Cristina Tufarelli*

Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK
* ct67@le.ac.uk

One of the fundamental questions of modern biology is to unravel how genes are switched on and off at the right time and in the correct tissues. It is well recognized that gene regulation depends on a dynamic balance between activating and repressing forces, and multiple mechanisms are involved in both gene silencing and activation. Work over the last decade has revealed that in some cases transcriptional silencing of specific genes is mediated by RNAs that specifically recruit repressing complexes to homologous DNA sequences. Examples of both cis and trans RNA driven transcriptional silencing have been reported. This review focuses on those examples of transcriptional gene silencing in which the RNA component seems to act uniquely in cis. Speculative models of how such cis acting transcripts may trigger transcriptional silencing are proposed. Future experimental testing of these and other mechanisms is important to gain a fuller understanding of how genes are regulated and to identify instances in which such mechanisms are defective, leading to disease. Understanding the basic molecular basis of these phenomena will provide us with invaluable tools for the future development of targeted therapies and drugs for those diseases in which they are faulty.

Keywords: antisense-RNA; CpG island; cytosine methylation; transcriptional silencing; cis effects

1. Introduction

One of the main goals of molecular biology is to understand how genes are regulated during development and differentiation so that only the right genes are expressed at the correct time and levels in the various tissues. DNA is packaged with histones and other protein in the nucleus of eukaryotes to form a dynamic complex called chromatin. It was soon recognized that this compact structure may constitute a challenge for gene expression by preventing the interaction of transcription factors to their cognate sequences (reviewed in Struhl 1999). Therefore, initially most of the effort was put into understanding how genes are activated from within such a repressive chromatin environment. However, it has now become clear that gene regulation is a dynamic process and that gene silencing as well as gene activation plays an important role in the correct regulation of gene expression.

Silenced and active chromatin can be distinguished by a series of different epigenetic characteristics. Two of the most commonly studied epigenetic marks found at silent chromatin are methylation of particular histone tails residues (predominantly H3-Lys9 and Lys27) and DNA methylation (mainly found in plants and vertebrates; Sims 2003; Freitag & Selker 2005). Recent studies in several model organisms from yeast to human are highlighting the presence of common molecular mechanisms underlying the set up of the epigenetic modifications associated with gene silencing. Of particular interest is the evidence that in some instances transcriptional silencing is mediated by an RNA component that gives the sequence specificity to precisely target particular DNA regions. Double stranded RNA (dsRNA) molecules are required to direct histone methylation and silencing in the yeast Schizosaccharomyces pombe (Hall et al. 2002; Volpe et al. 2002; Schramke & Allshire 2003) and the ciliate Tetrahymena termophila (Mochizuki et al. 2002). Similarly, RNA directed chromatin silencing has been observed in Arabidopsis (Chan et al. 2004) and Drosophila (Pal-Bhadra et al. 2002, 2004). In mammals, RNAs have been associated with chromatin silencing in the processes of imprinting (Verona et al. 2003) and X-inactivation (Boumil & Lee 2001), and more recently, they have been involved in the methylation and silencing of CpG islands at autosomal and non-imprinted locations (Tufarelli et al. 2003; Kawasaki & Taira 2004; Morris et al. 2004).

In the majority of the cases of transcriptional gene silencing studied to date, the RNA component is given by dsRNAs similar to the small interfering RNAs responsible for posttranscriptional gene silencing (also known as RNA interference), and indeed the two processes share common components (Matzke & Birchler 2005). A characteristic of chromatin silencing dsRNAs is their ability to act in trans (Bender 2004; Kawasaki & Taira 2004; Morris et al. 2004). However, some evidence points to the existence of cis acting mechanisms of RNA mediated chromatin silencing, suggesting that distinct but related molecular machineries for epigenetic silencing triggered by RNA may be in play (Seitz et al. 2003; Sleutels et al. 2003; Tufarelli et al. 2003; Noma et al. 2004).

Since their initial discovery, the field of small RNAs has been growing at a very fast speed and several review papers have been published regarding dsRNA directed DNA methylation in plants and other systems (e.g. Morey & Avner 2004; Kawasaki et al. 2005; Matzke & Birchler 2005). This review will focus on those examples of cis antisense transcription, where the sense and antisense-RNA (AS-RNA) are transcribed from the same locus. In particular, will be discussed those examples of transcriptional gene silencing of CpG islands associated with the presence of AS-RNAs transcribed through the CpG island, as seen at some imprinted loci and at the Xist/Tsix locus. In these cases, it is not clear whether a dsRNA component is involved, and based on the available experimental evidence, speculative mechanistic models of how the single stranded AS-RNA may act to trigger transcriptional silencing of the reversely transcribed CpG island in cis will be proposed.

2. Naturally occurring cis-antisense transcripts

The discovery of RNAi and of its involvement in posttranscriptional and transcriptional gene silencing has boosted the hunt within the fully sequenced human and mouse genomes for examples of sense/antisense gene pairs with a potential to form dsRNAs.

Examples of genes within genes in the human genome have been described in the early 1990s within the human genes neurofibromatosis type I (NF1; Cawthon et al. 1990, 1991; Viskochil et al. 1991), retinoblastoma susceptibility gene (RB; Herzog et al. 1996), and blood clotting factor VIII (Levinson et al. 1990, 1992). In particular, intron 22 of the human blood clotting factor VIII gene contains a bidirectional CpG island from which two internal genes are transcribed in opposite directions, F8A (Levinson et al. 1990) and F8B (Levinson et al. 1992). Initial analysis of human chromosome 22 sequence identified two genes that are found within the introns of other expressed genes (Dunham et al. 1999). More recently, a study in which an arrangement of genes within genes was sought, referred to as overlapping gene groups, found that a high proportion of these genes are associated with disease (Karlin et al. 2002). All these genes, with the exception of F8B, are transcribed in the opposite orientation to that of the gene within which they lie, and to date there is no evidence to suggest that they interfere with transcription of the sense gene or vice versa.

More recent genome wide analyses have revealed the existence of cis and trans sense-antisense gene pairs, depending on whether they are transcribed from the same or different loci, respectively (Lehner et al. 2002). The estimates of genes associated with cis-antisense transcripts within the human genome have been increasing with the optimization of the criteria used for the computational analysis. The initial prediction based on the study of RefSeq mRNAs and full length EMBL protein coding mRNAs was that 2% of the human genes are transcribed from both strands (Lehner et al. 2002). When the analysis was limited to expressed sequence tags (ESTs) of validated orientation, roughly the same percentage was obtained (Shendure & Church 2002). The prediction went up to 8% when polyadenylated ESTs spanning introns were considered (Yelin et al. 2003). The highest estimate of about 22% sense-antisense gene pairs among human transcripts comes from a recent study considering ESTs containing both a poly(A) signal and poly(A) tail, and ESTs with either of them if they had an annotated protein coding sequence (Chen et al. 2004). In mouse, about 15% of the genes appear to have cis-antisense transcripts (Kiyosawa et al. 2003). Interestingly, human-Fugu and human-mouse comparisons of conserved overlapping sense/antisense pairs have revealed that these gene pairs are evolutionarily conserved and that there is a selective pressure to maintain the distance between such gene pairs constant among species, implying that their presence has affected vertebrate genome evolution (Dahary et al. 2005).

Taken together these observations indicate that antisense transcripts are more widespread than previously thought and argue for their role in several processes including transcription, RNA stability, pre-mRNA processing, mRNA transport and translation (Lipman 1997; Kumar & Carmichael 1998; Vanhee-Brossolet & Vaquero 1998; Carmichael 2003). Transcriptional interference, RNA masking, dsRNA dependent mechanisms and antisense-induced DNA methylation have been proposed as possible mechanisms through which cis-AS-RNAs can exert their regulatory functions (reviewed in Lavorgna et al. 2004). cis oppositely transcribed genes can be present in one of four different genomic arrangements, 'tail to tail' (3' end overlap, figure 1a), 'head to head' (5' overlap, figure 1b), one transcript starting within an intron of the other transcript (figure 1c), and one transcript contained within the other (figure 1d; Lehner et al. 2002). When genes whose promoters are associated with CpG islands are considered, the latter two configurations (figure 1c,d) are consistent with a role of AS-RNA in DNA methylation and silencing of the sense gene promoter, while figure 1b would represent genes that share a bidirectional CpG island. Interestingly, the relative orientations of the sense-antisense gene pairs in figure 1c,d have been observed at some imprinted loci, at the Xist/Tsix locus and at few autosomal loci, where the presence of cis-antisense transcripts correlates with methylation and silencing of the sense gene CpG island. These examples will be discussed in detail in the following sections.

Figure 1: Relative organization of cis sense/antisense gene pairs as described by Lehner et al. 2002.

Figure 1: Relative organization of cis sense/antisense gene pairs as described by Lehner et al. 2002.

In each pair, the gene drawn above the line is transcribed from left to right and that drawn below the line from right to left. Transcription orientation is shown by bent arrows. Same shaded boxes represent exons of the same gene. The two oppositely transcribed genes can overlap at the 3'end (a) or they can overlap at the 5' end, being divergently transcribed from a common region (b). Alternatively, they can each contain the promoter region of the other gene within one of their introns (c), or they can have a 'gene within gene' arrangement with one gene completely contained within the other (d). For the purposes of this review that focuses mainly on genes associated with CpG islands, hatched boxes of arbitrary extent above each gene promoter indicate putative CpG islands. Note that in (b) a bidirectional CpG island is shared by the two oppositely transcribed genes.

As outlined above, cis-antisense transcripts in conjunction with silencing and methylation of the sense gene have been initially observed in mammals in the processes of imprinting and X-inactivation. It has been proposed that such antisense transcripts may be involved in silencing and methylation of the sense gene, but their exact role in these instances is not known. For example, it has been shown that imprinting of the Igf2r gene is lost when expression of its AS-RNA is abrogated by removing the intronic CpG island from which antisense transcription is initiated (Wutz et al. 1997). Similarly, targeted deletion of the Tsix gene, the Xist antisense gene, leads to non-random X inactivation (Lee & Lu 1999). However, the molecular mechanisms by which these cis-antisense transcripts act remain a matter for speculation.

The first example of AS-RNA associated with methylation of the sense gene CpG island was described at the imprinted Igf2r locus in 1997 by Wutz et al. Following this observation, AS-RNAs were soon identified at other imprinted loci, at the Xist locus on the X chromosome, and for few non-imprinted genes. Some of the experimentally identified examples of cis sense-antisense gene pairs in which transcription of at least one of the two genes runs through the CpG island of the conversely transcribed gene are listed in table 1.

It is clear at first glance from table 1 that the majority of AS-RNA transcripts are found at imprinted loci, though it has to be noted that the first cis-antisense described is at an imprinted locus biasing the initial search for such transcripts in imprinted regions of the genome. Interestingly, all the antisense transcripts described at imprinted loci are non-coding RNAs transcribed from the paternal allele, although their expression does not always correspond to silencing of the sense gene on the same allele. For example, both of the genes within the gene pairs MEST(Peg1)/MESTIT1, Igf2/Igf2as and Zfp127(MKRN3)/Zfp127as are expressed from the paternal allele (table 1 and references therein). The mechanisms of action of these antisense transcripts are not known, but it has been suggested that antisense transcription through the Igf2 promoters may regulate tissue specific levels of Igf2 (Constancia et al. 1998). Here will be described three gene pairs, Igf2r/Air, Nesp/Nespas and Kvlqt1/Lit1, showing mutually exclusive transcription.

The Igf2r/Air and Nesp/Nespas gene pairs are examples of the configuration represented in figure 1c whereby each gene starts within an intron of the oppositely transcribed gene, and show a correlation between antisense transcription and silencing and methylation of the sense gene. A common feature of these gene pairs is that both genes are associated with CpG islands showing reciprocal methylation and referred to as differentially methylated regions (or DMRs; figure 2a,b). There are two DMRs in the Igf2r locus, DMR1 associated with the promoter of the Igf2r and DMR2 found in intron 2 of the Igf2r gene and associated with the promoter of the antisense transcript Air (figure 2a). While DMR2 is methylated in the maternal germ line and remains so in all tissues (Stoger et al. 1993), methylation of DMR1 on the paternal allele occurs during implantation following repression mediated by Air transcription (Sleutels et al. 2002). The first evidence for a role of antisense transcription in imprinting of Igf2r came from a study reporting that imprinting of the Igf2r gene is lost when expression of its AS-RNA is abrogated by removing the intronic CpG island from which antisense transcription is initiated (Wutz et al. 1997). Transgenic experiments using fragments containing the Air promoter highlighted the possible requirement of oppositely firing CpG islands for imprinted DNA methylation of the Air CpG island (Sleutels & Barlow 2001). However, deletion of the Igf2r promoter leading to lack of sense gene expression did not affect imprinting of Air and other non-overlapping genes, suggesting that the AS-RNA acts in cis to silence the locus and arguing against the involvement of dsRNAs to establish imprinting of these genes (Sleutels et al. 2003). Truncation of Air transcripts by the insertion of a polyA site results in loss of imprinting of the overlapping Igf2r gene but also of other non-overlapping genes, confirming the involvement of the Air RNA in silencing (Sleutels et al. 2002). Although it is clear that transcription of the sense Igf2r RNA is not required for silencing and methylation of the Air CpG island on the maternal allele, the question of how the Air RNA silences the overlapping Igf2r and other non-overlapping imprinted genes within the cluster (Slc22a2 and Slc22a3) has not yet been answered. The more plausible model suggests that, similarly to the Xist RNA on the inactive X-chromosome, the Air RNA silences overlapping and non-overlapping genes in cis by coating a specific chromosomal region probably through the recruitment of repressor complexes (Rougeulle & Heard 2002; Sleutels et al. 2003; Morey & Avner 2004). It is, however, possible that two mechanisms are at play in this region and that silencing and methylation of the Igf2r CpG island is dependent on the transcriptional overlap, while silencing of the non-overlapping genes is independent of it (Sleutels et al. 2003).

Figure 2: Some sense/antisense gene pairs encompassing CpG islands.

Figure 2: Some sense/antisense gene pairs encompassing CpG islands and showing mutually exclusive expression.

Schematic diagrams (not to scale) of the organization of the mouse imprinted gene pairs (a) Igf2r/Air, (b) Nesp/Nespas and (c) Kcnq1/Kcnq1ot1. Paternally (black), maternally (grey) and biallelically (empty boxes) expressed genes are indicated. Exons are omitted for simplicity, except those in the Gnas locus in which different transcripts share common exons (b). Dotted lines in (b) indicate some of the splicing events. In (d) is shown the organization of the Xist (grey)/Tsix (black) locus with individual exons omitted for simplicity. CpG islands showing differential methylation are depicted as hatched boxes. Only the names of the genes referred to in the text are annotated. Bent arrows indicate the orientation of transcription.

In addition to the differential CpG island methylation, chromatin immunoprecipitation (ChIP) studies have revealed a different pattern of histone modifications associated with the two DMRs in tissues where Igf2r expression is imprinted (Hu et al. 2000; Fournier et al. 2002; Yang et al. 2003; Vu et al. 2004). In these tissues, DMR1 on the maternal allele is enriched in histone modifications that have been associated with active genes, such as histone H3 acetylated at residues K9 and K14 and methylated at residue K4, and acetylated histone H4, whereas on the paternal allele enrichment is found with antibodies to methylated K9 in histone H3, a mark for silent chromatin. The reciprocal pattern is observed at DMR2, with the paternal and maternal alleles enriched in modifications consistent with transcriptionally active or silent chromatin, respectively (Hu et al. 2000; Fournier et al. 2002; Yang et al. 2003; Vu et al. 2004). While the Air transcript is paternally expressed in all tissues analysed, biallelic expression of Igf2r is observed in brain (Hu et al. 1999). In brain, the Air promoter maintains the parent of origin specific patterns of DNA methylation and histone modifications, whereas the promoters of Igf2r on both alleles become indistinguishable, both showing high levels of histone acetylation (H3 and H4) and H3-K4 methylation, and low levels of H3-K9 methylation. (Fournier et al. 2002; Yang et al. 2003; Vu et al. 2004).

The other imprinted gene pair showing reciprocal methylation of the CpG island DMRs associated with the promoters of the two genes is the Nesp/Nespas pair within the Gnas/GNAS locus on mouse chromosome 2H and human chromosome 20q (figure 2b). This is a complex imprinted cluster, including maternally, paternally and biallelic expressed transcripts with shared exons, and three DMRs, one associated with the maternally expressed Nesp transcript, one with the paternally and divergently transcribed Nespas/Gnasxl RNAs and one with the paternally expressed ex1A transcript (Holmes et al. 2003). The maternally expressed Nesp transcript encodes the neuroendocrine secretory protein NESP55 (Ischia et al. 1997). Its paternally expressed antisense, Nespas, is a non-coding RNA expressed as a long unspliced transcript and as a number of alternative splicing isoforms, both in human and in mouse (Hayward & Bonthron 2000; Li et al. 2000; Wroe et al. 2000; Williamson et al. 2002). In contrast to what is observed with deletions of the Igf2r DMR, deletions of the NESP promoter DMR in two independent families with pseudohypoparathyroidism type Ib were shown to cause loss of maternal GNAS imprints (Bastepe et al. 2005). The NESP DMR is methylated during postimplantation, while the Nespas DMR is methylated during oogenesis (Liu et al. 2000; Coombes et al. 2003), suggesting that the Nesp DMR contains a cis acting element essential for maintenance but not establishment of the maternal imprints on the GNAS cluster (Bastepe et al. 2005). Nevertheless, these experiments cannot rule out the possibility that transcription of Nesp and/or the Nesp RNA itself is also important in maintaining the maternal imprints.

Although expression of Nesp and Nespas is non-complementary in mid-gestation mouse embryos and in adult interspecific mice (Li et al. 2000; Ball et al. 2001), reciprocal imprinting is observed in heart, suggesting a potential role of the antisense in regulating expression of the sense gene (Wroe et al. 2000). It would be interesting to further investigate the potential role of Nespas in regulating the sense gene by performing experiments in which the Nespas DMR is deleted or the transcripts itself truncated through the introduction of polyadenylation and transcription termination site, similarly to those reported for the Igf2r/Air gene pair.

The Lit1/LIT1 (or Kvlqt1ot or Kcnq1ot1) gene is a non-coding RNA transcribed antisense to Kvlqt1/KvLQT1 (or Kcnq1) within the KCNQ1OT1 imprinted subdomain on human 11p15.5 and mouse 7F (Lee et al. 1999b; Mitsuya et al. 1999; Smilinich et al. 1999; table 1 and figure 2c). This gene constitutes an example of a gene within a gene (figure 1d), as it does not extend to the promoter of the sense gene but is all contained within it (Mitsuya et al. 1999). The DMR of the locus (KvDMR1) corresponds to the CpG islands associated with the paternally expressed Lit1/LIT1 gene, which becomes methylated during oogenesis and is maintained differentially methylated in zygote and embryonic stem (ES) cells (Engemann et al. 2000). Targeted deletion of the human and mouse DMRs on the paternal allele results in lack of antisense Lit1 RNA expression and reactivation in cis of several imprinted genes normally exclusively maternally expressed (Horike et al. 2000; Fitzpatrick et al. 2002). More recent experiments using transient transfection assays suggest that, similarly to what is observed with the Air DMR, imprinting centre function of the KvDMR1 also requires the AS-RNA, as both deletion of promoter elements required for Lit1 expression and truncation of the Lit1 RNA by introduction of a polyadenylation site cause loss of bidirectional silencing (Thakur et al. 2004). Newly published studies have revealed that two differentially methylated regions (KvDMR1 on the maternal allele and the CpG islands of Cdkn1c on the paternal chromosome) are enriched in histone H3 trimethylated at residue K9 (Umlauf et al. 2004). Silencing of genes on the paternal allele does not require DNA methylation but displays repressive histone modifications such as trimethylation of residue K27 and dimethylation of residue K9 on histone H3 (Lewis et al. 2004; Umlauf et al. 2004). Consistently with this Eed-Ezh2 polycomb complex components are found associated with the paternally silenced genes, probably recruited by the Lit1 RNA (Umlauf et al. 2004). Indeed, expression in cis of the Lit1 RNA is required for establishment of the repressive histone modifications at a critical early stage in embryogenesis (Lewis et al. 2004). Interestingly Eed-Ezh2 is not found at the methylated KvDMR1 on the maternal chromosome despite the presence of both trimethyl H3-K27 and trimethyl H3-K9, suggesting that other complexes may be involved in methylation of H3-K27 and K9 of this DMR (Umlauf et al. 2004). All these data indicate that the non-coding Lit1 RNA is necessary for cis silencing of genes on the paternal chromosome, probably through mechanisms that include the coating in cis of regions of the paternal chromosome. It is not clear, however, if transcriptional overlap of the Kvlqt1 gene across KvDMR1, the CpG island of the Lit1 gene, is required for its silencing and methylation on the maternal allele.

(b) The Xist/Tsix sense-antisense gene pair

Xist/Tsix is a well characterized mouse sense/antisense gene pair expressing non-coding RNAs that are involved in X-chromosome inactivation, the process that leads to the silencing of one X-chromosome in mammalian female cells to achieve dosage compensation of X-linked genes between males and females (figure 2d; Willard & Carrel 2001). Xist and Tsix show a reciprocal pattern of expression and the CpG islands associated with the promoters of Xist and Tsix are methylated in a reciprocal manner (Courtier et al. 1995). The initiation of X-inactivation, which ultimately results in the methylation of many X-encoded CpG islands, requires the expression in cis of the non-coding RNA Xist which spreads and coats the inactive X chromosome (Lee & Jaenisch 1997). Although spreading of silencing on the inactive X chromosome requires the presence of the Xist RNA and its ability to coat in cis the inactive X chromosome (reviewed in Park & Kuroda 2001), initiation of X-inactivation and X chromosome choice rely on the ability to regulate the levels of expression of the Xist RNA itself. In mouse, Xist expression appears to be regulated by the expression of its AS-RNA Tsix (Lee & Lu 1999; Lee et al. 1999a, b). Consistent with this, deletion of the Tsix CpG island results in inactivation of the targeted allele in all cells, suggesting a role for Tsix in X-chromosome choice (Lee & Lu 1999). Further, truncation of the Tsix RNA by the insertion of a transcription termination site results in inactivation of the targeted allele, supporting the idea that antisense transcription and/or RNA play a primary role in downregulating Xist expression (Luikenhuis et al. 2001; Shibata & Lee 2004). Constitutive expression of Tsix driven by constitutive or inducible promoters prevents the targeted allele from being inactivated, indicating that to allow accumulation of Xist on the future inactive X chromosome Tsix needs to be down-regulated (Stavropoulos et al. 2001; Luikenhuis et al. 2001). Interestingly, Tsix mRNA expressed from the same locus but not overlapping Xist cannot rescue the Tsix knockout phenotype, indicating that transcription through Xist is necessary for its down regulation (Shibata & Lee 2004). Concomitant expression of Tsix and low levels of unstable Xist RNA prior to X-inactivation (Panning et al. 1997; Sheardown et al. 1997) have suggested a possible involvement of double stranded RNA in X-chromosome choice (Boumil & Lee 2001). Indeed, deletions upstream of the Xist promoter cause preferential inactivation of the X chromosome carrying the targeted allele but do not affect Tsix levels (Newall et al. 2001; Nesterova et al. 2003), supporting a model in which RNAi related mechanisms triggered by Xist/Tsix double stranded RNA may act in cis to repress Xist transcription (Nesterova et al. 2003).

Analyses of the chromatin structure at the Xist locus have been performed using allele specific assays in female cells. DNAse1 sensitivity studies have revealed an increase in sensitivity limited to the transcribed Xist locus on the inactive X (McCabe et al. 1999). ChIP assays have shown that on the inactive X expressing Xist, the promoter region is marked by acetylated H3 while H3-K4 methylation and H4 acetylation span the whole gene (Goto et al. 2002). Conversely, the repressed Xist promoter and gene on the active X is enriched in H3 methylated at residues K9 and K4 (Goto et al. 2002). Therefore, the chromatin modifications found reflect the expression status of Xist on the active and inactive alleles. Newly published data show that Xist expression in mouse cells displaying either imprinted or random X inactivation is accompanied by enrichment in H3-K4 methylation and RNApolII preinitiation complex (PIC) at the Xist promoter on the expressed allele (Navarro et al. 2005). In contrast, in ES cells, in which both X chromosomes are active, low levels of Xist expression can be explained by low levels of PIC at its promoter (Navarro et al. 2005). Truncation of the Tsix transcript in ES cells does not lead to PIC enrichment at the Xist promoter or to increased levels of Xist expression, indicating that in these cells Tsix transcription is not required to down-regulate Xist expression, and the authors propose that Tsix transcription renders the two alleles epigenetically equivalent prior to the onset of random X-inactivation (Navarro et al. 2005). However, a role for Tsix RNA in silencing and methylation of the Xist CpG island upon ES cell differentiation cannot be ruled out.

In conclusion, the data summarized here are consistent with the proposed potential dsRNA mediated mechanisms, but they do not exclude a possible co-transcriptional action of the single stranded Tsix RNA in silencing the Xist CpG island.

(c) AS-RNA at non-imprinted autosomal loci

Few experimentally identified cases of non-imprinted overlapping gene pairs spanning the promoter regions of the oppositely transcribed genes have been reported (table 1). For example, the presence of a non-coding polyadenylated AS-RNA transcript encompassing exon 1 and up to 1kb upstream of the haemochromatosis gene HFE has been described, and it has been shown that the AS-RNA transcripts can interfere in vitro with the expression of sense gene product (Thenie et al. 2001). However, it is not known whether the AS-RNA is acting to regulate HFE expression at a transcriptional or translational level.

AS-RNA transcription through the CpG island of a non-imprinted and autosomal tissue specific gene can lead to its silencing and methylation (Tufarelli et al. 2003). This phenomenon was observed in a patient with an inherited form of anaemia (alpha thalassaemia), in whom the decrease in expression of the erythroid specific alpha globin gene (HBA) is due to changes in chromatin structure, including the complete methylation of the alpha globin CpG island (Barbour et al. 2000). In this patient, a small interstitial deletion results in the juxtaposition of the intact erythroid specific alpha globin gene to a truncated, widely expressed gene (LUC7L), transcribed in the opposite orientation to that of the alpha globin gene (Tufarelli et al. 2001; figure 3). This results in a 'gene within gene' (figure 2d) configuration of the HBA gene on the deleted allele. In this patient and in a transgenic mouse model, methylation of the alpha globin CpG island is dependent upon the presence of AS-RNA transcripts (HBA-AS) transcribed in cis of the methylated gene from the promoter of the truncated LUC7L gene (Tufarelli et al. 2003). The cis nature of this AS-RNA mediated DNA methylation event is supported by the observation that the normal allele of the patient is not methylated (Barbour et al. 2000). In addition, F1 double hemizygous mice, generated by mating transgenic mice carrying constructs reproducing the AS-RNA mediated DNA methylation observed in the patient and mice containing a normal human alpha globin gene, display methylation only at the alpha globin gene in cis of the antisense transcripts (Tufarelli et al. 2003). The use of a differentiating mouse ES cell system revealed that methylation of the alpha globin CpG island is observed uniquely in the presence of AS-RNA transcripts and requires induction of differentiation in embryoid bodies (Tufarelli et al. 2003). The identification of AS-RNA mediated DNA methylation as a novel mechanism of human genetic disease indicates that this phenomenon is not specific to some imprinted genes or X-inactivation but it may be a more general mechanism by which associated genes become silenced during development, cell differentiation and malignancy progression. Moreover, the ES cell data clearly show that the machinery responsible for establishing DNA methylation via AS-RNA is normally present in the cells. Although a role for double stranded RNA cannot be excluded as both sense and AS-RNAs are expressed in undifferentiated ES cells, the observed cis effect points to a possible co-transcriptional effect triggered by the single stranded AS-RNA.

Figure 3: A disease associated deletion leads to expression of aberrant antisense RNA transcripts through a tissue specific CpG island.

Figure 3: A disease associated deletion leads to expression of aberrant antisense RNA transcripts through a tissue specific CpG island.

The diagrams show the relative organization of the erythroid specific alpha globin gene (HBA, light grey box) and the ubiquitous LUC7L (dark grey box) gene in the wild type allele and in the deleted allele. The deletion (striped box) removes the transcription termination site of the LUC7L gene and leads to the expression of antisense transcripts running through the HBA CpG island (dashed arrow). This results in silencing of the HBA gene in cis of the deletion and full methylation of its associated CpG island (black bar). Unmethylated (grey) and methylated (black) CpG islands are drawn as bars. Only the names of the genes described in the text are shown. Diagrams are not to scale.

There are several examples of naturally occurring AS-RNAs in higher eukaryotes, and in some situations, like the cases described above, overlapping transcription across CpG island promoter regions has been associated with their transcriptional silencing and methylation. It has been proposed that the RNA transcripts may be directly involved in the silencing process, but their mechanism of action remains largely unknown. RNA-directed DNA methylation, or RdDM, is a well-known process in plants where expression of dsRNA spanning the promoter region of genes leads to silencing and methylation of the homologous DNA sequences with mechanics highly related to those of RNA interference (e.g. Mette et al. 2000; see Mathieu & Bender 2004 for a recent review). RdDM in plants acts in trans, i.e. it does not require co-expression of sense and AS-RNA at the target site, and leads to methylation of all cytosines, not only those in the CpG or CpNpG sequences (Pelissier et al. 1999; reviewed in Matzke et al. 2001). It has been proposed that similar dsRNA mechanisms act in mammalian cells, and indeed recent reports have shown that introduction of short interfering RNAs (siRNAs) homologous to endogenous CpG island promoter sequences can lead to methylation and silencing in trans of the endogenous target promoters (e.g. Kawasaky & Taira 2004; Morris et al. 2004). However, the sense/antisense-RNA examples described in this review seem to function strictly in cis to silence and methylate CpG islands. Nevertheless, the involvement of dsRNAs in some or all of these cases cannot be excluded. It is possible that in mammalian cells the dsRNAs generated by these loci are very unstable or cannot diffuse to the other allele, or can act through the interaction with the nascent AS-RNAs that are expressed by one allele only. Interestingly, in S. Pombe nascent RNAs at the centromeric repeats recruit complexes containing components of the RNAi machinery through the interaction with RNA guides derived from siRNAs processed from dsRNAs expressed by the centromeric repeats themselves (Motamedi et al. 2004; Noma et al. 2004). The cis interaction of the complexes with the nascent RNA is critical for establishment of a silent chromatin state at centromeres (Motamedi et al. 2004), but it cannot be excluded that the recruited complexes at a given centromere may contain guide RNAs originating at different centromeres. Although it is important to determine whether this or similar mechanisms are at work in the mammalian systems reported here, their cis action would imply a thermodynamic component given by the sequences of the siRNAs, so that in the complexes only the sense strand is retained allowing the recognition of exclusively the nascent antisense RNA.

It is, therefore, tempting to speculate the existence of alternative mechanisms, that do not require the formation of dsRNAs, to explain how AS-RNAs running through a CpG island can trigger its cis silencing and methylation. One of the first questions to address is whether antisense transcription per se rather than the RNA itself can be causing the silencing. While this cannot be excluded, both sense and antisense intergenic transcription have been associated with gene activation (Gribnau et al. 2000; Bolland et al. 2004). In these cases, intergenic transcription is developmentally regulated and it has been proposed that transcription may be involved in the establishment of an open chromatin environment (Gribnau et al. 2000). As RNA polII complexes contain histone acetyl transferase activities, it seems unlikely that they would favour the formation of repressive chromatin when running through a CpG island. After all it is only transcription from the methylated CpG island that is blocked, as antisense transcription continues to occur through the methylated region. However, it is still possible that transcription is required to render the locus accessible to the DNA and histone methyl transferase containing complexes that may be recruited by the AS-RNA to lock the sense CpG island in a transcriptionally silenced state (see below).

Several options can be envisaged to explain RNA mediated cis-silencing in alternative to double stranded RNA mechanisms. The models put forward in figure 4 are based on the assumption that, at least in some instances, the observed cis effects are due to the ability of AS-RNA to co-transcriptionally interact with the locus from which it is transcribed. These models do not attempt to explain the cis action of the Xist RNA, but rather those CpG island silencing examples that seem to require the presence of AS-RNA which are complementary in sequence to the silenced CpG island, including the silencing of the Xist CpG island itself. It is proposed that transcription of AS-RNA could result in the formation of an RNA/DNA triple helix (figure 4a). Alternatively, the newly transcribed AS-RNA strand and its template DNA strand could form an RNA/DNA hybrid with the non-template strand forming a single stranded DNA loop (figure 4b). It is also possible that both sense and antisense-RNA could interact with their respective template strands to form RNA/DNA hybrids (figure 4c). Such unusual structures might attract DNA methyltransferases (Smith et al. 1991; Laayoun & Smith 1995), guiding them to the target sequence. These series of events could also play a part in the chromatin modification observed at the oppositely transcribed CpG islands through the recruitment of chromatin remodelling complexes and methyl CpG binding proteins (Jones et al. 1998; Nan et al. 1998; Razin 1998; Ng & Bird 1999; Wade et al. 1999; Zhang et al. 1999; see Freitag & Selker 2005 for a recent review). However, these models do not address the temporal order in which DNA methylation and histone modifications appear at the silenced locus, but can accommodate alternative possibilities, with DNA methylation preceding or following histone modifications depending on the local context.

Figure 4: Models of antisense RNA mediated CpG island methylation in cis.

Figure 4: Models of antisense RNA mediated CpG island methylation in cis.

The newly transcribed antisense RNA (dark grey arrow) driven by the antisense promoter (bent dark grey arrow) could interact with the DNA locus (black) from which it is transcribed to form specialized structures, such as an RNA/DNA triple helix (a), or an RNA/DNA hybrid and a DNA loop (b). Alternatively, both antisense and sense (light grey arrow) RNAs could form an RNA/DNA hybrid with their respective template strands (c).

These unusual structures could recruit (dashed arrows) by unknown mechanism the DNA methyltransferases and/or other repressive complexes (oval), leading to methylation (filled lollipops) and silencing (crossed arrow) of the sense promoter (bent light grey arrow) without preventing continued expression of the antisense RNA. Note that these models do not address the order of the silencing events, i.e. whether histone modifications precede or follow DNA methylation, but can accommodate either.

Bioinformatics tools have revealed a surprising number of sense/antisense gene pairs in the genome with a potential to be regulated by the cis mechanisms described above, suggesting that these mechanisms may play a more general role in normal regulation of gene expression and are not restricted to imprinted genes or to pathological situations. There are examples of random monoallelically expressed genes (e.g. immunoglobulin genes, T cell receptors genes, olfactory genes, etc., Chess 1998), but the mechanisms leading to their monoallelic expression remain elusive (see Goldmit & Bergman 2004 for a review). It is possible that in some cases monoallelic expression is achieved through the mechanisms described here. However, monoallelic gene expression is not necessarily the inevitable output of AS-RNA mediated silencing in cis, but these mechanisms may account for developmental stage and/or tissue specific gene silencing at some loci.

In summary, the available observations suggest that more than one RNA mediated mechanisms can lead to gene silencing and CpG island methylation. Based on the research summarized in this review, alternative mechanisms through which cis AS-RNA transcripts can lead to CpG island methylation have been proposed. Future work will aim to determine whether any of these mechanisms are at work to regulate expression of CpG island associated genes during development and differentiation, and if they contribute to altered gene expression in disease.

Acknowledgements

The author would like to thank Prof Doug Higgs and members of his lab for their continued help and support, and David Garrick for critical reading of the manuscript. C.T. is a recipient of a Royal Society Dorothy Hodgkin Fellowship. Research in the lab is also supported by a project grant from Cancer Research UK to C.T.

References

Ball, S.T., Williamson, C.M., Hayes, C., Hacker, T. & Peters, J. 2001 The spatial and temporal expression pattern of Nesp and its antisense Nespas, in mid-gestation mouse embryos. Mech. Dev. 100, 79-81 (doi:10.1016/S0925-4773(00)00490-1).

Barbour, V.M. et al. 2000 alpha-Thalassemia resulting from a negative chromosomal position effect. Blood 96, 800-807.

Bastepe, M., Frohlich, L.F., Linglart, A., Abu-Zahra, H.S., Tojo, K., Ward, L.M. & Juppner, H. 2005 Deletion of the NESP55 differentially methylated region causes loss of maternal GNAS imprints and pseudohypoparathyroidism type Ib. Nat. Genet. 37, 25-27 (doi:10.1038/ng1560).

Bender, J. 2004 Chromatin-based silencing mechanisms. Curr. Opin. Plant Biol. 7, 521-526 (doi:10.1016/j.pbi.2004.07.003).

Bolland, D.J., Wood, A.L., Johnston, C.M., Bunting, S.F., Morgan, G., Chakalova, L., Fraser, P.J. & Corcoran, A.E. 2004 Antisense intergenic transcription in V(D)J recombination. Nat. Immunol. 5, 630-637 (doi:10.1038/ni1068).

Boumil, R.M. & Lee, J.T. 2001 Forty years of decoding the silence in X-chromosome inactivation. Hum. Mol. Genet. 10, 2225-2232 (doi:10.1093/hmg/10.20.2225).

Campbell, C.E., Huang, A., Gurney, A.L., Kessler, P.M., Hewitt, J.A. & Williams, B.R.G. 1994 Antisense transcripts and protein binding motifs within the Wilms' tumour (WT1) locus. Oncogene 9, 583-595.

Carmichael, G.G. 2003 Antisense starts making more sense. Nat. Biotechnol. 21, 371-372 (doi:10.1038/nbt0403-371).

Cawthon, R. et al. 1990 Identification and characterization of transcripts from the neurofibromatosis 1 region: The sequence and genomic structure of EVI2 and mapping of other transcripts. Genomics 7, 555-565 (doi:10.1016/0888-7543(90)90199-5).

Cawthon, R.M. et al. 1991 cDNA sequence and genomic structure of EVI2B, a gene lying within an intron of the neurofibromatosis type 1 gene. Genomics 9, 446-460 (doi:10.1016/0888-7543(91)90410-G).

Chan, S.W., Zilberman, D., Xie, Z., Johansen, L.K., Carrington, J.C. & Jacobsen, S.E. 2004 RNA silencing genes control de novo DNA methylation. Science 303, 1336. (doi:10.1126/science.1095989).

Chen, J., Sun, M., Kent, W.J., Huang, X., Xie, H., Wang, W., Zhou, G., Shi, R.Z. & Rowley, J.D. 2004 Over 20% of human transcripts might form sense-antisense pairs. Nucleic Acids Res. 32, 4812-4820 (doi:10.1093/nar/gkh818).

Chess, A. 1998 Expansion of the allelic exclusion principle?. Science 279, 2067-2068 (doi:10.1126/science.279.5359.2067).

Constancia, M., Pickard, B., Kelsey, G. & Reik, W. 1998 Imprinting mechanisms. Genome Res. 8, 881-900.

Coombes, C., Arnaud, P., Gordon, E., Dean, W., Coar, E.A., Williamson, C.M., Feil, R., Peters, J. & Kelsey, G. 2003 Epigenetic properties and identification of an imprint mark in the Nesp-Gnasxl domain of the mouse Gnas imprinted locus. Mol. Cell Biol. 23, 5475-5488 (doi:10.1128/MCB.23.16.5475-5488.2003).

Courtier, B., Heard, E. & Avner, P. 1995 Xce haplotypes show modified methylation in a region of the active X chromosome lying 3' to Xist. Proc. Natl Acad. Sci. USA 92, 3531-3535.

Dahary, D., Elroy-Stein, O. & Sorek, R. 2005 Naturally occurring antisense: transcriptional leakage or real overlap?. Genome Res. 15, 364-368 (doi:10.1101/gr.3308405).

Dallosso, A.R., Hancock, A.L., Brown, K.W., Williams, A.C., Jackson, S. & Malik, K. 2004 Genomic imprinting at the WT1 gene involves a novel coding transcript (AWT1) that shows deregulation in Wilms' tumours. Hum. Mol. Genet. 13, 405-415 (doi:10.1093/hmg/ddh038).

Debrand, E., Chureau, C., Arnaud, D., Avner, P. & Heard, E. 1999 Functional analysis of the DXPas34 Locus, a 3' regulator of Xist expression. Mol. Cell Biol. 19, 8513-8525.

Dunham, I. et al. 1999 The DNA sequence of human chromosome 22. Nature 402, 489-495 (doi:10.1038/990031).

Eccles, M.R., Grubb, G., Ogawa, O., Szeto, J. & Reeve, A.E. 1994 Cloning of novel Wilms tumor gene (WT1) cDNAs; evidence for antisense transcription of WT1. Oncogene 9, 2059-2063.

Engemann, S., Strodicke, M., Paulsen, M., Franck, O., Reinhardt, R., Lane, N., Reik, W. & Walter, J. 2000 Sequence and functional comparison in the Beckwith-Wiedemann region: implications for a novel imprinting centre and extended imprinting. Hum. Mol. Genet. 9, 2691-2706 (doi:10.1093/hmg/9.18.2691).

Fitzpatrick, G.V., Soloway, P.D. & Higgins, M.J. 2002 Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1. Nat. Genet. 32, 426-431 (doi:10.1038/ng988).

Fournier, C., Goto, Y., Ballestar, E., Delaval, K., Hever, A.M., Esteller, M. & Feil, R. 2002 Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes. EMBO J. 21, 6560-6570 (doi:10.1093/emboj/cdf655).

Freitag, M. & Selker, E.U. 2005 Controlling DNA methylation: many roads to one modification. Curr. Opin. Genet. Dev. 15, 191-199 (doi:10.1016/j.gde.2005.02.003).

Goldmit, M. & Bergman, Y. 2004 Monoallelic gene expression: a repertoire of recurrent themes. Immunol. Rev. 200, 197-214 (doi:10.1111/j.0105-2896.2004.00158.x).

Goto, Y., Gomez, M., Brockdorff, N. & Feil, R. 2002 Differential patterns of histone methylation and acetylation distinguish active and repressed alleles at X-linked genes. Cytogenet. Genome Res. 99, 66-74 (doi:10.1159/000071576).

Gribnau, J., Diderich, K., Pruzina, S., Calzolari, R. & Fraser, P. 2000 Intergenic transcription and developmental remodeling of chromatin subdomains in the human beta-globin locus. Mol. Cell 5, 377-386 (doi:10.1016/S1097-2765(00)80432-3).

Hall, I.M., Shankaranarayana, G.D., Noma, K., Ayoub, N., Cohen, A. & Grewal, S.I. 2002 Establishment and maintenance of a heterochromatin domain. Science 297, 2232-2237 (doi:10.1126/science.1076466).

Hayward, B.E. & Bonthron, D.T. 2000 An imprinted antisense transcript at the human GNAS1 locus. Hum. Mol. Genet. 9, 835-841 (doi:10.1093/hmg/9.5.835).

Hernandez, A., Fiering, S., Martinez, E., Galton, V.A. & Germain, D.S. 2002 The gene locus encoding iodothyronin deiodinase type 3 (Dio3) is imprinted in the fetus and expresses antisense transcripts. Endocrinology 143, 4483-4486 (doi:10.1210/en.2002-220800).

Herzog, H., Darby, K., Hort, Y.J. & Shine, J. 1996 Intron 17 of the human retinoblastoma susceptibility gene encodes an actively transcribed G protein-coupled receptor gene. Genome Res. 6, 858-861.

Holmes, R., Williamson, C., Peters, J., Denny, P. & Wells, C. 2003 A comprehensive transcript map of the mouse Gnas imprinted complex. Genome Res. 13, 1410-1415 (doi:10.1101/gr.955503).

Horike, S., Mitsuya, K., Meguro, M., Kotobuki, N., Kashiwagi, A., Notsu, T., Schulz, T.C., Shirayoshi, Y. & Oshimura, M. 2000 Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome. Hum. Mol. Genet. 9, 2075-2083 (doi:10.1093/hmg/9.14.2075).

Hu, J.F., Balaguru, K.A., Ivaturi, R.D., Oruganti, H., Li, T., Nguyen, B.T., Vu, T.H. & Hoffman, A.R. 1999 Lack of reciprocal genomic imprinting of sense and antisense RNA of mouse insulin-like growth factor II receptor in the central nervous system. Biochem. Biophys. Res. Commun. 257, 604-608 (doi:10.1006/bbrc.1999.0380).

Hu, J.F., Pham, J., Dey, I., Li, T., Vu, T.H. & Hoffman, A.R. 2000 Allele-specific histone acetylation accompanies genomic imprinting of the insulin-like growth factor II receptor gene. Endocrinology 141, 4428-4435 (doi:10.1210/en.141.12.4428).

Ischia, R., Lovisetti-Scamihorn, P., Hogue-Angeletti, R., Wolkersdorfer, M., Winkler, H. & Fischer-Colbrie, R. 1997 Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity. J. Biol. Chem. 272, 11657-11662 (doi:10.1074/jbc.272.17.11657).

Jones, P.L., Veenstra, G.J., Wade, P.A., Vermaak, D., Kass, S.U., Landsberger, N., Strouboulis, J. & Wolffe, A.P. 1998 Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19, 187-190 (doi:10.1038/561).

Jong, M.T., Carey, A.H., Caldwell, K.A., Lau, M.H., Handel, M.A., Driscoll, D.J., Stewart, C.L., Rinchik, E.M. & Nicholls, R.D. 1999 Imprinting of a RING zinc-finger encoding gene in the mouse chromosome region homologous to the Prader-Willi syndrome genetic region. Hum. Mol. Genet. 8, 795-803 (doi:10.1093/hmg/8.5.795).

Jong, M.T., Gray, T.A., Ji, Y., Glenn, C.C., Saitoh, S., Driscoll, D.J. & Nicholls, R.D. 1999 A novel imprinted gene, encoding a RING zinc-finger protein, and overlapping antisense transcript in the Prader-Willi syndrome critical region. Hum. Mol. Genet. 8, 783-793 (doi:10.1093/hmg/8.5.783).

Karlin, S., Chen, C., Gentles, A.J. & Cleary, M. 2002 Associations between human disease genes and overlapping gene groups and multiple amino acid runs. Proc. Natl Acad. Sci. USA 99, 17008-17013 (doi:10.1073/pnas.262658799).

Kawasaki, H. & Taira, K. 2004 Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature 431, 211-217 (doi:10.1038/nature02889).

Kawasaki, H., Taira, K. & Morris, K.V. 2005 siRNA induced transcriptional gene silencing in mammalian cells. Cell Cycle 4, 442-448.

Kiyosawa, H., Yamanaka, I., Osato, N., Kondo, S. & Hayashizaki, Y. 2003 Antisense transcripts with FANTOM2 clone set and their implications for gene regulation. Genome Res. 13, 1324-1334 (doi:10.1101/gr.982903).

Kumar, M. & Carmichael, G.G. 1998 Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes. Microbiol. Mol. Biol. Rev. 62, 1415-1434.

Laayoun, A. & Smith, S.S. 1995 Methylation of slipped duplexes, snapbacks and cruciforms by human DNA(cytosine-5)methyltransferase. Nucleic Acids Res. 23, 1584-1589.

Lavorgna, G., Dahary, D., Lehner, B., Sorek, R., Sanderson, C.M. & Casari, G. 2004 In search of antisense. Trends Biochem. Sci. 29, 88-94 (doi:10.1016/j.tibs.2003.12.002).

Lee, J.T. & Jaenisch, R. 1997 The (epi)genetic control of mammalian X-chromosome inactivation. Curr. Opin. Genet. Dev. 7, 274-280 (doi:10.1016/S0959-437X(97)80138-4).

Lee, J.T. & Lu, N. 1999 Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell 99, 47-57 (doi:10.1016/S0092-8674(00)80061-6).

Lee, J.T., Davidow, L.S. & Warshawsky, D. 1999 Tsix, a gene antisense to Xist at the X-inactivation centre. Nat. Genet. 21, 400-404 (doi:10.1038/7734).

Lee, M.P., DeBaun, M.R., Mitsuya, K., Galonek, H.L., Brandenburg, S., Oshimura, M. & Feinberg, A.P. 1999 Loss of imprinting of a paternally expressed transcript, with antisense orientation to KVLQT1, occurs frequently in Beckwith-Wiedemann syndrome and is independent of insulin-like growth factor II imprinting. Proc. Natl Acad. Sci. USA 96, 5203-5208 (doi:10.1073/pnas.96.9.5203).

Lee, Y.J., Park, C.W., Hahn, Y., Park, J., Lee, J., Yun, J.H., Hyun, B. & Chung, J.H. 2000 Mit1/Lb9 and Copg2, new members of mouse imprinted genes closely linked to Peg1/Mest(1). FEBS Lett. 472, 230-234 (doi:10.1016/S0014-5793(00)01461-7).

Lehner, B., Williams, G., Campbell, R.D. & Sanderson, C.M. 2002 Antisense transcripts in the human genome. Trends Genet. 18, 63-65 (doi:10.1016/S0168-9525(02)02598-2).

Levinson, B., Kenwrick, S., Lakich, D., Hammonds, G. & Gitschier, J. 1990 A transcribed gene in an intron of the human factor VIII gene. Genomics 7, 1-11 (doi:10.1016/0888-7543(90)90512-S).

Levinson, B., Kenwrick, S., Gamel, P., Fisher, K. & Gitschier, J. 1992 Evidence for a third transcript from the human factor VIII gene. Genomics 14, 585-589 (doi:10.1016/S0888-7543(05)80155-7).

Lewis, A., Mitsuya, K., Umlauf, D., Smith, P., Dean, W., Walter, J., Higgins, M., Feil, R. & Reik, W. 2004 Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation. Nat. Genet. 36, 1291-1295 (doi:10.1038/ng1468).

Li, T., Vu, T.H., Zeng, Z.L., Nguyen, B.T., Hayward, B.E., Bonthron, D.T., Hu, J.F. & Hoffman, A.R. 2000 Tissue-specific expression of antisense and sense transcripts at the imprinted Gnas locus. Genomics 69, 295-304 (doi:10.1006/geno.2000.6337).

Lipman, D.J. 1997 Making (anti)sense of non-coding sequence conservation. Nucleic Acids Res. 25, 3580-3583 (doi:10.1093/nar/25.18.3580).

Liu, J., Yu, S., Litman, D., Chen, W. & Weinstein, L.S. 2000 Identification of a methylation imprint mark within the mouse Gnas locus. Mol. Cell Biol. 20, 5808-5817 (doi:10.1128/MCB.20.16.5808-5817.2000).

Luikenhuis, S., Wutz, A. & Jaenisch, R. 2001 Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells. Mol. Cell Biol. 21, 8512-8520 (doi:10.1128/MCB.21.24.8512-8520.2001).

Malik, K.T.A., Wallace, J.I., Ivins, S.M. & Brown, K.W. 1995 Identification of an antisense WT1 promoter in intron 1: implications for WT1 gene regulation. Oncogene 11, 1589-1595.

Malik, K., Salpekar, A., Hancock, A., Moorwood, K., Jackson, S., Charles, A. & Brown, K.W. 2000 Identification of differential methylation of the WT1 antisense regulatory region and relaxation of imprinting in Wilms' tumor. Cancer Res. 60, 2356-2360.

Mathieu, O. & Bender, J. 2004 RNA-directed DNA methylation. J. Cell. Sci. 117, 4881-4888 (doi:10.1242/jcs.01479).

Matzke, M.A. & Birchler, J.A. 2005 RNAi-mediated pathways in the nucleus. Nat. Rev. Genet. 6, 24-35 (doi:10.1038/nrg1500).

Matzke, M., Matzke, A.J. & Kooter, J.M. 2001 RNA: guiding gene silencing. Science 293, 1080-1083 (doi:10.1126/science.1063051).

McCabe, V., Formstone, E.J., O'Neill, L.P., Turner, B.M. & Brockdorff, N. 1999 Chromatin structure analysis of the mouse Xist locus. Proc. Natl Acad. Sci. USA 96, 7155-7160 (doi:10.1073/pnas.96.13.7155).

Mette, M.F., Aufsatz, W., van der Winden, J., Matzke, M.A. & Matzke, A.J. 2000 Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19, 5194-5201 (doi:10.1093/emboj/19.19.5194).

Mise, N., Goto, Y., Nakajima, N. & Takagi, N. 1999 Molecular cloning of antisense transcripts of the mouse Xist gene. Biochem. Biophys. Res. Commun. 258, 537-541 (doi:10.1006/bbrc.1999.0681).

Mitsuya, K. et al. 1999 LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids. Hum. Mol. Genet. 8, 1209-1217 (doi:10.1093/hmg/8.7.1209).

Mochizuki, K., Fine, N.A., Fujisawa, T. & Gorovsky, M.A. 2002 Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena. Cell 110, 689-699 (doi:10.1016/S0092-8674(02)00909-1).

Moore, T., Constancia, M., Zubair, M., Bailleul, B., Feil, R., Sasaki, H. & Reik, W. 1997 Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2. Proc. Natl Acad. Sci. USA 94, 12509-12514 (doi:10.1073/pnas.94.23.12509).

Morey, C. & Avner, P. 2004 Employment opportunities for non-coding RNAs. FEBS Lett. 567, 27-34 (doi:10.1016/j.febslet.2004.03.117).

Morris, K.V., Chan, S.W., Jacobsen, S.E. & Looney, D.J. 2004 Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305, 1289-1292 (doi:10.1126/science.1101372).

Motamedi, M.R., Verdel, A., Colmenares, S.U., Gerber, S.A., Gygi, S.P. & Moazed, D. 2004 Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119, 789-802 (doi:10.1016/j.cell.2004.11.034).

Nakabayashi, K. et al. 2002 Identification and characterization of an imprinted antisense RNA (MESTIT1) in the human MEST locus on chromosome 7q32. Hum. Mol. Genet. 11, 1743-1756 (doi:10.1093/hmg/11.15.1743).

Nan, X., Ng, H.-H., Johnson, C.A., Laherty, C.D., Turner, B.M., Eisenman, R.N. & Bird, A. 1998 Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386-389 (doi:10.1038/30764).

Navarro, P., Pichard, S., Ciaudo, C., Avner, P. & Rougeulle, C. 2005 Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation. Genes Dev. 19, 1474-1484 (doi:10.1101/gad.341105).

Nesterova, T.B., Johnston, C.M., Appanah, R., Newall, A.E., Godwin, J., Alexiou, M. & Brockdorff, N. 2003 Skewing X chromosome choice by modulating sense transcription across the Xist locus. Genes Dev. 17, 2177-2190 (doi:10.1101/gad.271203).

Newall, A.E., Duthie, S., Formstone, E., Nesterova, T., Alexiou, M., Johnston, C., Caparros, M.L. & Brockdorff, N. 2001 Primary non-random X inactivation associated with disruption of Xist promoter regulation. Hum. Mol. Genet. 10, 581-589 (doi:10.1093/hmg/10.6.581).

Ng, H.H. & Bird, A. 1999 DNA methylation and chromatin modification. Curr. Opin. Genet. Dev. 9, 158-163 (doi:10.1016/S0959-437X(99)80024-0).

Noma, K., Sugiyama, T., Cam, H., Verdel, A., Zofall, M., Jia, S., Moazed, D. & Grewal, S.I. 2004 RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing. Nat. Genet. 36, 1174-1180 (doi:10.1038/ng1452).

Pal-Bhadra, M., Bhadra, U. & Birchler, J.A. 2002 RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila. Mol. Cell 9, 315-327 (doi:10.1016/S1097-2765(02)00440-9).

Pal-Bhadra, M., Leibovitch, B.A., Gandhi, S.G., Rao, M., Bhadra, U., Birchler, J.A. & Elgin, S.C. 2004 Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303, 669-672 (doi:10.1126/science.1092653).

Panning, B., Dausman, J. & Jaenisch, R. 1997 X chromosome inactivation is mediated by Xist RNA stabilization. Cell 90, 907-916 (doi:10.1016/S0092-8674(00)80355-4).

Park, Y. & Kuroda, M.I. 2001 Epigenetic aspects of X-chromosome dosage compensation. Science 293, 1083-1085 (doi:10.1126/science.1063073).

Pelissier, T., Thalmier, S., Kempe, D. & Sanger, H.L. 1999 Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA methylation. Nucleic Acids Res. 27, 1625-1634 (doi:10.1093/nar/27.7.1625).

Razin, A. 1998 CpG methylation, chromatin structure and gene silencing--a three-way connection. EMBO 17, 4905-4908 (doi:10.1093/emboj/17.17.4905).

Rivkin, M., Rosen, K.M. & Villa-Komaroff, L. 1993 Identification of an antisense transcript from the IGF-II locus in mouse. Mol. Reprod. Dev. 35, 394-397 (doi:10.1002/mrd.1080350413).

Rougeulle, C. & Heard, E. 2002 Antisense RNA in imprinting: spreading silence through Air. Trends Genet. 18, 434-437 (doi:10.1016/S0168-9525(02)02749-X).

Rougeulle, C., Cardoso, C., Fontes, M., Colleaux, L. & Lalande, M. 1998 An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nat. Genet. 19, 15-16.

Runte, M., Huttenhofer, A., Gross, S., Kiefmann, M., Horsthemke, B. & Buiting, K. 2001 The IC-SNURF-SNRPN transcript serves as a host for multiple small nucleolar RNA species and as an antisense RNA for UBE3A. Hum. Mol. Genet. 10, 2687-2700 (doi:10.1093/hmg/10.23.2687).

Schramke, V. & Allshire, R. 2003 Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing. Science 301, 1069-1074 (doi:10.1126/science.1086870).

Seitz, H., Youngson, N., Lin, S.P., Dalbert, S., Paulsen, M., Bachellerie, J.P., Ferguson-Smith, A.C. & Cavaille, J. 2003 Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene. Nat. Genet. 34, 261-262 (doi:10.1038/ng1171).

Sheardown, S.A. et al. 1997 Stabilization of Xist RNA mediates initiation of X chromosome inactivation. Cell 91, 99-107 (doi:10.1016/S0092-8674(01)80012-X).

Shendure, J. & Church, G.M. 2002 Computational discovery of sense-antisense transcription in the human and mouse genomes. Genome Biol. 3, RESEARCH0044.1-0044.14 (doi:10.1186/gb-2002-3-9-research0044).

Shibata, S. & Lee, J.T. 2004 Tsix transcription- versus RNA-based mechanisms in Xist repression and epigenetic choice. Curr. Biol. 14, 1747-1754 (doi:10.1016/j.cub.2004.09.053).

Sims, R.J., 3rd, Nishioka, K. & Reinberg, D. 2003 Histone lysine methylation: a signature for chromatin function. Trends Genet. 19, 629-639 (doi:10.1016/j.tig.2003.09.007).

Sleutels, F. & Barlow, D.P. 2001 Investigation of elements sufficient to imprint the mouse Air promoter. Mol. Cell Biol. 21, 5008-5017 (doi:10.1128/MCB.21.15.5008-5017.2001).

Sleutels, F., Zwart, R. & Barlow, D.P. 2002 The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415, 810-813.

Sleutels, F., Tjon, G., Ludwig, T. & Barlow, D.P. 2003 Imprinted silencing of Slc22a2 and Slc22a3 does not need transcriptional overlap between Igf2r and Air. EMBO J. 22, 3696-3704 (doi:10.1093/emboj/cdg341).

Smilinich, N.J. et al. 1999 A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome. Proc. Natl Acad. Sci. USA 96, 8064-8069 (doi:10.1073/pnas.96.14.8064).

Smith, S.S., Kan, J.L., Baker, D.J., Kaplan, B.E. & Dembek, P. 1991 Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase. J. Mol. Biol. 217, 39-51 (doi:10.1016/0022-2836(91)90609-A).

Stavropoulos, N., Lu, N. & Lee, J.T. 2001 A functional role for Tsix transcription in blocking Xist RNA accumulation but not in X-chromosome choice. Proc. Natl Acad. Sci. USA 98, 10232-10237 (doi:10.1073/pnas.171243598).

Stoger, R., Kubicka, P., Liu, C.G., Kafri, T., Razin, A., Cedar, H. & Barlow, D.P. 1993 Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell 73, 61-71 (doi:10.1016/0092-8674(93)90160-R).

Struhl, K. 1999 Fundamentally different logic of gene regulation in eukaryotes and prokaryotes. Cell 98, 1-4 (doi:10.1016/S0092-8674(00)80599-1).

Thakur, N., Tiwari, V.K., Thomassin, H., Pandey, R.R., Kanduri, M., Gondor, A., Grange, T., Ohlsson, R. & Kanduri, C. 2004 An antisense RNA regulates the bidirectional silencing property of the kcnq1 imprinting control region. Mol. Cell Biol. 24, 7855-7862 (doi:10.1128/MCB.24.18.7855-7862.2004).

Thenie, A.C., Gicquel, I.M., Hardy, S., Ferran, H., Fergelot, P., Le Gall, J.Y. & Mosser, J. 2001 Identification of an endogenous RNA transcribed from the antisense strand of the HFE gene. Hum. Mol. Genet. 10, 1859-1866 (doi:10.1093/hmg/10.17.1859).

Tufarelli, C., Hardison, R., Flint, J. & Higgs, D.R. 2001 Characterization of a widely expressed gene (LUC7-LIKE; LUC7L) defining the centromeric boundary of the human alpha-globin domain. Genomics 71, 307-314 (doi:10.1006/geno.2000.6394).

Tufarelli, C., Stanley, J.A., Garrick, D., Sharpe, J.A., Ayyub, H., Wood, W.G. & Higgs, D.R. 2003 Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. Nat. Genet. 34, 157-165 (doi:10.1038/ng1157).

Tufarelli, C., Hardison, R., Miller, W., Hughes, J., Clark, K., Ventress, N., Frischauf, A.M. & Higgs, D.R. 2004 Comparative analysis of the alpha-like globin clusters in mouse, rat, and human chromosomes indicates a mechanism underlying breaks in conserved synteny. Genome Res. 14, 623-630 (doi:10.1101/gr.2143604).

Umlauf, D., Goto, Y., Cao, R., Cerqueira, F., Wagschal, A., Zhang, Y. & Feil, R. 2004 Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes. Nat. Genet. 36, 1296-1300 (doi:10.1038/ng1467).

Vanhee-Brossollet, C. & Vaquero, C. 1998 Do natural antisense transcripts make sense in eukaryotes?. Gene 211, 1-9 (doi:10.1016/S0378-1119(98)00093-6).

Verona, R.I., Mann, M.R. & Bartolomei, M.S. 2003 Genomic imprinting: intricacies of epigenetic regulation in clusters. Annu. Rev. Cell Dev. Biol. 19, 237-259 (doi:10.1146/annurev.cellbio.19.111401.092717).

Viskochil, D., Cawthon, R., O'Connell, P., Xu, G., Stevens, J., Culver, M., Carey, J. & White, R. 1991 The gene encoding the oligodendrocyte-myelin glycoprotein is embedded within the neurofibromatosis type 1 gene. Mol. Cell Biol. 11, 906-912.

Volpe, T.A., Kidner, C., Hall, I.M., Teng, G., Grewal, S.I. & Martienssen, R.A. 2002 Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833-1837 (doi:10.1126/science.1074973).

Vu, T.H., Li, T. & Hoffman, A.R. 2004 Promoter-restricted histone code, not the differentially methylated DNA regions or antisense transcripts, marks the imprinting status of IGF2R in human and mouse. Hum. Mol. Genet. 13, 2233-2245 (doi:10.1093/hmg/ddh244).

Wade, P.A., Gegonne, A., Jones, P.L., Ballestar, E., Aubry, F. & Wolffe, A.P. 1999 Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat. Genet. 23, 62-66.

Willard, H.F. & Carrel, L. 2001 Making sense (and antisense) of the X inactivation center. Proc. Natl Acad. Sci. USA 98, 10025-10027 (doi:10.1073/pnas.191380198).

Williamson, C.M., Skinner, J.A., Kelsey, G. & Peters, J. 2002 Alternative non-coding splice variants of Nespas, an imprinted gene antisense to Nesp in the Gnas imprinting cluster. Mamm. Genome 13, 74-79 (doi:10.1007/s00335-001-2102-2).

Wroe, S.F., Kelsey, G., Skinner, J.A., Bodle, D., Ball, S.T., Beechey, C.V., Peters, J. & Williamson, C.M. 2000 An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus. Proc. Natl Acad. Sci. USA 97, 3342-3346 (doi:10.1073/pnas.050015397).

Wutz, A., Smrzka, O.W., Schweifer, N., Schellander, K., Wagner, E.F. & Barlow, D.P. 1997 Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature 389, 745-749 (doi:10.1038/39631).

Yang, Y., Li, T., Vu, T.H., Ulaner, G.A., Hu, J.F. & Hoffman, A.R. 2003 The histone code regulating expression of the imprinted mouse Igf2r gene. Endocrinology 144, 5658-5670 (doi:10.1210/en.2003-0798).

Yelin, R. et al. 2003 Widespread occurrence of antisense transcription in the human genome. Nat. Biotechnol. 21, 379-386 (doi:10.1038/nbt808).

Yun, J., Park, C.W., Lee, Y.J. & Chung, J.H. 2003 Allele-specific methylation at the promoter-associated CpG island of mouse Copg2. Mamm. Genome 14, 376-382 (doi:10.1007/s00335-002-2252-x).

Zhang, Y., Ng, H.-H., Erdjument-Bromage, H., Tempst, P., Bird, A. & Reinberg, D. 1999 Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Gene. Dev. 13, 1924-1935 (doi:10.1101/gad.13.18.2388).

In this comprehensive review by Christina Tufarelli, we find increasing evidence for the roles played by paired sense-antisense RNAs of a non-coding type in DNA methylation and histone modifications during cis gene transcription, and especially during cell differentiation involving paternal and maternal allelic marking.

1. Frenster JH, and Hovsepian JA,
"DNA-DNA Tetraplex Model of Paired Sense-Antisense RNA Synthesis".

2.  Frenster JH, and Hovsepian JA, "Ultrastructure  of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei", Molec. Biol. Cell, vol. 15, suppl., p. 450a (November, 2004).

3. Kioussis D, "Gene regulation: Kissing Chromosomes",  Nature vol. 435, no. 7042, pp. 579-580 (June 2,  2005).

4. Frenster JH, and Hovsepian JA, "Ultrastructure of Euchromatin Contact Points between the Closed Loops of Adjacent Interphase Chromosomes", Molec. Biol. Cell, vol. 16, suppl., p. 1280a (December, 2005).

5. Xu N., Tsai C-L, and Lee J.T., "Transient Homologous Chromosome Pairing Marks the Onset of X Inactivation",   Science, 311: 1149-1152, (February 24, 2006).

6. Frenster JH, and Hovsepian JA, "Kissing Chromosomes and Paired Sense-Antisense RNA Synthesis", Cold Spring Harbor Symposium on Quantitative Biology, vol. 71, page 62, May 31-June 5, 2006.

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"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).