.7 kb 33 33 33 Dlk1 Mico1 2 kb Unspliced n.d. In silico predicted precursor (12) miRNA 33 33 33 Mico1os 2 kb Unspliced n.d. In silico predicted precursor miRNA Gtl2lt Genomic size: Hypothetical transcript; might contain all mmu-mir: 34-36 208 kb ncRNAs listed below 770/673/493/337/540/665/431/433/1 43 43-45 Gtl2 1.9-7 kb Several splice n.d. 27/434/136/341/1188/370/882/379/4 11/299/380/1197/323/758/329/494/6 Genomic size: variants 43,§ 30.7 kb mainly (N) 79/1193/543/495/667/376c/654/376b 40 40 /376a/300/381/487b/539/544/352/13 Rtl1as n.d. Processed miRNAs involved 4/668/485/435/154/496/377/541/409/ in trans silencing 34,37-40 412/369/41 of Rtl1 47 47 47 MBII-48(5), MBII-49(4), ImsnoRNA1, Rian 5.4 kb Spliced n.d. 47,‡ MBII-426(1), ImsnoRNA2, MBII- Genomic size: mainly (N) 343(1), ImsnoRNA3, MBII-78(1), 6.1 kb 15,41,42 40 40 40 MBII-19(3) Mirg 1.3 kb Spliced n.d. Genomic size: 14.5 kb The six well-studied imprinted clusters and their associated ncRNAs are listed with the macro ncRNA name, sizes of unspliced and spliced variants, genomic size if different from the longest unspliced version, and cellular localisation [note that nuclear localisation was sometimes inferred from RNA FISH (see Glossary, Box 1) data that 1 2 3 did not exclude export to the cytoplasm]. Silencing function and hosted small ncRNAs are indicated. References: (Wutz et al., 1997), (Lyle et al., 2000), (Seidl et al., 4 5 6 7 8 9 10 2006), (Sleutels et al., 2002), (Watanabe et al., 2008), (Smilinich et al., 1999), (Pandey et al., 2008), (Mancini-Dinardo et al., 2006), (Terranova et al., 2008), (Shin et 11 12 13 14 15 16 17 al., 2008), (Landers et al., 2004), (Gerard et al., 1999), (Le Meur et al., 2005), (Cavaille et al., 2000), (Huttenhofer et al., 2001), (Skryabin et al., 2007), (Ding et 18 19 20 21 22 23 al., 2005), (de los Santos et al., 2000), (Wevrick and Francke, 1997), (Chamberlain and Brannan, 2001), (Wroe et al., 2000), (Williamson et al., 2002), (Holmes et 24 25 26 27 28 29 30 al., 2003), (Li et al., 2000), (Royo et al., 2006), (Liu et al., 2000), (Pachnis et al., 1984), (Pachnis et al., 1988), (Brannan et al., 1990), (Schmidt et al., 1999), 31 32 33 34 35 36 37 (Cai and Cullen, 2007), (Moore et al., 1997), (Labialle et al., 2008), (Seitz et al., 2004), (da Rocha et al., 2008), (Tierling et al., 2006), (Lagos-Quintana et al., 38 39 40 41 42 43 44 2002), (Houbaviy et al., 2003), (Kim et al., 2004), (Seitz et al., 2003), (Cavaille et al., 2002), (Xiao et al., 2006), (Schuster-Gossler et al., 1998), (Schmidt et al., 45 46 47 2000), (Miyoshi et al., 2000), (Davis et al., 2005), (Hatada et al., 2001). C, cytoplasmic; ImsnoRNA, snoRNAs at the Irm locus; MBII, mouse brain 2; mmu-mir, Mus musculus microRNA; N, nuclear; n.d., not determined. *Renamed from Air by HUGO nomenclature committee. Provisional name. By RNA FISH. By whole-mount in situ hybridisation. DEVELOPMENT Development 136 (11) REVIEW 1777 Fig. 2. Embryonic and extra- A Lineage relationships in embryonic development embryonic tissues in vivo and in (a) 3.5 dpc blastocyst (b) 12.5 dpc embryo and extra-embryonic tissues vitro. (A) Lineage relationships during embryonic development. (a)A Decidua basalis PYS (maternal) blastocyst at 3.5 days post-coitum Inner cell mass VYS Trophectoderm (ICM) Amnion Giant cells (dpc). The trophectoderm (blue) Primitive contributes to the parietal yolk sac Trophoblast giant cells PYS endoderm Maternal (PYS) and the placenta of the 12.5 dpc Parietal endoderm blood embryo as depicted in b. The inner cell vessels Visceral endoderm BI VYS mass (ICM, green) gives rise to the Extra-emb. mesoderm Amnion (ectoderm+mesoderm) embryo proper and contributes to the Labyrinth visceral yolk sac (VYS) and amnion Spongiotrophoblast BI: Blood island extra-embryonic membranes. The Umbilical cord primitive endoderm (red) differentiates into the endoderm layer of the PYS B In vitro models to study imprinted expression and VYS. (b) A 12.5 dpc embryo and Pre-implantation development Post-implantation development its extra-embryonic tissues. The embryo (green) is surrounded by the amnion, Embryo Extra-embryonic Embryo Extra-embryonic which consists of ICM-derived Trophectoderm Extra-embryonic membranes In vivo Inner cell mass Embryo* ectoderm and mesoderm. The middle Primitive endoderm Umbilical cord, **Placenta extra-embryonic membrane is the VYS, Undifferentiated Undifferentiated Differentiated Differentiated which consists of ICM-derived In vitro ES cells TS, XEN cells ES cells TS, XEN cells mesoderm (green) and endoderm (red). *Embryo: tissue-specific **Placenta: maternal The outer membrane, the PYS, is lost imprinted expression contamination after 13.5 dpc and consists of ICM- derived parietal endoderm (red) and trophoblast giant cells (blue), which are not ICM derived. The placenta consists of distinct layers: the inner labyrinth (green), the spongiotrophoblast (blue) and giant cells (blue). The outermost part of the placenta, the deciduas basalis (pink), is derived from maternal tissue. The intermingling of maternal blood vessels with the placenta is indicated. (B) In vitro model systems to study genomic imprinting. Undifferentiated embryonic stem (ES) cells are considered to mimic the blastocyst ICM, whereas undifferentiated trophoblast stem (TS) and extra-embryonic endoderm (XEN) cells mimic the blastocyst trophectoderm and primitive endoderm, respectively (Rossant, 2007). The differentiated derivatives of ES, TS and XEN cells might provide models for studying imprinting in post-implantation embryonic and extra-embryonic tissues, but care should be taken to ensure that the in vitro situations recapitulate what is observed in vivo. same elements, there are two possible explanations for this methylation can therefore offer insights into how this epigenetic phenomenon: either the ICE or the ncRNA acts differently in modification is attracted to specific sequences and how it is used these two tissues to repress genes; or the placenta allows to inhibit ncRNA transcription and insulator function. In the spreading of the basic mechanism that operates in embryonic maternal germline, the DNA methyltransferase-like protein tissue. The mouse placenta is a highly invasive organ, and a DNMT3L, in concert with the DNA methyltransferase DNMT3A, complete separation of embryonic and maternal tissue is not are crucial players in the establishment of ICE germline DNA possible (Fig. 2A). This maternal contamination means that the methylation (Bourc’his and Proudhon, 2008). The subsequent placenta might not be a reliable tissue for analysing imprinted maintenance of ICE methylation requires the DNMT1 family of expression and epigenetic modifications. The placenta is an extra- DNA methyltransferases (Hirasawa et al., 2008). Additional embryonic tissue, which means that it is an embryonic tissue that proteins, such as the Krüppel-associated box zinc-finger protein does not contribute to the embryo itself. If ‘placental-specific’ ZFP57, are also required for acquiring ICE methylation in the imprinted expression were a general feature of extra-embryonic Pws/As cluster and for maintaining ICE methylation in Dlk1 (as tissues, it might be more easily analysed in the extra-embryonic well as in three other imprinted clusters not shown in Fig. 1), but membranes (amnion, parietal and visceral yolk sac), which can be play no role in the Igf2 and Igf2r clusters (Li et al., 2008). isolated without the presence of contaminating maternal tissue Although the exact mechanism by which ZFP57 acts is unknown, (Fig. 2A). The imprinted expression of some genes in the Igf2 and this finding raises the possibility that each ICE requires different Kcnq1 imprinted clusters has been demonstrated in the visceral additional factors for the acquisition and maintenance of germline yolk sac (Davis et al., 1998; Frank et al., 1999); however, as the DNA methylation. Exactly how de novo DNA methylation lineages of the extra-embryonic membranes and placenta differ, enzymes recognise ICE sequences is unclear. Many ICEs contain it remains to be determined whether the ‘placental-specific’ a run of tandem direct repeats that have been suggested to form a imprinted expression pattern is conserved in all extra-embryonic secondary structure that induces DNA methylation (Neumann and tissues. Barlow, 1996). Tandem direct repeat sequences from the Igf2r and Kcnq1 cluster ICEs are able to induce maternal germline DNA methylation represses a cis-acting repressor methylation in a transgenic model (Reinhart et al., 2006). The role As described above, deletion experiments show that only the of these repeats in the endogenous Igf2r ICE is not yet known; unmethylated ICE is active in inducing the silencing of flanking however, the in vivo deletion of a subset of tandem repeats from protein-coding genes, either by activating a ncRNA promoter or the Kcnq1ot1 ICE or of direct repeats that flank the Igf2 ICE did by forming an insulator (see also Fig. 1). Thus, the ICE can be not change ICE DNA methylation (Lewis et al., 2004; Mancini- viewed as a cis-acting repressor and DNA methylation as a Dinardo et al., 2006). By contrast, a mouse strain-specific loss of modification to repress this repressor. The analysis of ICE methylation was observed following the deletion of a repeat DEVELOPMENT Eset G9a Ezh2 1778 REVIEW Development 136 (11) region in the paternally imprinted Rasgrf1 cluster located on chromosome that carries the methylated ICE. Thus, in imprinted mouse chromosome 9 (not shown in Fig. 1) (Yoon et al., 2002). clusters, DNA methylation acts by repressing a repressor of Thus, further work is needed to determine exactly how the imprinted protein-coding genes. methylation machinery in oocytes targets ICE sequences. Although the analysis of imprinted genes highlights one of the Histone modifications associated with imprinted few reported cases of a functional role for DNA methylation in gene clusters gene silencing, it should be noted that its silencing effect is The previous section described how DNA methylation directly directed towards the ncRNA. As shown in Fig. 1, imprinted regulates ICE activity, but does not directly silence imprinted protein-coding genes are expressed from the parental protein-coding genes. Here, we discuss current progress in A Igf2r cluster Fig. 3. Allele-specific histone modifications at the Igf2r and 11.5 dpc placenta Kcnq1 clusters. Active and repressive Plg Slc22a3 Slc22a2 Slc22a1 Igf2r Mas1 histone modifications on the maternal or paternal allele are shown for the (A) Me Igf2r and (B) Kcnq1 clusters for embryo and placenta. For simplicity, histone G9a ? modifications are combined into three ? ? groups: repressive constitutive Airn heterochromatin (H3K9me2/3, G9a H4K20me3), repressive facultative heterochromatin (H3K27me3) and 13.5 dpc embryo - MEFs Suv4-20H active euchromatin (H3K9ac, Plg Slc22a3 Slc22a2 Slc22a1 Igf2r Mas1 H3K4me2/3). In both clusters, only the indicated positions were assayed in the Me placenta, with the exception of one Suv4-20H ChIP-chip mapping of H3K27me3 (orange lines). (A) In the Igf2r cluster, an unbiased continuous genome-wide location (ChIP-chip) analysis was Airn performed in the embryo, with the indicated positions showing B Kcnq1 cluster enrichment. (B) In the Kcnq1 cluster, all 9.5 dpc - 13.5 dpc placenta positions shown in the placenta were Ascl2 Cd81 Trpm5* Slc22a18 Nap1/4 Tnfrsf23 Tnfrsf26* Tspan32 Tssc4 Kcnq1 Cars* Tnfrsf22* Osbpl5 also assayed in the embryo, and only Cdkn1c Phlda2 the indicated positions show allele- specific enrichment. Blue shaded Me ellipses indicate histone-modifying enzymes, and the solid blue arrows 8 8 G9a G9a extending from the ellipses indicate the involvement of the respective enzyme in the setting or maintaining of a histone mark. Both Airn (A) and Kcnq1ot1 Kcnq1ot1 (B) ncRNAs bind to histone- modifying enzymes. Dashed arrows (B) Suz12 indicate binding of Kcnq1ot1 ncRNA to G9a chromatin. Gene expression marked by 9.5 dpc - 13.5 dpc embryo an asterisk is inferred from the presence of active histone PR-SET7 Slc22a18 Nap1/4 Tnfrsf23 Ascl2 Cd81 Trpm5* Tnfrsf26* Cars* Tnfrsf22* modifications (Mikkelsen et al., 2007). Tspan32 Tssc4 Kcnq1 Cdkn1c Phlda2 Osbpl5 Suv4-20H References: Igf2r in the placenta Me (Nagano et al., 2008); Igf2r in the embryo (Regha et al., 2007); Kcnq1 in the placenta: all histone modifications without a numbered reference (Umlauf et al., 2004); others: (Green et al., Kcnq1ot1 2007), (Lewis et al., 2004), 3 4 (Mikkelsen et al., 2007), (Pandey et Key: 5 al., 2008), (Pannetier et al., 2008), 6 7 Combined K27me3/K9me3/K20me3 Repressive facultative heterochromatin (H3K27me3) (Regha et al., 2007), (Umlauf et al., Active euchromatin (H3K9ac, H3K14ac, H3K4me2/3) Repressive constitutive heterochromatin (H3K9me2/3, H4K20me3) 2004), (Wagschal et al., 2008). ac, acetylation; H3, histone 3; H4, histone No information Biallelic silent Biallelic expression Imprinted expressed Imprinted silent Me Methylated ICE 4; ICE, imprint control element; K, Transcript orientation *No imprinted expression Imprinted macro ncRNA Unmethylated ICE lysine; me2, dimethylation; me3, Full, weak or no DNA methylation ? Possible maternal contamination trimethylation. H3K27me3 enrichment placenta vs. liver, no allele-specific information Eset 1,2 1,2 2,8 2,8 1,2,7 1,2 1,2 2 2 2,8 2 2 2,7 2 2 2 7 2 DEVELOPMENT Development 136 (11) REVIEW 1779 understanding the potential roles played by histone modifications in general function for histone modifications has not yet been restricting expression of macro ncRNAs to one parental allele and identified. Repressive H3K9me3 modifications are regulated by imprinted protein-coding genes to the other allele. Recent studies three known histone methyltransferases, SUV39H1, SUV39H2 have shown that the ICE carries histone modifications that are (also known as KMT1A and KMT1B) and ESET (KMT1E, specific to the DNA-methylated or the DNA-unmethylated allele SETDB1), whereas the repressive H4K20me3 modification is (Fig. 3 and Table 1). Genome-wide sequencing and oligonucleotide regulated by SUV4-20H1 and SUV4-20H2 (KMT5B and KMT5C) tiling-array analyses have been used to show that the DNA- (Table 3). The repressive H3K9me3 mark is maintained and even methylated ICE is marked by focal repressive histone modifications enhanced on the ICE in embryonic fibroblasts that lack SUV39H1 of the type found in constitutive centromeric and telomeric and SUV39H2, whereas the repressive H4K20me3 is reduced in heterochromatin (Table 3), such as H3K9me3, H4K20me3 and the embryonic cells that lack SUV4-20H1 and SUV4-20H2 without presence of HP1 (Mikkelsen et al., 2007; Regha et al., 2007). The removing either the repressive H3K9me3 mark or DNA methylation discovery of focal repressive heterochromatin changed our general (Pannetier et al., 2008; Regha et al., 2007). The ESET understanding of chromatin, which has traditionally been classified methyltransferase was found to bind to the Igf2r ICE; however, its as either heterochromatin or euchromatin and considered to role could not be tested directly because ESET-deficient cells are not represent, respectively, transcriptionally repressed and active regions viable at any developmental stage (Dodge et al., 2004; Regha et al., (Huisinga et al., 2006). As the ICE is often located within 2007). Thus, suitable genetic systems are not yet available to test the transcribed genes (Fig. 3), it is now clear that focal heterochromatin role of the repressive H3K9me3 and H4K20me3 modifications in can exist inside actively transcribed regions without blocking RNA regulating ICE activity. polymerase II (RNAPII) elongation. In the Igf2r and Kcnq1 clusters, In contrast to the lack of a defined role for histone-modifying the repressive H3K27me3 mark is present on the methylated ICE enzymes in regulating ICE activity, several reports describe a role only in undifferentiated ES cells. In the Igf2r cluster, H3K27me3 is for these enzymes in regulating placental, but not embryonic, absent from embryonic fibroblasts (Fig. 3A), but in the Kcnq1 imprinted expression. The Polycomb group protein EED, which cluster it is present in both embryo and placenta (Fig. 3B) (Latos et is required for repressive H3K27me3 modifications, has been al., 2009; Lewis et al., 2006; Lewis et al., 2004; Umlauf et al., 2004). shown to repress the paternal allele of four out of 18 tested The unmethylated ICE lacks repressive modifications but carries imprinted genes in embryos 7.5 days post-coitus (dpc), which active histone modifications, such as H3K4me and H3/H4 mainly consist of extra-embryonic tissue at this stage (Mager et acetylation. The presence of active and repressive histone al., 2003). The affected genes were located in three different modifications on the same DNA sequence in diploid cells that imprinted clusters, in which the majority of genes maintained modify different parental chromosomes can be used to identify an correct imprinted expression. This indicates that EED does not ICE (Fig. 3). The usefulness of this approach was demonstrated in a play a general role in regulating imprinted expression, but is genome-wide study of diploid ES cells that identified short regions attracted to specific genes. The G9A (KMT1C, EHMT2) histone that carry both repressive H3K9me3 and active H3K4me3 lysine methyltransferase, which dimerises with G9A-like protein modifications on the ICE of the six imprinted clusters shown in Fig. (GLP; KMT1D, EHMT1) to induce repressive H3K9me2 1 (Mikkelsen et al., 2007). modifications (Table 3), is necessary for the paternal repression The histone modification profiles established so far show that of some genes in the Kcnq1 and Igf2r clusters in the placenta, but repressive marks are associated with the DNA-methylated ICE, not in the embryo (Nagano et al., 2008; Wagschal et al., 2008). whereas active histone marks are associated with the unmethylated As mentioned above, in embryonic tissue, repressive ICE. Although it has proven to be relatively straightforward to heterochromatin (H3K9me3, H4K20me3, HP1), but not assign a function to DNA methylation in regulating ICE activity, a repressive H3K27me3, modifies the DNA-methylated ICE in a Table 3. Epigenetic modifications associated with imprinted genes in the mouse genome Gene expression Modification Modifying enzyme or complex Modifications associated with De novo DNA methylation, DNMT3A, DNMT3B, DNMT3L me repressed genes and/or pericentric i.e. CpG to CpG and telomeric heterochromatin DNA methylation of hemi- DNMT1, UHRF1 methylated DNA HP1 H3K27me3 PRC2 containing EED, EZH2 and SUZ12 H3K9me2 G9A (KMT1C, EHMT2); functions as a heterodimer with GLP (KMT1D, EHMT1) H3K9me3 SUV39H1 (KMT1A), SUV39H2 (KMT1B), ESET (KMT1E, SETDB1) H4K20me1 PR-SET7 (KMT5A, SETD8) H4K20me3 SUV4-20H1 (KMT5B), SUV4-20H2 (KMT5C) Modifications associated with H3K4me2 and H3K4me3 Multiple lysine methyltransferases (KMT2A-G) expressed genes Acetyl groups at several lysines in Multiple lysine acetyltransferases (KAT1-13) the tail of histone H3 (K9, K14, K18, K23) and H4 (K5, K8, K16) CpG, CG dinucleotide; DNMT, DNA methyltransferase; EED, embryonic ectoderm development; ESET, ERG-associated protein with a SET domain 7; EZH2, enhancer of zeste homolog 2; GLP, GATA-like protein 1; H3, histone 3; H4, histone 4; HP1, heterochromatin protein 1; K, lysine; KAT, lysine acetyltransferase; KMT, lysine me methyltransferase; me2, dimethylation; me3, trimethylation; CpG, methylated CG dinucleotide; PRC2, Polycomb repressor complex 2; PR-SET7, PR/SET domain- containing protein; SUV, suppressor of variegation; SUZ12, suppressor of zeste 12; UHRF1, ubiquitin-like containing PHD and RING finger domains 1. References: (Allis et al., 2007a; Allis et al., 2007b; Kouzarides, 2007). DEVELOPMENT 1780 REVIEW Development 136 (11) focal manner. Not all of these repressive modifications have been located in nuclear domains that are characterised by a high density mapped throughout the imprinted clusters in the placenta, but of repressive H3K27me3 and by a lack of active histone repressive H3K9me2/3 and H3K27me3 marks were found at the modifications and RNAPII (Terranova et al., 2008). In summary, the promoters of silenced mRNA genes in the Igf2r cluster (Nagano evidence so far indicates that the Airn and Kcnq1ot1 ncRNAs induce et al., 2008) (Fig. 3A). By contrast, these repressive marks were imprinted expression by an RNA-directed targeting mechanism in found to be more widespread in the placenta on the chromosome the placenta that only affects genes that show placental-specific that carries the silenced mRNA genes in the Kcnq1 cluster (Fig. imprinted expression (Fig. 1). We present a model (Fig. 4A) 3B). In one study in the placenta (Nagano et al., 2008), both active according to which the ncRNA expressed from the unmethylated and repressive histone modifications were found on genes that ICE is maintained at the site of transcription and associates with showed placental-specific imprinted expression. Although this chromatin in cis. The ncRNA could localise throughout the cluster might indicate the existence of ‘bivalent’ domains (Bernstein et (Fig. 4A, Kcnq1ot1) or to specific promoters by looping (Fig. 4A, al., 2006), care should be taken in interpreting these results owing Airn), and might subsequently attract specific histone modifications to the risk of maternal tissue contamination in placental samples that repress the transcription of multiple genes located at some (Fig. 2). distance from the ncRNA gene itself. In summary, the analysis of histone modifications shows that the same active and repressive histone modifications that correlate with expressed and silent genes also modify imprinted genes in an allele- specific manner. Further work is needed to determine which A Placenta: large silent domain, RNA-directed silencing modifications reflect the cause as opposed to the consequence of Kcnq1 cluster imprinted expression. Although there is currently no indication that Tspan32 Tssc4 Slc22a18 Nap1/4 Tnfrsf23 Cdkn1c Ascl2 Cd81 Kcnq1 Kcnq1ot1 Kcnq1ot1 Phlda2 Osbpl5 histone modifications co-operate with DNA methylation to restrict macro ncRNA expression to one parental allele, there is emerging data that, in the placenta, histone modifications might play a role in repressing imprinted mRNA genes in cis. As discussed in the next Loop? Igf2r cluster section, there might even be a link between macro ncRNA function Slc22a2 Igf2r Slc22a3 Airn Airn and the establishment of histone modifications. In the placenta, ncRNAs might target repressive B Embryo: promoter-specific/regulator transcription interference histone marks Slc22a18 In both placental and embryonic tissue, the repression of multiple Kcnq1 Kcnq1ot1 Kcnq1ot1 Cdkn1c Kcnq1 cluster Phlda2 genes in the Igf2r and Kcnq1 clusters on the paternal chromosome depends on the Airn and Kcnq1ot1 macro ncRNAs (Mancini- Dinardo et al., 2006; Shin et al., 2008; Sleutels et al., 2002). However, the mechanism by which these ncRNAs induce repression is unknown. One significant open question is whether it is the Igf2r cluster Igf2r Airn Airn ncRNA itself, or the act of its transcription, that is required for silencing (Pauler et al., 2007). Two recent studies indicate that the Airn and Kcnq1ot1 ncRNAs are themselves directly involved in silencing genes in the placenta. Kcnq1ot1 was found to localise Key: physically to several silent genes on the paternal allele that lay Transcription factor 3D interaction Transcription factor binding site hundreds of kb away from the Kcnq1ot1 promoter (Pandey et al., ncRNA Silent imprinted allele Blocked enhancer interaction 2008). This finding is supported by RNA/DNA FISH, which showed partial overlap between the Kcnq1ot1 RNA and the flanking Fig. 4. Models of gene silencing by imprinted macro ncRNAs. imprinted genes in the Kcnq1 cluster in the trophectoderm cells of Imprinted macro ncRNAs might use different modes of silencing in early embryos, which contribute to the placenta (Terranova et al., embryonic and placental tissues. Only the paternal chromosome is 2008). Furthermore, Kcnq1ot1 also directly interacts with Polycomb shown (blue bar). The positions and names of silent imprinted genes group proteins, which are necessary for establishing the repressive are indicated in black font and by pale red boxes. Imprinted macro H3K27me3 mark, and with G9A, which is involved in setting the ncRNAs are indicated in blue font and by blue wavy arrows. (A) In the repressive H3K9me2 mark (Pandey et al., 2008). Together, this placenta, Kcnq1ot1 in the Kcnq1 cluster is transcribed from the indicates that in the placenta the Kcnq1ot1 ncRNA localises to paternal allele and localises to the whole imprinted domain by an chromatin and targets histone methyltransferases to the whole unknown mechanism, inducing the recruitment of repressive histones, imprinted cluster. Notably, embryos that are deficient for G9A and which leads to gene silencing. In the Igf2r cluster, Airn either locates to the silent Slc22a3 promoter, or the promoter forms a three-dimensional for the Polycomb proteins EZH2 and RNF2 show a loss of paternal (3D) loop (indicated by a blue arch) to the Airn gene, similarly recruiting repression for some of the placental-specific imprinted genes in the repressive histone modifications that silence Slc22a3. In both cases, the Kcnq1 cluster (Terranova et al., 2008; Wagschal et al., 2008). ncRNA itself is involved in the silencing process. (B) In embryonic Similarly, in the placenta, the Airn ncRNA in the Igf2r cluster lies in tissues, only a few genes are silenced by the ncRNA in the Kcnq1 and close proximity to the silent Slc22a3 promoter and has been shown Igf2r clusters. No localisation of the ncRNA to chromatin has been to bind G9A. In addition, G9a-null embryos show a loss of placental reported in the embryo. It is therefore possible that the transcription of imprinted expression for Slc22a3, but maintain Igf2r imprinted the ncRNA is sufficient to silence all genes by interfering with the expression (Nagano et al., 2008). An RNA FISH study of the Airn binding of essential transcription factors (black ellipse), thereby and Kcnq1ot1 ncRNAs in TS cells and in preimplantation inducing gene silencing by interrupting enhancer interactions (dashed trophectoderm cells has also shown that both these ncRNAs are arrows). DEVELOPMENT Development 136 (11) REVIEW 1781 Is ncRNA transcription more important in References Allis, C. D., Berger, S. L., Cote, J., Dent, S., Jenuwien, T., Kouzarides, T., embryonic cells? Pillus, L., Reinberg, D., Shi, Y., Shiekhattar, R. et al. (2007a). New As described above, recent work suggests that in the placenta it is the nomenclature for chromatin-modifying enzymes. Cell 131, 633-636. ncRNA transcript itself that mediates gene silencing. However, Allis, C. D., Jenuwein, T. and Reinberg, D. (2007b). Overview and concepts. In Epigenetics (ed. C. D. Allis, T. Jenuwein, D. Reinberg and M. Caparros), pp. 23- imprinted ncRNAs exert different effects in the mouse embryo than 61. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. in the placenta, as only a subset of the genes in the imprinted gene Amaral, P. P. and Mattick, J. S. (2008). Noncoding RNA in development. Mamm. clusters show imprinted expression in embryonic and adult somatic Genome 19, 454-492. Barlow, D. P. and Bartolomei, M. S. (2007). Genomic imprinting in mammals. In tissue (Fig. 1, Fig. 4B). In the Igf2r cluster, the Airn ncRNA represses Epigenetics (ed. C. D. Allis, T. Jenuwein, D. Reinberg and M. Caparros), pp. 357- Igf2r in embryos, but represses Igf2r, Slc22a2 and Slc22a3 in the 375. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. placenta. In the Kcnq1 cluster, the Kcnq1ot1 ncRNA silences Kcnq1, Bell, A. C. and Felsenfeld, G. (2000). Methylation of a CTCF-dependent Cdkn1c, Slc22a18 and Phlda2 in the embryo, but an additional six boundary controls imprinted expression of the Igf2 gene. Nature 405, 482-485. Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., genes in the placenta. A further indication of the differences between Fry, B., Meissner, A., Wernig, M., Plath, K. et al. (2006). A bivalent chromatin the embryo and the placenta is that G9A and EED, which are required structure marks key developmental genes in embryonic stem cells. Cell 125, for repressive H3K9me2 and H3K27me3 modifications, respectively, 315-326. Bielinska, B., Blaydes, S. M., Buiting, K., Yang, T., Krajewska-Walasek, M. and for the imprinted expression of some placental genes, appear to Horsthemke, B. and Brannan, C. I. (2000). De novo deletions of SNRPN exon play no role in the imprinted expression in the Igf2r and Kcnq1 1 in early human and mouse embryos result in a paternal to maternal imprint clusters in embryonic tissues (Mager et al., 2003; Nagano et al., 2008; switch. Nat. Genet. 25, 74-78. Wagschal and Feil, 2006). We have proposed previously that the Airn Bourc’his, D. and Proudhon, C. (2008). Sexual dimorphism in parental imprint ontogeny and contribution to embryonic development. Mol. Cell. Endocrinol. ncRNA might silence Igf2r because of transcriptional interference, 282, 87- 94. and that Airn might act solely by transcription per se (Pauler et al., Braidotti, G., Baubec, T., Pauler, F., Seidl, C., Smrzka, O., Stricker, S., Yotova, 2007). According to this model, ncRNA transcription either interferes I. and Barlow, D. P. (2004). The Air noncoding RNA: an imprinted cis-silencing transcript. Cold Spring Harb. Symp. Quant. 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I., overlap with a promoter (Fig. 4B). However, it is possible to propose Horsthemke, B., Bachellerie, J. P., Brosius, J. and Huttenhofer, A. (2000).Identification of brain-specific and imprinted small nucleolar RNA genes a transcriptional interference mode of silencing for this ncRNA by exhibiting an unusual genomic organization. Proc. Natl. Acad. Sci. USA 97, postulating the existence of crucial cis-regulatory elements that are 14311-14316. overlapped by Kcnq1ot1. Although there is less evidence to support a Cavaille, J., Seitz, H., Paulsen, M., Ferguson-Smith, A. C. and Bachellerie, J. P. (2002). Identification of tandemly-r epeated C/D snoRNA genes at the transcriptional interference model for Kcnq1ot1, the lack of imprinted human 14q32 domain reminiscent of those at the Prader- widespread repressive chromatin marks on genes in this cluster that Willi/Angelman syndrome region. Hum. Mol. 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Journal
Development – The Company of Biologists
Published: Jun 1, 2009