Regulation of the mammalian epigenome by long noncoding RNAs

Biochimica et Biophysica Acta 1790 (2009) 936–947

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Review

Regulation of the mammalian epigenome by long noncoding RNAs Joanne Whitehead b, Gaurav Kumar Pandey a, Chandrasekhar Kanduri a,⁎ a b

Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, 75185 Uppsala, Sweden Mechanics and Genetics of Embryonic and Tumoral Development, Institut Curie/CNRS UMR168, 75005 Paris, France

a r t i c l e

i n f o

Article history: Received 6 August 2008 Received in revised form 9 October 2008 Accepted 12 October 2008 Available online 30 October 2008 Keywords: Noncoding RNA Chromatin Epigenetic Antisense RNA Gene regulation

a b s t r a c t Genomic analyses have demonstrated that although less than 2% of the mammalian genome encodes proteins, at least two thirds is transcribed. Many nontranslated RNAs have now been characterized, and several long transcripts, ranging from 0.5 to over 100 kb, have been shown to regulate gene expression by modifying chromatin structure. Functions uncovered at a few well characterized loci demonstrate a wide diversity of mechanisms by which long noncoding RNAs can regulate chromatin over a single promoter, a gene cluster, or an entire chromosome, in order to activate or silence genes in cis or in trans. In reviewing the activities of these ncRNAs, we will look for common features in their interactions with the chromatin modifying machinery, and highlight new experimental approaches by which to address outstanding issues in ncRNA-dependent regulation of gene expression in development, disease and evolution. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Chromatin is the result of the intimate association between histone proteins and DNA, and provides an epigenetic regulatory framework through which to decode the information stored in the genome in a spatially and temporally regulated manner. This epigenetic information encoded in chromatin is stable, and once established, is replicated faithfully through subsequent cell divisions. Our understanding of the role of such epigenetic control of mammalian gene expression has developed largely out of studies of dosage compensation and genomic imprinting. These two phenomena, now considered paradigms of epigenetic regulation of gene expression, are characterized by the differential regulation, within a single cell, of two alleles which are identical at the DNA sequence level, and thus provide experimental systems with ideal internal controls. By comparing the active and repressed alleles of genes subject to X chromosome inactivation or imprinting, researchers have demonstrated the importance of covalent DNA modification in the form of methylation at CpG dinucleotides, and of a wide variety of post-translational histone modifications, including methylation and acetylation of the histone tails, in determining chromatin conformation [1–4]. A combinatorial use of these modifications serves to precisely regulate the accessibility of the DNA to binding factors, in order to control the probability of transcription of the embedded genes [5]. Characterization of these remarkable epigenetically regulated loci soon led to a more general understanding of chromatin dependent control of gene transcription, which acts throughout the genome to establish tissue specific gene ⁎ Corresponding author. Tel.: +46739600450; fax: +4618558931. E-mail address: [email protected] (C. Kanduri). 0304-4165/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2008.10.007

expression patterns, and to regulate other vital genome functions such as replication, repair and recombination [6]. The prevailing view has been that active and inactive chromatin domains arise mainly as the result of interaction between trans-acting protein factors and cis-acting gene regulatory elements, leading to the hierarchical recruitment of chromatin remodelling complexes [7,8]. However, it is becoming increasingly apparent that RNA constitutes an important component of chromatin and that RNA has a functional role in organizing the chromatin structure and epigenetic memory by acting as an interface with the chromatin modifying machinery [9]. With recent high-throughput analyses suggesting that the vast majority of the mammalian genome is transcribed, while most remains untranslated, it is thought that these numerous noncoding (nc) RNAs have important biological functions in development and differentiation through organizing chromatin into domains of active or repressive structure [10]. Although recent evidence implicates ncRNAs in indexing genetic information through the introduction of DNA and histone modifications, much remains to be deciphered of the underlying mechanisms. Recent studies characterizing the molecular mechanisms which establish and maintain X inactivation and imprinting show how these loci have again provided evidence for an additional layer of complexity in determining chromatin context, based on long nontranslated RNA transcripts. Further evidence from additional loci is now emerging, strengthening the idea that these RNA dependent chromatin modifying mechanisms may represent ubiquitous control parameters. Despite relatively few loci yet investigated in sufficient detail, already a wide diversity of interactions between long noncoding RNAs and different chromatin modifying complexes have been described. ncRNAs can act at on domains ranging in size

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from a single promoter to an entire chromosome, and they can function in cis or in trans to establish chromatin conformations which either activate or repress transcription. The functional significance of several classes of small ncRNAs in regulating chromatin organization has been reviewed elsewhere [9] so here we restrict our discussion to the long ncRNAs. We will look for commonalities in their modes of action, investigate their developmental context, and suggest new approaches to the study of their myriad roles in mammalian disease and evolution. 2. Antisense ncRNAs regulate overlapping genes Global transcriptome analysis has revealed that the mammalian genome encodes a surprising abundance of antisense transcripts, such that an estimated 72% of transcripts in the mouse genome are overlapped by a transcript from the opposite strand [11]. This suggests a high potential for regulatory interactions between overlapping transcripts, and indeed many sense/antisense pairs were found to be coregulated, both concordantly and reciprocally. Coexpression may result from shared enhancers within an expression domain, while reciprocal expression suggests that transcription from one strand serves to inhibit expression of the overlapping partner. The tissuespecific expression patterns of sense/antisense pairs and the abundance of coexpressed pairs [11] together point to a highly contextdependent system of epigenetic silencing via antisense transcription. Data from several model systems supports two general mechanisms by which antisense transcription suppresses expression from the overlapping promoter, referred to as transcriptional interference and RNA interference [12], although no evidence yet shows that these mechanisms act at the level of chromatin, at least in the case of mammals. Here we will focus on emerging data indicating that antisense RNAs can also silence genes through chromatin remodelling pathways. The first evidence linking antisense RNA transcription with chromatin structure was discovered as a part of novel disease mechanism that leads to an inherited form of α-thalassemia via silencing of the HBA2 gene by a cis acting RNA [13]. It was shown in patients carrying a deletion resulting in truncation of the widely expressed gene LUC7L and its juxtaposition to a structurally normal HBA2 gene, that transcription from the LUC7L promoter across the HBA2 promoter results in CpG methylation and silencing of HBA2. Interestingly, this antisense RNA-mediated methylation is restricted to a single CpG island in the HBA2 promoter, while CpG islands within the gene body remain unmethylated, showing a highly localized effect. Intriguingly, the authors recapitulated this phenomenon experimentally using the ubiquitin C promoter to drive antisense transcription across HBA2, and showed that methylation of the promoter does not specifically require LUC7L sequences. Direct silencing of HBA2, through mutation of the promoter or transcription factor binding sites, is insufficient to cause CpG island methylation [13]. Nevertheless, use of an alternative promoter to drive antisense transcription does not allow us to differentiate between de novo methylation due to transcription across the HBA2 promoter and that due to functional sequence motifs in the antisense RNA. A recent investigation has documented a similar mode of action at the p15 cyclin-dependent kinase inhibitor locus, encoding a tumour suppressor implicated in leukemia [14]. In vivo epigenetic silencing by promoter methylation of p15 is restricted to tumour cells, and is correlated with antisense transcription across the p15 promoter. Using an in vitro defined system in HCT116 cells in which an exogenous p15 gene was juxtaposed to the cytomegalovirus (CMV) promoter, it was shown that antisense transcription from the CMV promoter is also correlated with the silencing of p15, as well as with enrichment of repressive histone modifications such as H3K9me2, but without CpG promoter methylation. However, methylation of the p15 promoter did occur in differentiated but not undifferentiated embryonic stem (ES)

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cells in response to antisense transcription, demonstrating the context-specific nature of chromatin remodelling responses. Intriguingly, this study also demonstrated that the antisense RNA derived from the artificial construct also silenced the endogenous p15 gene in a dicer-independent manner, indicating that the CMV-driven antisense RNA has both cis and trans silencing effects, which do not act through known double stranded RNA pathways. While it cannot be ruled out that the CMV promoter drives transcription at an artificially high level, thus inducing non-physiological effects, the ability of the exogenous antisense RNA to silence the endogenous p15 gene provides strong evidence that it is the antisense RNA itself, and not the transcriptional process, which is important in this case. It is possible that antisense transcription through the p15 promoter and its first exon generates functional RNA secondary structures which in turn recruit cell type-specific chromatin modifying machinery, leading to transcriptional silencing. Although this study implicates a role for the antisense transcript in epigenetic silencing of p15, mechanistic details remain to be investigated more thoroughly. It is currently unclear whether epigenetic silencing of the human HBA2 and p15 promoters by antisense RNAs share overlapping mechanisms. The phenomenon of X chromosome inactivation (XCI) in female mammals represents another paradigm of antisense ncRNA mediated regulation of the sense partner [15,16]. XCI is part of a dosage compensation mechanism by which X-linked genes of female mammals are downregulated to maintain expression at a comparable level with those in the male, and the establishment and fine-tuning of this process is primarily the result of interplay between two ncRNAs, Xist and Tsix [17]. Mammalian dosage compensation is controlled to a great extent by the X-inactivation center (Xic), which is a complex locus on the X chromosome encoding several nontranslated RNA genes, among which Xist, Tsix and Xite have been functionally implicated in the counting, choice and silencing steps of XCI [18,19]. There are two forms of XCI: imprinted, in which the paternallyderived X chromosome is silenced in the mouse preimplantation embryo and trophectodermal lineages, and random, in which silencing takes place on either X chromosome in the postimplantation embryo proper. Xist has been shown to mediate both forms of XCI through exploiting lineage-specific silencing mechanisms. For example, mice lacking DNA methylation show stable maintenance of imprinted X-inactivation but not of random X-inactivation [20]. At the onset of random XCI, expression of Tsix antisense ncRNA is crucial for the choice of X chromosome to inactivate, through regulation of its sense counterpart Xist. At this point, Xist is upregulated on the future inactive X chromosome (Xi) and its expression coincides with coating and silencing the chromosome in cis, while on the future active X chromosome (Xa), Tsix antagonizes Xist's ability to initiate silencing [21,22]. Thus XCI is regulated by the opposing actions of sense and antisense transcripts in cis. Tsix is also responsible for the paternally restricted expression of Xist during imprinted XCI [23]. Investigations by Navarro et al. have provided more insight into the functional role of Tsix in the transition from imprinted to random Xist expression, which occurs in the inner cell mass (ICM) of the blastocyst [24]. Biallelic activation of Tsix at the implantation stage of female mouse embryonic development erases the epigenetic marks that characterize imprinted Xist expression through inducing the active chromatin-specific dimethylation of lysine 4 of histone H3 (H3K4me2) throughout the Xist locus, which renders the two Xist alleles epigenetically indistinguishable and competent for random X-inactivation. This study also proposed a secondary role for Tsix in modulating the level of H3K4me2 specifically at the Xist promoter [24]. However, it was not determined whether the observed activity of Tsix is due to recruitment of the histone methyltransferase complex by the RNA itself or by the transcribing RNA polymerase complex, leaving open the question of which aspect of transcription drives chromatin remodelling over the Xist locus.

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In contrast, a later study by Lee et al. presented an alternative mechanism to explain Tsix mediated Xist regulation during the onset of random XCI [25]. This study demonstrated that on the future Xi, loss of Tsix expression causes a transient heterochromatic state involving H3K4me2, H3K27 trimethylation (H3K27me3) and H3K9 acetylation (H3K9ac), which precedes induction of Xist expression. In addition, this study also documented that on the future Xa, Tsix RNA itself directs DNA methylation over the Xist promoter through depositing the methyltransferase DNMT3a, indicating that transcriptional silencing of Xist is at least in part regulated by the Tsix RNA itself. However, in mouse embryonic stem (ES) cells with a homozygous Tsix deletion, DNA methylation of the Xist promoter occurred in surviving cells but not in dead floating cells, indicating that DNA methylation can occur in a Tsix independent manner [25]. Consistent with this notion, male ES cells, in which low level expression of the single Xist allele becomes silenced upon differentiation, can maintain Xist repression independently of Tsix [26]. A recent investigation documented that male ES cells lacking both the polycomb group protein Eed and Tsix showed Xist hyperactivation, while either single mutant did not, indicating a synergy between the molecular actions of polycomb proteins and Tsix RNA [27]. The above examples show that antisense transcription across a promoter, through various context-specific means, can epigenetically silence the sense promoter, and suggest that promoter methylation may be a common, although not universal, feature of these regulatory mechanisms. Based on these examples, we propose the following models as a basic framework from which to further explore the RNA-directed DNA methylation of promoters due to antisense transcription from an overlapping gene (Fig. 1).

A) Antisense transcription through the sense promoter causes occlusion of the basal transcription machinery and/or lineagespecific activators, allowing the recruitment of chromatin modifying machinery containing DNA methyltransferases, which then methylate the sense promoter. This mechanism would help to explain the locally restricted activity of methyltransferases to the promoter CpG island but not neighbouring CpG islands, as seen at the HBA2 locus. This model represents a permissive rather than active mechanism, however, which depends on methylation being the default state in the absence of transcription factor or activator binding, and is therefore not consistent with the observation that at the HBA2 locus, promoter silencing does not lead to methylation [13]. That an antisense p15 transcript can silence its sense promoter in trans also argues against this model acting as the primary mechanism. B) Alternatively, the antisense transcript is retained close to the sense promoter region through sequence homology, and forms functional secondary structures which recruit the chromatin modifying machinery, leading to methylation of the sense promoter. This model is supported by the observation that silencing of the endogenous p15 locus is less efficient than that of the artificial construct expressing the antisense transcript, consistent with a diffusion parameter for the transcript to find its complementary endogenous target [14]. The localized methylation seen at the HBA2 promoter could in this case be explained by the particular location of sequence homology, used to direct the methylation machinery in a precise, restricted manner. Further experimental support for this model could be generated by demonstrating colocalization of an antisense transcript with the sense promoter.

Fig. 1. Models explaining the epigenetic silencing of a sense promoter due to an antisense RNA or its transcriptional process. (A) Antisense transcription through the sense promoter prevents binding of the basal transcription machinery and/or lineage-specific factors, allowing the chromatin remodelling machinery harbouring DNA methyltransferases to initiate CpG methylation of the sense promoter. (B) The antisense transcript remains localized to the chromatin, where secondary structures recruit the chromatin remodelling machinery to methylate the sense promoter.

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The two models presented here need not be mutually exclusive. Indeed, the different kinetics of silencing of pl5 in cis and in trans could represent a contribution of transcription machinery occlusion by antisense transcription, which facilitates an RNA-dependent recruitment of methyltransferases. 3. Antisense ncRNAs silence chromosomal domains One of the salient features of the genomic loci exhibiting parent of origin-specific expression patterns is the prevalence of ncRNAs within these gene clusters [28–30]. Genomic imprinting is a cis acting epigenetic process by which a subset of autosomal genes is expressed in a parent of origin-specific manner. So far, more than 80 such genes have been identified in mammals and they are generally grouped in clusters. A typical imprinted cluster consists of several protein coding genes and at least one noncoding RNA which invariably shows reciprocal expression to the coding genes. These ncRNAs can range in size up to more than a hundred kilobases (kb). In most cases, the promoters of these ncRNAs map to differentially methylated imprinting control regions (ICRs) located in the intron of a protein coding gene, and direct transcription in an antisense orientation to the coding gene. The majority of these long ncRNAs are expressed only from the paternal chromosome due to methylation of their promoters on the maternal chromosome [31,32]. Paternal expression of these long antisense transcripts is correlated with the repression in cis of genes spread over as much as several hundred kb on either side of the transcript [33]. This bidirectional control of the expression of flanking genes raises the important question of how these long antisense RNAs differ in their execution of silencing mechanisms, as compared to the shorter antisense RNAs such as Tsix, whose effects are primarily restricted to the overlapping gene. Although several long antisense ncRNAs have been detected in imprinted clusters, only two of them, Air and Kcnq1ot1, have been functionally implicated in imprinted gene regulation [34–37]. We discuss here the epigenetic regulatory mechanisms uncovered thus far by exploiting these two long ncRNAs as model systems. The Kcnq1 imprinted domain spans a one megabase region on distal mouse chromosome 7 (Fig. 2A), and is orthologous to the Beckwith Weidemann Syndrome (BWS) region on human chromo-

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some 11 [38]. It contains the paternally expressed ncRNA gene Kcnq1ot1 and 9 (mouse) or 4 (human) maternally expressed protein coding genes, which in the mouse are interspersed with biallelically expressed genes [39]. Based on chromosomal translocations and CpG methylation changes in BWS patients, a differentially methylated imprinting control region was identified in intron 10 of the Kcnq1 gene [31]. The Kcnq1 ICR is methylated on the maternal chromosome, but on the paternal chromosome it is unmethylated and serves as a promoter for the antisense Kcnq1ot1 transcript. The length of Kcnq1ot1 is thought to be in the range of 60 kb [34], indicating that Kcnq1ot1 does not span across the promoter of the sense Kcnq1 gene. Genetic studies have shown that a paternally inherited Kcnq1 ICR deletion results in the activation of all genes which are normally paternally silenced, including Kcnq1, indicating a crucial role for the ICR in establishing or maintaining parent of origin-specific expression of genes throughout the domain [40]. Using experimentally welldefined constructs in human cells, it has been documented that the Kcnq1 ICR acts as a bidirectional silencer dependent on the Kcnq1ot1 antisense RNA [37,41–43]. Consistent with these observations is that targeted deletion of regions encompassing the Kcnq1ot1 promoter or truncation of Kcnq1ot1 by the addition of a transcription termination signal in transgenic mice also result in the activation of paternal alleles [34,40], supporting a critical role for the antisense Kcnq1ot1 or the act of transcription in bidirectional silencing. Several lines of recent evidence indicate that Kcnq1ot1 carries out its transcriptional silencing through epigenetic regulation of chromatin structure. Using episome-based constructs in the human placentalderived JEG-3 cell line, it has been shown that the Kcnq1 ICR, encompassing the Kcnq1ot1 promoter, carries out transcriptional silencing of flanking reporter genes through inducing DNA methylation, and that this spreading of DNA methylation over flanking reporter genes is itself methylation-sensitive, as specific methylation of the ICR prevents the methylation of flanking sequences [43]. This suggests that production of Kcnq1ot1, which could be repressed by methylation of its promoter, is involved in transcriptional silencing by spreading DNA methylation. By employing a set of primers covering the promoters of imprinted and nonimprinted genes on either side of the Kcnq1ot1 promoter, it was demonstrated that genes situated up to 450 kb from the ICR carry epigenetic modifications specific to

Fig. 2. Physical maps of imprinted gene clusters in the mouse. Expressed genes are green and silenced genes are red. (A) The one megabase Kcnq1 domain, regulated by the antisense Kcnq1ot1 transcript, contains 10 imprinted genes. (B) The Igf2r cluster, regulated by the antisense Air transcript, contains 5 genes, 4 of which are imprinted.

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repressive chromatin on the paternal chromosome in placental tissue. In the embryo proper, on the other hand, the promoters of genes only within 200 kb of the ICR are enriched with repressive histone marks [44,45]. Such allele-specific patterns of histone modifications correlate with imprinting status in these tissues. Paternal inheritance of the Kcnq1 ICR deletion results in loss of these modifications, indicating that Kcnq1ot1 could play an important role in establishing the paternal heterochromatin structure throughout the domain [45]. The functional role of Kcnq1ot1 RNA or its transcription in establishing repressive histone modifications was definitively demonstrated by exploiting the episome-based system, wherein mutation of cis regulatory elements crucial for assembly of the transcription machinery at the Kcnq1ot1 promoter resulted in loss of heterochromatinization over the flanking regions [46]. Further insight into the kinetics of chromatin modification in relation to Kcnq1ot1 transcription was obtained by propagating episomal plasmids for various lengths of time in JEG-3 cells [46]. These studies revealed that within two days post-transfection, the overlapping reporter gene promoter is silenced and deficient in basal transcription machinery, and becomes enriched with H3K4me2, H3K9me and H3K9ac, whereas the nonoverlapping promoter shows transcriptional activity and is enriched only with H3K4me2 and H3K9ac. However, by 8 days post-transfection, both promoters are substantially silenced and enriched with H3K9me3, H3K27me3 and deacetylated H3K9. Based on these observation, it was speculated that the silencing of overlapping gene occurs by both occlusion of basal transcription machinery and ncRNA mediated heterochromatin formation whereas silencing of nonoverlapping gene occurs only by ncRNA-mediated heterochromatin formation. The functional role of repressive histone modifications in transcriptional gene silencing in the Kcnq1 domain was further reinforced by the observations that mice with a mutated EED, which establishes H3K27 methylation in cooperation with Ezh2, and G9a, which is responsible for mono- and di-methylation of H3K9, are unable to maintain imprinting at a subset of genes in the Kcnq1 domain [47,48]. It is apparent from the accumulated evidence that the Kcnq1ot1 RNA plays a critical role in establishing chromatin-dependent imprinting patterns throughout the Kcnq1 domain, yet the investigations described above were not able to distinguish between functions of the RNA itself and of the transcriptional process. Recently it has been demonstrated that Kcnq1ot1 harbours a specific domain involved in transcriptional silencing by promoting interaction of the transcript with the chromatin [35]. In particular, this study has implicated highly conserved repeat motifs in the establishment or maintenance of transcriptional silencing by targeting the locus to the perinucleolar region, demonstrating a specific role for the Kcnq1ot1 RNA itself. We cannot, however, conclusively rule out additional functional roles for the act of Kcnq1ot1 transcription in establishing or maintaining silencing. Air is another long antisense ncRNA that takes part in domain-wide transcriptional gene silencing, in this case at the Igf2r cluster located on proximal chromosome 17 in mouse (Fig. 2B). The Igf2r locus spans about 400 kb and harbours four imprinted genes: Igf2r, Air, Slc22a2 and Slc22a3 [49]. The Air promoter maps to a differentially methylated imprinting control region (DMR2) in intron 2 of the Igf2r gene [50]. DMR2 is methylated on the maternal chromosome but unmethylated on paternal chromosome, where it serves as a promoter for the Air transcript, oriented in an antisense direction across the 5′ part of the Igf2r gene. Air is poorly spliced and the full 108 kb transcript is exclusively localized in the nucleus, while its splice variants, ranging from 0.5 to 1.5 kb, are cytoplasmic [51], suggesting that the function of transcriptional silencing is restricted to the full length Air transcript. The production of Air on the paternal chromosome is linked to the silencing in extraembryonic tissues of the three other imprinted genes, which are located on both sides of DMR2 [52]. In embryonic tissues, only the overlapping Igf2r gene is silenced, demonstrating that

tissue-specific imprinting mechanisms act at this gene cluster. In mice with either paternally deleted DMR2 or truncation of the Air transcript to 3 kb through insertion of a transcription termination signal, all three genes are activated [36], suggesting that either Air RNA or the transcription of Air plays a critical role in imprinting the Igf2r locus. A recent investigation analysed the chromatin structure of the Igf2r locus in relation to Air transcription in mouse embryonic fibroblasts using ChIP on chip technology [53]. The silent maternal Air promoter and paternal Igf2r promoter, but not their respective gene bodies, harbour similar repressive histone modifications (H4K20me3 and H3K9me3) as well as HP1 association. Conversely, the transcribed paternal Air and maternal Igf2r promoters contain active chromatin modifications, revealing the presence of adjacent active and inactive chromatin domains even within the region overlapped by the antisense transcript. This provides evidence in support of localized targeting or recruitment functions for the transcript itself, rather than effects simply due to transcription through the locus. The genes which are silenced tissue-specifically (Slc22a2 and Slc22a3) are enriched with H3K27me3 on both chromosomes not only at the promoter regions but also throughout the gene body, indicating that their silencing can also occur independently of Air. It is not clear what determines focal heterochromatin on the maternal Air and paternal Igf2r promoters. Both promoters are encompassed within differentially methylated regions, but the maternal Air allele acquires methylation at DMR2 in the oocyte, while the paternal Igf2r allele becomes methylated at DMR1 during postimplantation development. Since CpG methylation is locally restricted to promoter regions, it is likely that methylation plays an important role in the focal maintenance of heterochromatin at both loci, leading to the observed similarities in chromatin modification profiles. We propose that continuous transcription on one strand of both parental chromosomes prevents the spreading of repressive histone modifications into the gene body. The CpG methylation of the Igf2r promoter is reminiscent of what has been described above at the single gene-level due to overlapping antisense transcription. Several models have been proposed to explain the mode of action of Kcnq1ot1 and Air [49], and based on the available evidence, we suggest that both Air and Kcnq1ot1 mimic the mode of action of Xist in that production of the long antisense ncRNAs is followed by hierarchical recruitment of heterochromatic modifications throughout the imprinted domains. 4. Noncoding RNAs mediate chromosomal silencing Xist-mediated chromosome-wide transcriptional silencing on one X chromosome during female mammalian embryogenesis has become a paradigm for understanding RNA-mediated transcriptional silencing through chromatin structure regulation. We have already discussed at length the functional role of the antisense ncRNA Tsix in restricting the expression of Xist to the future Xi through regulating chromatin structure at the Xist promoter, and we discuss here how Xist functions to maintain chromosome-wide transcriptional silencing on the inactive X chromosome. During mouse preimplantation development, the onset of Xist expression on the paternal chromosome at the 4 cell stage [54] suggests that differential chromatin marks have already been established in the gametes. Xist expression coincides with the accumulation of Xist RNA along the future inactive X chromosome and the adoption of a heterochromatic configuration devoid of RNA polymerase II (RNAPII) [55]. This exclusion of the transcription machinery from the paternal X chromosome represents the first step in gene silencing in imprinted XCI, and is followed by hierarchical changes in chromatin modifications, including hypoacetylation of H4, hypomethylation of H3K4, trimethylation of H3K9 and H3K27, and accumulation of histone variant macroH2A [55]. This demonstrates

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Fig. 3. Cartoon showing the epigenetic content of the active (Xa) and inactive (Xi) X-chromosomes. On Xi, Xist transcription and its accumulation along the X-chromosome leads to RNAPII occlusion, followed by hierarchical recruitment of the epigenetic machinery responsible for histone H3 Lys27 and Lys9 trimethylation, histone H2A Lys119 ubiquitination and CpG methylation, leading to transcriptional silencing. Silencing of Xist on Xa due to the antisense Tsix RNA or its transcription allows expression of X-linked genes.

that a wide variety of chromatin modifying enzymes act in synergy to establish X chromosome inactivation (Fig. 3). The inactive paternal X chromosome remains silenced in the trophectodermal lineage, but is reactivated in the inner cell mass of the blastocyst, which gives rise to the embryo proper. This occurs shortly after uterine implantation, through activation of Tsix transcription on both X chromosomes as discussed above, to set the stage for random XCI. During the initiation of random XCI, the expression of Tsix and Xist are restricted to Xa and Xi, respectively. By using an in vitro ES cell model system, it has been demonstrated that transient pairing of Xic domains occurs shortly after the onset of differentiation [56]. Although the nature of this molecular cross-talk between the paired X chromosomes is unknown, the pairing is required for the choice of X chromosome to become inactive, leading to the mutually exclusive expression of Tsix and Xist. Further details of the molecular events which serve to establish random XCI in the ICM have been investigated using ES cell cultures. Chromatin modifications established during random XCI are similar to those seen for imprinted XCI, while in addition DNA methylation occurs at the CpG islands associated with repressed genes along the Xi [20]. Once the Xist-mediated epigenetic memory is established, it

is maintained through subsequent cell divisions, and although the initiation phase of silencing is reversible and Xist-dependent, maintenance of silencing is stable and Xist-independent [57]. The PRC1 and PRC2 complexes, which mediate chromosome-wide ubiquitination of H2A and methylation of H3 respectively, are not necessary for the establishment of epigenetic memory during XCI [58], but contribute to the Xist-independent maintenance of silencing. In addition, the PRC2 complex has been shown to play critical role in the maintenance of imprinted XCI during differentiation of the extra-embryonic lineage [59]. By conditionally deleting Xist in differentiated cells such as mouse embryonic fibroblasts, it has been documented that although Xist is not absolutely essential for maintenance of XCI, it nevertheless contributes to its stability, as loss of Xist results in reduction of the macroH2A content of Xi [60]. This study also demonstrated that Xist RNA, histone deacetylation and DNA methylation act synergistically to ensure stable chromosome replication through subsequent cell divisions. The randomly inactivated X chromosome in the embryo is composed of constitutive heterochromatin and is late replicating, in contrast to the early replication of the paternally inactivated extraembryonic X chromosome [61]. The primary difference

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between these two chromatin conformations is CpG methylation, which is less important for the maintenance of the paternally inactivated X in extraembryonic tissues [20], perhaps because the trophectodermal lineage contributes only to transient tissues, so unlike in somatic lineages, silencing does not require the long term stabilization provided by methylation. These results indicate that transcriptional inactivity does not dictate the replication timing patterns, so the late replication of the embryonic Xi could be due to an additional layer of heterochromatic modifications whose identity remains elusive to date. Chromosome-wide silencing by Xist is clearly not simply a consequence of the transcriptional process, as coating of X chromosome by the RNA can be visualized during the initiation of XCI. The Xist transcript harbours a silencing domain at its 5′ end containing a repeated A-rich sequence, and several chromatin-localizing regions which are spread throughout the transcript in a redundant fashion [62]. The A-rich repeats have been proposed to carry out silencing through organizing secondary structures, although no RNA binding factor has yet been shown associate specifically with this silencing domain. In ES cells recruitment of the PRC1 and PRC2 complexes is not dependent on the Xist silencing domain, suggesting that these complexes are recruited, directly or indirectly, by other functional sequences within Xist. A recent investigation demonstrated that the formation of the RNAPII deficient nuclear compartment during initiation of XCI does not require the Xist silencing domain, but that this domain functions to relocate the associated chromatin to this compartment [63]. During the initiation of XCI, the depletion of RNAPII as a first step in the initiation procedure could occur due to physical exclusion of the transcription machinery by Xist ribonucleoprotein (RNP) complexes. Alternatively, hypomethylation of H3K4 and H3K36 by the Xist–RNP complexes could inhibit initiation or elongation of transcription, facilitating the establishment of epigenetic memory by RNP complexes associated specifically with the Xist silencing domain, and leading to relocation of the silenced genes to the RNAPII depleted nuclear compartment. Most ncRNAs have been shown to carry out their biological functions through specific secondary structures, but as it is difficult to predict potential associations with RNP complexes based on primary sequence, there is a need for targeted strategies to define the various additional functional sequences within Xist. It is interesting to note that some of the functions of Xist RNA which have been defined in ES cells have now been recapitulated for Kcnqt1ot1 RNA in an episomal system [35], indicating that alternative models could be exploited in defining the functions and associated RNP complexes of specific sequence motifs. Intriguingly, it has recently been demonstrated that some aspects of XCI are regulated by double stranded RNAs formed from the Xist and Tsix antisense transcripts through a dicer-dependent siRNA pathway [64], suggesting the many aspects of XCI remain to be revealed. 5. Noncoding RNAs regulate chromatin structure in trans Expression of homeobox (Hox) transcription factors, which specify the positional identities of the cells within a developing embryo, is regulated in a spatial and temporal manner by complex interactions of two opposing groups of histone modifying enzymes, the trithorax group (TrxG) and the polycomb group (PcG) of proteins [65]. TrxG proteins act by maintaining the open chromatin structure of active genes through methylation of H3K4, while PcG proteins maintain the inactive status of genes through methylation of H3K27. In Drosophila melanogaster, both TrxG and PcG proteins have been shown to regulate Hox gene expression via interaction with polycomb/trithorax responsive elements (PRE/TRE), and there is evidence for the involvement of ncRNAs through promoting the binding of TrxG proteins [65,66]. In mammals, however, the mechanisms underlying the actions of polycomb and trithorax homologues in regulating Hox

gene expression are unclear. Mammalian Hox genes are clustered into four chromosomal loci, HoxA–HoxD. An elegant study in human primary fibroblasts by Chang and colleagues provided insight into the functional role of ncRNAs in HOX gene regulation [67]. They show that a significant portion of the HOX loci encode ncRNAs, and that a 2 kb transcript, HOTAIR (HOX intergenic antisense RNA), located at the boundary of two chromatin domains with mutually exclusive modifications in the HOXC locus, represses the expression in trans of genes in the HOXD cluster. Depletion of HOTAIR by siRNA resulted in the activation of HOXD genes in trans but not HOXC genes in cis. Interestingly, the trans regulatory property of HOTAIR is not a consequence of small RNA molecules derived from HOTAIR, and the entire 2 kb HOTAIR RNA plays a critical role in establishing H3K27me3 enriched chromosomal domains through interaction with PRC2 complex members such as Suz12 and Ezh2 [67]. HOTAIR is the first example of a natural ncRNA acting in trans to regulate a specific chromatin domain. It is currently unclear whether sequence complementarity between HOTAIR and the HOXD locus localizes the transcript to its target, or if this specific interaction is due to protein– protein interactions between RNP complexes bound to HOTAIR and transcription factors bound at the HOXD locus. Further elucidation of the components and interactions underlying this intriguing process will provide great insight into ncRNA mediated distant transcriptional silencing mechanisms. 6. Noncoding RNAs establish active chromatin structure The PRE/TRE elements regulating Drosophila homeobox genes are maintained by default in a silent state through the binding of PcG proteins. Switching to an active state is carried out in a developmentally regulated manner through the recruitment of TrxG proteins, and once established, is maintained faithfully through subsequent cell divisions. Although these cis regulatory elements have long been known to regulate the precise spatial and temporal expression of homeobox gene clusters, it has only recently been shown that ncRNAs are transcribed from PRE/TRE elements, and two investigations have now implicated these ncRNAs in regulating the epigenetic switch. Schmitt et al. demonstrated that transcription of ncRNAs through PRE/TRE elements alters the accessibility of DNA sequence motifs important for TrxG and PcG binding: transcription through PRE/TRE elements enables TrxG binding, which counteracts the PcG mediated silencing and leads to the activation of downstream Hox genes [68]. Sanchez-Elsner et al. suggested that several ncRNAs transcribed from PRE/TRE elements situated 5′ of the Drosophila homeobox gene Ubx, bind to and recruit the TrxG protein Ash1 to the PRE/TRE elements and to the Ubx promoter, thus inducing Ubx transcription [66]. Interestingly, Ash1-mediated activation of the Ubx promoter involves the formation of DNA/RNA hybrids between the TRE transcript and its corresponding DNA sequence, indicating that TRE ncRNAs may remain anchored to their coding sequence, where they serve as an interface between regulatory motifs and TrxG proteins. Dosage compensation in Drosophila represents another notable example of long ncRNA-mediated transcriptional activation. As in mammals, it also involves modulation of X chromosome activity such that it restores the balance of X-linked gene expression between males and females, but in flies this is achieved through hyperactivation of the single X chromosome in males. The ribonucleoprotein dosage compensation complex (DCC) coordinates X-linked gene activation in male flies by regulating chromatin structure. Protein components of the DCC include three structural MSL proteins, the H4K16 acetyltransferase MOF, and the H3S10 kinase JIL [69]. The two long ncRNA components of the DCC, roX1 and roX2, show low sequence similarity yet are functionally redundant. Flies are completely viable with a single roX gene, but display dramatically reduced viability and mislocalization of MSL proteins when both roX1 and roX2 are mutated, indicating that these ncRNAs play a crucial role in targeting the MSL

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proteins to X-linked genes. Importantly, roX RNAs produced from an autosomal transgene can restore viability and localization of the DCC complex, indicating that roX RNAs can function in trans and carry strong X chromosome recognition elements [70]. roX1 is a 3.7 kb transcript and the major splice variant of roX2 is about 500 bp. roX1 contains 5′ functional elements and a 3′ conserved stem loop structure, and artificial constructs containing only these two elements are able to mimic roX1-mediated X chromosome activation in male flies [71]. Several high affinity chromatin entry sites have been implicated in DCC-mediated X chromosome hyperactivation [72], but it is not clear how the spreading of DCC complexes along the chromosome from these points is accomplished. It has been suggested that roX RNAs could induce conformational changes in MSL1/MSL2 such that they can efficiently recognize numerous sites throughout the X chromosome [73]. Alternatively, spreading of DCCs could occur via RNA–RNA interactions between roX RNAs and nascent RNA products of X-linked genes, or through RNA–DNA hybrids formed at the X chromosome entry sites by roX RNAs. In mammals not many studies have demonstrated a role for ncRNA in gene regulation through recruiting transcriptional activators. However, a study by Feng et al. provides some insight into this phenomenon using Dlx genes as a model system [74]. Dlx genes encode homeodomain proteins required for the differentiation and migration of interneurons during vertebrate brain development. Previously it has been demonstrated that the Dlx-2 protein interacts with two ultraconserved intergenic enhancer sequences ei and eii, and transcriptionally activates the flanking Dlx-5/6 genes [75]. Interestingly, these enhancer regions encode two noncoding RNAs Evf-1 and Evf-2 [74,76]. Evf-2, an alternatively spliced form of Evf-1, is transcribed from ei and harbours a highly conserved region at its 5′ end. Interaction between the Evf-2 RNA and the Dlx-2 protein is crucial for the transcriptional activation of Dlx-5/6, which is mediated through the conserved 5′ sequence [74]. Interestingly, ectopic expression of complete Evf-2 or just the 5′ sequence resulted in transcriptional activation of Dlx-5/6 genes, suggesting that the ncRNA rather than the transcriptional process exerts the regulatory function. This study also suggested that Evf-2 mediated transcriptional activation does not involve RNA/DNA hybrids, but it is not yet known whether Dlx-2 requires a conserved primary sequence motif or a particular secondary structure of Evf-2 RNA for their interaction, although Dlx-2 is also capable of binding naked DNA in mobility shift assays [75]. Based on these observations, it has been proposed that Evf-2 RNA binds to the Dlx-2 protein, leading to stabilization of Dlx2, binding of the Evf-2–Dlx-2 complex to the enhancers, and activation of Dlx-5/6 (Fig. 4). It is yet not clear how the Evf-2–Dlx-2 complex is specifically targeted to the enhancers. 7. Long ncRNAs reflect evolutionary plasticity Comparison of mutation and variation rates in domains of differing chromatin conformation in human cells has revealed that closed chromatin domains generally have a higher mutation rate, likely reflecting inaccesibility to DNA repair mechanisms, yet show stronger selection at synonymous sites than open chromatin domains [77]. This trend is consistent with selection at the level of RNA base pairing within loci at which the RNA transcript is retained by the chromatin or forms secondary structures. Analysis of the human and mouse genomes has shown that while short ncRNAs, particularly miRNAs and snoRNAs, can be highly conserved between species, long ncRNAs tend to retain lower homology, although often contain short embedded conserved regions [78]. This may reflect the multiplicity of targets of miRNAs and snoRNAs which impose strong contraints on sequence variation. Long RNAs, in contrast, can consist of multiple motifs, each with a single target or secondary structure, and as such, may tolerate higher overall variation. Detailed evolutionary comparisons of long ncRNAs loci support these general conclusions.

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The X chromosome provides an interesting model in which to study the evolution of noncoding RNAs in mammals and their effects on chromatin structure. The monotremes have an independently evolved sex chromosome system, for which mechanisms of dosage compensation are unknown, and no Xist homolog has been detected in the platypus genome [79,80]. The long arm and the proximal short arm of the X chromosome are common to all therian mammals, with the distal short arm being translocated from an autosome in an early eutherian, after the divergence of the marsupial lineage [81]. Marsupials display imprinted X chromosome inactivation in somatic tissues [82,83], and the silent paternal X chromosome is associated with deacetylated histones [79]. The mechanisms underlying this imprinted X inactivation are not known, but silencing is carried out in the absence of a Xist noncoding RNA, and without differential DNA methylation [79]. Methylation of CpG islands at the promoters of silenced X-linked genes is common to all eutherians, and the ancient domains of the X chromosome show more complete silencing than the X-added region [84]. The Xist gene appears to have evolved in eutherian mammals from the Lnx3 gene, with multiple lineage- and species-specific alterations, particularly insertions and deletions of repeat sequences [84,85]. Xist RNA coats the silent X chromosome in all eutherian species examined. A comparison of Xist sequences from seven eutherian species revealed significant divergence of intron/exon structure and intron length, frequent expansion or contraction of repeats, and very low sequence homology throughout the locus [84]. However, specific regions of RNA are quite conserved, particularly the A repeat, which in mouse localizes silenced genes to a compartment of the nucleus which is depleted of RNA polymerase II [63]. The use of the Tsix antisense transcript to inhibit maternal Xist expression is a mechanism apparently restricted to rodents, in which X inactivation is imprinted in the placenta and random in the embryo. Significant variation is found even within the rodent lineage, as rats express a Tsix transcript which is quite divergent from that of the mouse [86], and Tsix has not yet been detected in voles, whose Xist gene has very low sequence homology with mouse [87]. Cows also show imprinted placental and random embryonic X inactivation [88], but a bovine Tsix transcript has not been detected [89]. Human X inactivation is random in both placenta and embryo, and the human Tsix locus has degenerated into a pseudogene missing the regulatory CpG island [89]. This is consistent with a role for Tsix in erasing imprinted X inactivation [24], a function specific to the rodent lineage. These comparative studies of mammalian X chromosome inactivation suggest the successive acquisition of multiple layers of ncRNA dependent regulation, built upon an ancestral imprinted X inactivation mechanism. Conservation of the A repeat region of Xist in eutherians suggests that recruitment of the chromatin remodelling machinery and translocation to a transcriptionally silent nuclear compartment may be common to eutherians, although this awaits experimental validation in other species. Comparative analyses of other noncoding RNA loci support a species-dependent variety of chromatin regulatory mechanisms. The mouse Kcnq1 locus consists of 10 imprinted genes, which are under the control of the Kcnq1 ICR. The human locus is similarly organized, although imprinting of some of the genes has been lost in the cluster, and as in the mouse, shows reciprocal allelic expression of Kcnq1 and the antisense Kcnq1ot1. Gene order is conserved back to marsupials, and although the tammar wallaby shares with eutherian mammals the imprinted expression of Igf2 and Ins on the neighbouring cluster, Cdkn1c (the only gene in the Kcnq1 cluster analysed to date) is biallelically expressed [90]. Sequence analysis revealed no Kcnq1ot1 promoter or CpG island in the wallaby, yet a long antisense transcript is nevertheless expressed from within intron 10 of the Kcnq1 gene in the late gestation placenta [39]. A functional role for this antisense transcript has not yet been tested, nor have the allelic expression

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Fig. 4. Model describing the long noncoding RNA Evf-2 mediated transcriptional activation of Dlx-5 and Dlx-6. Evf-2 RNA interaction with Dlx-2 protein results in the stabilization of Dlx-2 and binding to the ei and eii enhancers, leading to the activation of the flanking Dlx-5 and Dlx-6 genes.

patterns of Kcnq1 or the Kcnq1ot1 homolog been determined. Despite the coexpression and functional antagonism of the Igf2 and Cdkn1c gene clusters, imprinting at these syntenic loci has evolved at different times, and the expression of the noncoding Kcnq1ot1 transcript predates imprinting of Cdkn1c. As such, the ancestral Kcnq1ot1 may have been used to antagonize Cdkn1c at a critical time in placental development, only later being co-opted for long range imprinted gene expression control. The Igf2r domain, which harbours four imprinted genes including the antisense Air transcript in the mouse, shows a more complicated evolutionary history, with imprinting arising in a therian mammal ancestor and largely disappearing in primates. The Igf2r promoter CpG island is conserved across all mammals, yet it is not differentially methylated in the nonimprinted platypus, nor in two imprinted opossum species [91,92]. Monotremes and marsupials both lack the Igf2r intron 2 CpG island (DMR2) which harbours the Air promoter, and neither platypus nor opossum show any detectable antisense transcription at this locus [92]. All eutherian mammals examined to date show imprinted Igf2r expression and allele-specific CpG island methylation [93,94], despite Air expression being unique to rodents,

until we reach the primate lineage, in which the differential methylation of DMR2 and imprinted expression of Igf2r are absent in the lemur, tree shrew and the majority of humans [95]. Surprisingly, however, Igf2r and Slc22a2 imprinting has been detected as a polymorphic trait in humans, despite lack of Air, and Slc22a3, which is silenced by Air in mouse [36], is thought to be imprinted in the first trimester human placenta [96]. These findings suggest that differentiation between the parental chromosomes at this locus may not have been entirely lost early in the primate lineage, despite allelic transcriptional control being absent or tightly restricted. In the mouse, neither DMR2 methylation nor Air transcription correlates perfectly with the silencing of Igf2r, as both features are allele-specific in all tissues, yet Igf2r expression is biallelic in the brain. Histone modification patterns, on the other hand, correlate well with allelic expression at this locus: in mouse, trimethylated H3K4 is always found at the silent maternal Air promoter, and acetylated H3 and H4 at the active Igf2r promoter. No differential histone modifications have been found in human tissues [95], although this has yet to be examined in the rare human tissues showing Igf2r and Slc22a2/3 imprinting. Thus, despite detailed characterization of the mouse Igf2r

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locus revealing a crucial role for Air in the regulation of imprinted Igf2r, Slc22a2 and Slc22a3 expression, a wide diversity of gene regulatory mechanisms, many of which are Air-independent, can be found across different mammalian tissues and species. Comparisons between and within the three branches of mammals reveal insight into the closely intertwined evolution of reproductive strategies and of the physiology of the maternal–fetal interface, and the resulting changing demands on maternal resources. One can imagine a scenario of parental conflict driven evolution of imprinted gene expression in embryonic and placental development, in which long noncoding RNAs represent a rich source of functional plasticity in modulating species specific developmental demands. Once in place, these chromatin regulatory mechanisms may then be further coopted for other tissue specific regulatory functions. 8. Outlook The recent uncovering of copious amounts of ncRNA transcription in mammals by high throughput analysis, and the characterization of functional roles for some of these ncRNAs in various biological processes, have highlighted long ncRNAs as important regulators of cell differentiation, development and disease. In this review we have discussed the mechanistic pathways that are being utilized by long ncRNAs in transcriptional regulation ranging from the control of a single gene to an entire chromosome, with a special emphasis on chromatin, as the medium through which all genome functions are transacted. One contentious issue commonly arising in discussion of the mode of action of ncRNAs is whether it is the RNA itself or the transcriptional process which directs chromatin modification, yet there is no reason to suppose these mechanisms to be mutually exclusive. The act of transcription may involve direct interactions between the RNA polymerase complex or associated regulatory factors and the chromatin modification machinery, or could facilitate accessibility to chromatin remodelling factors indirectly due to the opening of the double strand. The RNA itself can exert effects through sequence complementarity for localization to its target, and through secondary structure formation to recruit specific protein factors. Even where specific functions for the RNA transcripts have been documented, such as for HOTAIR, Evf-2, Kcnq1ot1 and Xist, this does not rule out additional effects signalled by the transcriptional process, and in the majority of cases, experimental evidence does not distinguish between these two types of effect. Such ambiguities will require sophisticated molecular methods to resolve the chromatin modifying mechanisms acting at each ncRNA target locus. Inactivating the endogenous antisense promoter while expressing the transcript exogenously may determine whether antisense transcription in situ is necessary for inducing chromatin modifications. It has been noted, however, that the generic act of transcription is unlikely to be able to direct a limited repertoire of chromatin remodelling complexes in a sufficiently precise manner to many distinct genomic loci without taking advantage of the sequencespecific information inherent to RNA transcripts [10]. Several functional motifs have been identified within long ncRNAs, such as the Xist A repeat or the Kcnq1ot1 silencing domain, yet no common regulatory sequence motifs between chromatin modifying ncRNAs have yet been described. Even functionally similar ncRNAs, such as TRE transcripts of the Drosophila Ubx locus, do not show any homology at the primary sequence level [66], although this does not preclude homology at the level of secondary structure. Currently there is no evidence of conserved secondary structures between the well characterized long ncRNAs Xist, Kcnq1ot1 and Air, despite many functional similarities, but definitive experimental methods for assessing secondary structure formation in vivo present technical challenges. Recent developments in computational tools for predicting RNA secondary structures and identifying conserved functional

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elements will undoubtedly prove useful in informing experimental design, and reciprocally, developments in experimental methods will aid in refining computational algorithms [97,98]. The high rate of sequence variation in comparison to protein coding or infrastructural RNA genes, and lack of sequence homology between similarly acting ncRNAs, shows either relaxed constraints at the primary sequence level, or strong positive selection, which may be related to phenotypic variation (between individuals as well as species), and which may be a significant force in evolutionary adaptive radiation [10,78,99]. The diversity of chromatin based regulatory mechanisms reviewed here provides further evidence in support of an emerging understanding of a vast network of RNA based coordination of genomic processes, which has facilitated an exponential increase in organismic complexity to be derived from a basic set of protein coding genes [100,101]. This has been attributed to the use of massively parallel processing functions, mediated by diverse species of RNA to dynamically coordinate complex combinations of genomic functions [99]. In this context it is interesting to note that linking the regulatory functions of ncRNAs to the transcriptional process alone would provide only a linear increase in phenotypic complexity, while harnessing the sequence information embedded in RNA transcripts for regulatory functions is consistent with the observed exponential increase in eukaryotic complexity. One aspect of long ncRNA directed chromatin remodelling which has not been well explored is how the RNA communicates with the chromatin in imposing modifications. Is the RNA itself which associates specifically with the target locus and recruits the chromatin modifying complexes, or are additional protein factors assembled with the RNA into ribonucleoprotein complexes required for targeting? How is a particular combination of chromatin remodelling enzymes and cofactors recruited to each locus? In the case of chromatin remodelling over a single promoter, one can imagine direct interactions in the form of RNA–RNA or RNA–chromatin hybrids, but the longer ncRNAs having multiple target sites presumably have more complex targeting and recruitment mechanisms. Xist, for example, contains sequences essential for chromatin localization embedded within the transcript, but the complement of proteins with which it colocalizes to the chromatin during remodelling has yet to be determined. Further characterization of the role of ncRNAs in regulating chromatin structure in development and disease will require sensitive experimental tools able to capture the dynamic interactions between many macromolecular components. One such set of tools based on fluorescence resonance energy transfer (FRET) can be used to monitor molecular dynamics in living cells, with single molecule resolution. FRET techniques have already been adapted for the visualization in real time of RNA folding [102], RNA–DNA hybrid formation [103] and RNA–protein interactions [104,105], but these powerful tools have yet to be exploited in the context of chromatin dynamics. Their systematic application to the analysis of ncRNAs promises to provide an exciting new perspective on the epigenome. While there may be generic components of the long ncRNAdirected chromatin remodelling machinery, it is clear from the examples described here that many mechanistic details are cell type specific, and it is likely that further variations will be uncovered as more tissue types and developmental stages are investigated. Despite the evidence for tissue specific chromatin modifying mechanisms, it is not yet known whether ncRNAs are themselves driving cell fate decisions and differentiation, and such variation between tissues and species highlights the need to select model systems carefully. In contrast to studying the chromatin composition of somatic cell lines, which represent the output of developmental epigenetic processes, the use of controlled in vitro differentiation of embryonic stem cell cultures has proven to be an excellent experimental system in which to uncover the dynamics of X chromosome inactivation. As we have seen at the Xist/Tsix locus,

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the transient expression of regulatory RNAs can lead to stably transmitted changes in gene expression via chromatin modification, even though the RNAs themselves may be dispensable in mature cell lines. The dynamical processes associated with establishing chromatin conformation can only be fully explored by using such developmentally relevant model systems. Could we adapt similarly appropriate models to the further investigation of other developmentally regulated ncRNAs? The development of in vitro differentiation models of the appropriate developmental stages will undoubtedly prove to be of great benefit in revealing the subtle dynamics of these RNA dependent chromatin remodelling processes in development and differentiation. The demonstration that transient ncRNA expression can trigger stable changes in chromatin structure suggests that spurious activation of a regulatory RNA can lead to heritable gene expression changes in clonal derivatives, suggests new lines of investigation in studies of tumour development. We have seen the pathological outcome of inappropriate antisense transcription in the case of the HBA2 in thalassemia, and p15 in leukemia. Given that at least 70% of the genome can be transcribed, the multitude of karyotypic rearrangements seen almost universally in cancer cells [106] must be accompanied by many such alterations in the transcriptome, potentially leading to a vast array of long range and heritable effects on gene expression in a transformed cell. Acknowledgements This work was supported by the grants from Swedish Research Council (VR-NT), Swedish Medical Research Council (VR-M) and the Swedish Cancer Research Foundation (Cancerfonden) to CK. CK is a Senior Research Fellow supported by VR-M. References [1] W. Reik, A. Collick, M.L. Norris, S.C. Barton, M.A. Surani, Genomic imprinting determines methylation of parental alleles in transgenic mice, Nature 328 (1987) 248–251. [2] S. Saitoh, T. Wada, Parent-of-origin specific histone acetylation and reactivation of a key imprinted gene locus in Prader–Willi syndrome, Am. J. Hum. Genet. 66 (2000) 1958–1962. [3] A.D. Riggs, X inactivation, differentiation, and DNA methylation, Cytogenet. Cell Genet. 14 (1975) 9–25. [4] P. Jeppesen, B.M. Turner, The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression, Cell 74 (1993) 281–289. [5] T. Jenuwein, C.D. Allis, Translating the histone code, Science 293 (2001) 1074–1080. [6] V. Morales, C. Giamarchi, C. Chailleux, et al., Chromatin structure and dynamics: functional implications, Biochimie 83 (2001) 1029–1039. [7] T. Agalioti, S. Lomvardas, B. Parekh, J. Yie, T. Maniatis, D. Thanos, Ordered recruitment of chromatin modifying and general transcription factors to the IFN[beta] promoter, Cell 103 (2000) 667–678. [8] T. Ragoczy, M.A. Bender, A. Telling, R. Byron, M. Groudine, The locus control region is required for association of the murine beta-globin locus with engaged transcription factories during erythroid maturation, Genes Dev. 20 (2006) 1447–1457. [9] E. Bernstein, C.D. Allis, RNA meets chromatin, Genes Dev. 19 (2005) 1635–1655. [10] J.S. Mattick, I.V. Makunin, Non-coding RNA, Hum. Mol. Genet. 15 (Spec No 1) (2006) R17–29. [11] S. Katayama, Y. Tomaru, T. Kasukawa, et al., Antisense transcription in the mammalian transcriptome, Science 309 (2005) 1564–1566. [12] G. Lavorgna, D. Dahary, B. Lehner, R. Sorek, C.M. Sanderson, G. Casari, In search of antisense, Trends Biochem. Sci. 29 (2004) 88–94. [13] C. Tufarelli, J.A. Stanley, D. Garrick, et al., Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease, Nat. Genet. 34 (2003) 157–165. [14] W. Yu, D. Gius, P. Onyango, et al., Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA, Nature 451 (2008) 202–206. [15] C. Rougeulle, P. Avner, The role of antisense transcription in the regulation of X-inactivation, Curr. Top. Dev. Biol. 63 (2004) 61–89. [16] J.T. Lee, L.S. Davidow, D. Warshawsky, Tsix, a gene antisense to Xist at the X-inactivation centre, Nat. Genet. 21 (1999) 400–404. [17] J. Lee, L.S. Davidow, D. Warshawsky, Tsix, a gene antisense to Xist at the X-inactivation centre, Nat. Genet. 21 (1999) 400–404. [18] J.A. Erwin, J.T. Lee, New twists in X-chromosome inactivation, Curr. Opin. Cell Biol. 20 (2008) 349–355.

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