Non-coding RNA in fly dosage compensation

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1 Review TRENDS in Biochemical Sciences Vol.31 No.9 Non-coding RNA in fly dosage compensation Xinxian Deng 1,2 and Victoria H. Meller 2 1 Department of Biological Science, Wayne State University, Detroit, MI 48201, USA 2 Department of Biology, Tufts University, Medford, MA 02155, USA Dosage compensation modulates global expression of an X chromosome and is necessary to restore the balance between X-chromosome and autosome expression in both sexes. A central question in the field is how this regulation is directed. Large non-coding RNAs, such as Xist in mammals and rox in flies, have pivotal roles in targeting chromosome-wide modification for dosage compensation. Several recent studies in Drosophila provide new insight into the principles of X-chromosome recognition and the function of non-coding RNA in this process. Coordinated regulation of closely linked genes The need to execute complex developmental programs and adapt rapidly to changing conditions requires coordinated expression of many genes. Whereas eukaryotic regulation is typically thought of in terms of local elements that regulate a single gene, coordinated expression of closely linked genes also occurs. The clustering of genes with similar expression patterns in yeast, Drosophila, Caenorhabditis elegans, mice and humans suggests that regional gene control is widespread [1,2]. The mechanisms by which this is accomplished are varied, but usually involve stably maintained changes in chromatin biochemistry or architecture. Sex chromosomes provide an extreme example of coordinated regulation of linked genes. Many organisms have a single X chromosome in males and two in females. The X is rich in genetic information, and carries genes that are required for both sexes. By contrast, the Y often has little genetic information. The resulting imbalance in X- linked and autosomal genes that occurs in one sex is fatal to mammalian embryos and Drosophila larvae. Dosage compensation is a regulatory mechanism that has evolved to modulate expression of an X chromosome and restore balanced expression between X chromosomes and autosomes. Modulation occurs by directed chromatin modifications targeting the X chromosome. One of the fundamental questions in dosage compensation is how this regulation is directed at a chromosome-wide level. A novel class of large regulatory RNAs that exclusively coat the X chromosome in flies and mammals is central to this process. Here, we focus on recent advances in understanding the function of rox1 and rox2, non-coding RNAs that have central roles in directing dosage compensation of the X chromosome in flies. Corresponding author: Meller, V.H. (vmeller@biology.biosci.wayne.edu). Available online 4 August Large non-coding RNAs in mammalian dosage compensation Equalization of X-linked gene expression in mammals involves silencing one of the two X chromosomes in females. The large non-coding X inactive-specific transcript (Xist) RNA is central to this process. Xist is transcribed from the X inactivation center (Xic), an X-linked locus that counts the number of X chromosomes, chooses one to remain active and silences the inactive X chromosome [3]. Because silencing of both X chromosomes would be lethal, this process is restricted to chromatin located in cis to a single Xic allele. Knock-out mutations preventing Xist transcription render a chromosome constitutively active. Translocation of a Xic-containing segment of the X chromosome to an autosome (or autosomal insertion of Xist transgenes) can silence autosomal genes flanking the insertion site [4,5]. Thus, the Xic is sufficient to induce silencing on the chromosome that it is situated on. An early step in Xist-mediated silencing is the sequential recruitment of two Polycomb group complexes [6 8], which modify histones to repress transcription. Establishment of irreversible silencing, a process that involves DNA methylation, takes several more days [9]. Xist coats the inactive X throughout the life of females but is dispensable for the continued maintenance of silencing, revealing that X inactivation is epigenetically stable and self-perpetuating [10 12]. Redundant segments of Xist serve to direct the RNA to the X chromosome and modify chromatin, but a series of tandem repeats (Xist repeat A) that fold into short stem loops are necessary for silencing chromatin [6,13,14]. Dosage compensation in Drosophila Fruit flies must also address unequal X-chromosome dosage between males and females, with one and two X chromosomes, respectively. This is accomplished by a complex of proteins and RNA that binds to hundreds of sites along the male X chromosome (Figure 1a). Although there is general agreement that this complex is responsible for dosage compensation, disagreement about how compensation is accomplished remains. Two recent studies provide strong evidence that binding of this complex to the X chromosome of Drosophila males increases transcription from almost all X-linked genes [15,16]. Five protein components of this complex, collectively known as the Malespecific lethal (MSL) proteins [17], are: MSL1, MSL2, MSL3, Maleless (MLE) and Males absent on first (MOF) (Figure 1a). Mutation of any single msl results in male lethality, but none are essential in females. MSL1, an /$ see front matter ß 2006 Elsevier Ltd. All rights reserved. doi: /j.tibs

2 Review TRENDS in Biochemical Sciences Vol.31 No Figure 1. The MSL complex. (a) The MSL complex, comprising five proteins and rox RNA, binds to hundreds of sites along the male X chromosome and mediates dosage compensation. MSL1, MSL2, MSL3 and MOF interact directly with each other [18,60,62]. MLE and JIL-1 are enriched on the male X chromosome but are thought to have a more peripheral association and also bind to autosomal sites [27,56]. (b) rox1 functional domains. The line represents the rox1 gene. rox1 DNaseI hypersensitive site (DHS; white box) and rox box (white oval) are indicated. The rox1 transcript (arrow) contains 5 0 elements (gray box) and a 3 0 stem loop (black box). The region of rox1 RNA between these elements has no known function. Internal deletions and studies with mutated transgenes suggest that 5 0 elements are important for X localization but the 3 0 end might function in chromatin modification. acidic protein, can interact with MSL2, MSL3 and MOF through distinct domains [18]. MSL2 regulates assembly of the complex and is only expressed in males [17]. MSL3 carries a MRG [Mortality factor on chromosome 4 (MORF4)-related gene] domain necessary for X localization, and both MOF and MSL3 have chromo-barrel domains (CBDs), which can interact with nucleic acids in vitro [19,20]. The MSL3 CBD is necessary for transcriptional up-regulation [21]. Whereas MSL1, MSL2 and MSL3 are thought to provide structural and regulatory functions, MLE is an ATPase and RNA/DNA helicase, and MOF is a histone acetyltransferase [22 24]. MOF acetylates histone H4 on Lys16, a modification that precisely co-localizes with the MSL complex on the male X chromosome. When targeted to a promoter in a cell-free system, MOF can relieve the repression of chromatin on transcription [25]. The enzymatic activity and substrate specificity of MOF is dramatically improved by simultaneous contact with MSL1 and MSL3 [26]. A sixth protein in the complex, JIL-1 histone kinase, binds to chromatin throughout the genome but is enriched on the male X chromosome [27]. Although JIL-1 is essential in both sexes, hypomorphic JIL-1 alleles show more dramatic reductions in male viability [28]. Partial loss-offunction JIL-1 alleles reduce expression of a compensated white allele in males [29]. Loss of JIL-1 leads to ectopic spreading of heterochromatin proteins into euchromatic regions, an effect that is more pronounced on the X chromosome in both sexes [30]. This implicates JIL-1 in maintaining active chromatin, but also hints at a difference between chromatin of the X chromosome and autosomesthatdoesnotrelyonthepresenceofthemsl complex. Enrichment of H4 acetylated Lys16 (H4Ac16) is found throughout several X-linked genes regulated by the MSL complex, suggesting a potential role in enhancement of transcriptional elongation [31]. Genome-wide analyses of the MSL-binding pattern in Drosophila cell lines and embryos detected MSL proteins preferentially enriched in coding sequences and at the 3 0 end of most X-linked genes [32,33]. By contrast, the binding of MSL to promoters is rare. Although RNA polymerase II is found at most MSL1-binding genes, the binding profile of the two proteins is dissimilar because the polymerase is enriched in promoter regions [32]. These findings suggest that the MSL complex relies on transcription-coupled remodeling to establish the mature pattern of association with the X chromosome, a concept that has been incorporated into the following models of X-chromosome recognition. Non-coding RNAs direct localization of the MSL proteins The RNA components of the MSL complex are large noncoding RNAs from two X-linked genes, RNA on the X 1 and 2 (rox1 and rox2, respectively) [34,35]. Both rox RNAs assemble with the MSL proteins and co-localize to hundreds of sites on the male X chromosome [36] (Figure 1a). Incorporation of rox RNAs into the MSL complex depends on MLE activity, which might be necessary to integrate the rox RNAs and MSL proteins [36]. RNase treatment releases MLE from larval polytene chromosomes and MOF from the X chromosome in SL-2 cells, suggesting that these proteins interact with RNA in vivo [19,37]. rox1 and rox2 RNAs are crucial but functionally redundant for localization of the MSL proteins to the X chromosome [38]. Flies are perfectly normal with a single wild-type

3 528 Review TRENDS in Biochemical Sciences Vol.31 No.9 rox gene, but rox1 rox2 males display dramatically reduced viability and mislocalization of the MSL proteins at ectopic autosomal sites, the heterochromatic chromocenter and the fourth chromosome [38,39]. It is plausible that large regulatory RNAs, such as Xist and rox, arose as a mechanism to recruit proteins that are already present in chromatin-modification complexes and direct them to dosage compensation [40]. Despite the striking parallels between the roles of Xist and of the rox transcripts, there are notable differences. A rox transgene inserted on an autosome can restore male viability and X localization of the dosage-compensation proteins, indicating that unlike Xist rox RNAs can travel from their site of transcription to another chromosome [38]. The rox RNAs are non-coding RNAs that direct activation, rather than silencing, of target genes. How the rox RNAs ensure exclusive localization of the MSL complex to the X chromosome is a question of considerable interest. Recent studies of rox function, recruitment of the MSL complex and elucidation of the mature MSL-binding pattern on the X chromosome provide an entry point to examine this mystery. Structure function studies of rox The rox1 RNA is 3.7 kilobases (kb) and the major rox2 splice form is 500 nucleotides [34,35,41]. rox1 is strongly expressed 2 h after egg laying in both sexes but is lost from females midway through embryogenesis [42]. By contrast, rox2 is first expressed 6 h after egg laying and is strictly limited to males. Expression of rox1 and rox2 seems to be identical in male larvae and adults. Despite differences in embryonic expression and low sequence similarity, rox1 and rox2 RNAs are functionally redundant [38]. rox1 and rox2 contain two conserved DNA regions: a weakly conserved DNA sequence of 200 base pairs (bp) within each rox gene attracts the MSL proteins and forms a malespecific DNaseI hypersensitive site (DHS) [43,44] (Figure 1b). However, rox alleles and transgenes lacking this site have full function [39,45,46]. The rox DHS is not required for male-specific transcription of the endogenous rox genes, but it can function as an enhancer that drives transcription of transgenes or reporters in males and repression in females [46,47]. Although the ability of these sequences to attract the MSL proteins is striking, their functional role remains speculative. At the 3 0 end of rox1 and rox2 is a highly conserved 30-bp sequence termed the rox box [48] (Figure 1b). Deletion of this sequence does not reduce the activity of rox transgenes, and its function remains unclear [43,45]. The only discrete region of rox1 necessary for RNA function is 59 bases and near the 3 0 end that folds into a stem loop [45] (Figure 1b). rox1 transgenes with this region deleted display sharply reduced rescue of rox1 rox2 males despite substantial recruitment of the MSL proteins to the X chromosome. By contrast, rox1 transgenes carrying small deletions (300 bp) that remove any other portion of the transcript still rescue rox1 rox2 males [45]. Nevertheless, the 5 0 end of rox1 does contain elements that are necessary for RNA function, but these are thought to be redundant (Figure 1b). rox1 alleles with internal deletions display incremental reductions in activity as the 5 0 end is gradually lost [39]. Loss of 5 0 sequences has a particularly strong effect on MSL localization. Remarkably, an internal deletion of 2.35 kb that retains the 3 0 end and 0.9 kb of 5 0 sequence supports 100% male survival, indicating that most of the rox1 transcript is non-essential for RNA function (Figure 1b). Not only are both ends of rox1 necessary, but they must also be present in a single transcript [38].An attractive conclusion is that the essential feature of these RNAs is the ability to simultaneously bind to different molecules. It would be intriguing if the rox transcripts were found to have distinct domains for X localization and gene regulation, as does Xist. Although functional domains within rox2 RNA have not yet been identified, rox2 produces a multitude of alternative splice forms with reduced activity [41]. It is puzzling that so many splice forms with limited rox activity are produced, and tempting to speculate that the plethora of weakly functional rox2 molecules reveals a system for moderating the activity of the MSL proteins, thus, finetuning the level of X activation. Evolving models for MSL recruitment to the X chromosome Whereas mutation of msl1 or msl2 prevents all chromatin binding by the remaining MSL proteins, removal of any other MSL protein leads to binding of partial complexes to a limited number of high-affinity sites on the X chromosome [17]. The two sites that most avidly bind to the MSL proteins under these conditions are found within the rox1 and rox2 genes [49]. Not only do the rox genes recruit the MSL proteins, but they also enable the spreading of MSL complexes into the flanking chromatin from autosomal insertions of rox1 or rox2 [49]. These observations suggest that, like Xist, the rox genes are strong X-recognition elements (Figure 2a). However, rox RNA produced from an autosomal transgene can rescue a rox1 rox2 X chromosome, revealing that the location of rox genes on the X chromosome is non-essential [38]. It has also been proposed that the MSL complex initially binds to the 35 high-affinity sites from which it spreads into the flanking chromatin [49] (Figure 2b). This is challenged by recent studies that indicate all X-derived DNA fragments greater than 40 kb can recruit the MSL proteins to autosomal insertion sites, regardless of the presence or absence of a high-affinity MSL site in the fragment [50,51] (Figure 2c). Furthermore, X-to-autosome translocations or transpositions never induce spreading of the MSL complex into flanking autosomal regions (Figure 2c), even when the fragment from the X carries a rox gene. Autosomal fragments inserted on the X chromosome show no ectopic binding of the MSL complex [51]. Although this seems to counter the spreading of the MSL complex from rox transgenes, it has been noted that most translocation stocks have been maintained in culture for decades and rearrangements that permit spreading of the MSL complex into autosomal chromatin are likely to have been selected against [22]. Collectively, these observations indicate that the X chromosome is marked with a fine-grained distribution of recognition elements for MSL binding (Figure 2c). What, then, is the role of X-linked high-affinity sites and MSL spreading from the rox genes? Although they are

4 Review TRENDS in Biochemical Sciences Vol.31 No local concentrations of the MSL complex during establishment of compensation in early embryos. Most studies of MSL localization rely on immuno-staining of larval polytene chromosome preparations. These detect the mature pattern of MSL distribution long after the stage in which long-range elements might be most important. Co-transcriptional loading of MSL proteins Active transcription of a transgene inserted on the X chromosome can attract the MSL complex to a region that is normally free of detectable MSL proteins [54]. This suggests that some aspect of transcription, such as changes in chromatin structure, or transit of the transcriptional machinery, enables MSL binding. Genome-wide chromatin immunoprecipitation analyzed on gene chip (ChIP-chip) in Drosophila embryos and male cell lines reveals a mature MSL-binding pattern with almost all binding on the X chromosome and 90% within expressed genes [32,33,55] (Figure 3a). Highly expressed and essential genes are frequent MSL targets, but binding to intergenic regions and genes that are untranscribed or transcribed at Figure 2. Hypothetical models for X recognition by the MSL complex. (a) The rox genes might function as strong elements in cis to direct recognition. In this model, the MSL complex will spread from rox1 and rox2 to coat the entire X chromosome. (b) Several X-linked sites with high affinity for MSL binding might function in cis to mark the X chromosome and direct MSL complexes into the flanking chromatin. (c) Densely distributed recognition elements might be located throughout the X chromosome to enable recognition of translocated or transposed X-chromosome fragments by the MSL complex. MSL binding is indicated by red. Rearrangements involving X and autosomal sequence would not enable spreading of the MSL complex into the flanking autosomal regions, represented by the sharp boundary at translocation break-points (middle panel). Fragments of the X chromosome inserted on an autosome could recruit MSL proteins regardless of the presence or absence of a MSL high-affinity site (bottom panel). unnecessary for the identification of X chromatin, the presence of strong cis-acting elements on the X chromosome might facilitate establishment of MSL binding. Ectopic expression of MSL2 in females reveals a hierarchy of X-linked sites showing differential MSL-binding affinity [52]. Several newly characterized MSL-binding sites from the X chromosome are found to have a wide range of affinities for the MSL proteins [50,53]. Autosomal insertions of these sites rarely display secondary bands of MSL spreading into autosomal chromatin but, when spreading is observed, it relies on nearby autosomal sites with affinity for the MSL proteins. Collectively, these observations favor a model in which densely distributed sites with a range of MSL affinities work cooperatively to recruit the MSL complex to the X chromosome [24] (Figure 2c). Initial binding of the MSL complex to high-affinity sites might assist recognition of low-affinity sites nearby. In the absence of a strong site, numerous closely spaced, weak sites collaborate to recruit the MSL complex. Thus, recognition of the X chromosome would occur by integration of numerous densely clustered, strong and weak, X-linked signals that function at short and medium distances to cooperatively accumulate the MSL complex. Because both rox genes are situated on the X chromosome and are capable of directing long-distance spreading of the MSL complex, the rox genes can facilitate this process by raising Figure 3. A speculative model for co-transcriptional loading of the MSL complex. (a) Mature pattern of the MSL complex binding to a section of the X chromosome. Binding is biased towards the 3 0 end of coding sequences, illustrated by color intensity. An active gene (black arrow) and formally active gene (gray arrow) retain MSL binding, but an inactive gene (gray box without arrow) is less likely to attract MSL binding. The red vertical bar indicates a hypothetical X-recognition element between genes. (b) Model for co-transcriptional spreading of the MSL complex from a rox gene (red and black stripes). Genes bound by the MSL complex are shown in red. An inactive gene not bound by the MSL complex is shown in gray. Upon activation of transcription, the promoter is recruited to a compartment near the nuclear membrane (arrow shows movement into the orange compartment near the nuclear membrane). (c) In this compartment, we hypothesize that enrichment for MSL proteins and rox RNA promotes co-transcriptional loading of the MSL complex. Genes closely linked to rox are likely to occupy the same compartment upon activation and, thus, will be exposed to high levels of the MSL complex during transcription. This leads to the appearance of linear spreading along a chromosome from a source of rox transcripts.

5 530 Review TRENDS in Biochemical Sciences Vol.31 No.9 low levels is rare [32,33,55]. MSL target genes in male salivary glands are a subset of those in embryos, suggesting that a pattern established at an early stage is maintained throughout development [55]. The mature MSL-binding pattern is not invariant; subtle changes of MSL binding that correlate with transcription have been observed between different cell lines and different tissues, suggesting dynamic MSL recruitment [33,54]. Transcription alone is not sufficient to recruit the MSL complex. Some active X-linked genes are not bound by the MSL proteins, and a few visible gaps in MSL binding to polytene chromosomes contain puffed regions with strong RNA polymerase II binding [32,52,55,56]. All ChIP-chip assays have identified MSL-binding sites in regions lacking annotated genes; these might represent cis-acting DNA elements. It is plausible that the mature pattern of MSL binding on the X chromosome is achieved by the combinatorial effect of cis-acting DNA elements and co-transcriptional loading of the MSL complex (Figure 3b,c). An intriguing study of MOF and MSL3 in Drosophila embryos and cells has identified an association between components of the nuclear pore complex and the MSL complex [57]. Surprisingly, RNAi depletion of these nuclear pore components reduces MSL complex bound to the X chromosome and down-regulates several dosagecompensated genes. Interactions between nuclear pore proteins and promoters are observed in yeast, which suggests involvement of the pore in transcription [58]. This raises the possibility that the MSL complex gains access to transcribed regions when activated genes move to a particular subnuclear domain, such as the nuclear pore, during transcription (Figure 3b,c). Recruitment of linked genes to a common site upon activation might account for the odd ability of the MSL complex to spread discontinuously, but a great distance from, autosomal insertions of rox (Figure 3c). The role of rox RNA in targeting In the absence of rox RNAs, the MSL proteins still colocalize, but in an abnormal pattern (Figure 4a). Low levels of H4Ac16 are detected at these sites of MSL binding [59] (X. Deng and V.H. Meller, unpublished). This suggests that a protein only MSL complex has weak acetyltransferase activity (MOF; Figure 4a). How do rox RNAs direct targeting the MSL proteins to the X chromosome? rox transcripts might provide a scaffold for arrangement of the MSL proteins, thus, changing the conformation of the complex (Figure 4b). A rearranged complex might have increased affinity for the cis-acting DNA elements on the X chromosome and higher MOF activity (Figure 4b). Rearrangement might also change binding properties to facilitate X recognition. For example, if a MSL complex containing rox has enhanced binding cooperativity, it would preferentially accumulate on densely distributed DNA elements on the X chromosome at the expense of scattered DNA elements on autosomes (Figure 4b). The role of rox RNAs might be to recruit MLE to the MSL complex [42] (Figure 3c). MLE does not interact directly with the other MSL proteins and MLE association with the X chromosome is RNase sensitive [37,60]. It has Figure 4. Model of rox RNA-directed MSL localization. (a) In the absence of rox RNA, the MSL proteins bind to numerous DNA elements with a range of affinities for the MSL complex, which is represented by the colour intensity of the vertical bars, on the X chromosome and autosomes (A). The X chromosome has densely spaced DNA elements whereas the autosomes have scattered DNA elements. The acetyltransferase activity of MOF in the protein-only complex is low (pink). The single X chromosome is not effectively dosage compensated. (b) rox RNAs provide scaffolds for the arrangement of MSL proteins and change complex conformation and/or activity. This might increase the enzymatic activity of MOF (now in red). If the MSL complex containing rox has enhanced cooperative binding, the densely spaced DNA elements located on the X chromosome will be favored at the expense of scattered autosomal DNA elements. MSL proteins will become exclusively X-linked and MOF enzymatic activity might also be enhanced, leading to effective compensation. been proposed that the MLE ATPase and/or helicase could mediate the specific interaction between the MSL rox complex and DNA or RNA of the X chromosome [61]. Finally, rox RNAs might direct proper targeting of the MSL complex by interacting with as yet unidentified proteins. The observation that nuclear pore proteins are required for MSL X localization in cultured cells raises the possibility that the rox RNAs facilitate the recruitment of transcribed genes to a region where increased expression is possible, or where co-transcriptional loading of the MSL complex can occur. Concluding remarks rox1 and rox2 are members of an intriguing group of noncoding RNAs that are implicated in chromatin modifications. These transcripts have powerful and far-reaching roles in the epigenetic establishment of correct genome expression. Further analysis of rox RNA functional domains and elucidation of the structure of the MSL complex will reveal the protein RNA interactions that produce the mature MSL complex. Identification of X-chromosome sequence elements that recruit the MSL complex will be necessary to understand selective recognition of the X chromosome. The rox RNAs provide an opportunity to explore the mechanism of RNA-directed chromatin regulation in a model organism in which many of the interacting proteins are already known.

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