InteractiveFly: GeneBrief

RNA on the X 2: Biological Overview | References

BIOLOGICAL OVERVIEW

Dosage compensation in Drosophila involves a global activation of genes on the male X chromosome. The activating complex (MSL-DCC) consists of male-specific-lethal (MSL) proteins and two long, noncoding roX RNAs. The roX RNAs are essential for X-chromosomal targeting, but their contributions to MSL-DCC structure and function are enigmatic. Conceivably, the RNA helicase MLE, itself an MSL subunit, is actively involved in incorporating roX into functional DCC. This study determined the secondary structure of roX2 and mapped specific interaction sites for MLE in vitro. Upon addition of ATP, MLE disrupted a functionally important stem loop in roX2. This RNA remodeling enhanced specific ATP-dependent association of MSL2, the core subunit of the MSL-DCC, providing a link between roX and MSL subunits. Probing the conformation of roX in vivo revealed a remodeled stem loop in chromatin-bound roX2. The active remodeling of a stable secondary structure by MLE may constitute a rate-limiting step for MSL-DCC assembly (Maenner, 2013).

Since the discovery of Xist RNA as a crucial epigenetic regulator involved in mammalian X inactivation about 20 years ago, an increasing number of long, noncoding (lnc) RNAs have been found associated with chromatin-modifying complexes (Lee, 2012 and Moran, 2012). Their hypothetical functions include roles as versatile scaffolds for protein complexes and in targeting associated regulators to genomic loci, either as nascent RNA or through R-loop or triple helix formation. Considering the conformational flexibility and structural diversity of RNA, its regulatory potential is enormous. However, a detailed mechanism of action has only been resolved in a few cases (Kugel, 2012; Maenner, 2013 and references therein).

The highly elaborate dosage compensation systems that evolved to compensate for sex chromosome monosomy provide instructive examples for regulatory functions of lnc RNAs. The inactivation of one X chromosome in female mammals is orchestrated by the interplay of at least three lnc RNAs. In Drosophila melanogaster, dosage compensation involves doubling the transcription of many X-linked genes. This activation is achieved by a ribonucleoprotein dosage compensation complex (DCC) consisting of five male-specific-lethal (MSL) proteins and two lnc RNAs, roX1 and roX2 (for 'RNA-on-the-X'). These RNAs are considered scaffolds for the proper assembly of the MSL proteins. The MSL-DCC only forms in male flies due to the male-specific expression of two key components: the core subunit MSL2 and the roX RNAs. The remaining protein subunits-MSL1, MSL3, the acetyltransferase MOF (Males-absent-on-the-first), and the RNA helicase MLE (Maleless)-are also expressed in female cells and have additional functions outside of the MSL-DCC (Maenner, 2013).

Genome-wide mapping of the chromosomal interactions revealed that most of the MSL-DCC interacts with the bodies of actively transcribed genes on the X chromosome. According to a popular model, the X-specific targeting of the MSL-DCC involves an initial recognition of a limited number of 'chromosomal entry' or 'high-affinity' sites ('CES' or 'HAS,' respectively), which are distributed along the X. Accordingly, the complex transfers from these sites to active genes in the nuclear vicinity. Once tethered to the active chromatin, the MSL-DCC activates transcription through acetylation of lysine 16 of histone H4 (H4K16ac) by MOF, which is thought to unfold the chromatin fiber and facilitate the production of mature mRNAs (Maenner, 2013).

RoX1 and roX2 RNAs differ greatly in size (3.7 kb versus 600 nt) and show very little sequence similarity. Despite these differences, each alone can support the assembly of a functional MSL-DCC. Inactivation of both roX genes abolishes dosage compensation and is lethal for males. Exploring the common denominator between the two RNAs by phylogenetic comparison and functional analyses, Kuroda, Park, and colleagues discovered a series of conserved sequence motifs (GUUNUNCG), the 'roX-boxes,' which reside in the 3' ends of the roX RNAs (Kelley, 2008; Park, 2007; Park, 2008). In silico analysis of structural motifs predicts that prominent roX-boxes participate in the formation of stable stem-loop structures (SLroX). The integrity of SLroX structure is essential for roX function. Furthermore, expressing an RNA consisting solely of six tandem repeats of SLroX2 suffices to recruit the MSL proteins to the X chromosome and acetylate H4K16 on the X in the absence of endogenous roX RNA. These data highlight the importance of the SL for roX RNA function, but the underlying molecular mechanism remains unclear. Although four of the five MSL proteins are able to bind RNA in vitro, so far no specific interaction of any protein with SLroX has been demonstrated (Maenner, 2013).

Several lines of evidence suggest a primary role for the RNA helicase MLE in incorporating roX into the MSL-DCC. RoX1 is initially expressed in preblastoderm embryos of both sexes, where it is stabilized by maternal MLE until dosage compensation is established. At later developmental stages, MLE is required for steady-state assembly and targeting the MSL-DCC to the X chromosome. Inactivating the ATPase activity of MLE by point mutation (MLE^GET) impairs X-chromosomal targeting of the DCC. A mutation that selectively inactivates the helicase function greatly limits the spreading of the MSL-DCC onto the active gene bodies (Maenner, 2013).

It is unclear where in the nucleus roX RNAs and MSL proteins assemble to form a functional DCC. This may happen at the site of roX gene transcription. Another possibility is that HASs are sites of MSL-DCC formation. It was recently found in a genome-wide study of chromosomal interactions that MLE strongly enriched at some 240 HAS sites on the X, in close proximity to MSL2 (Straub, 2013). Since the specific chromatin immunoprecipitation (ChIP) methodology applied in this study emphasizes direct chromatin interactions, it was consider that MLE may affect the assembly or reorganization of the MSL-DCC at HAS. In line with this argument, the recent ChIRP (chromatin isolation by RNA purification) profiling of chromatin interactions also found roX2 RNA enriched at HAS (Maenner, 2013).

The current study suggests that the assembly of the ribonucleoprotein MSL-DCC is initiated by an obligatory, energy-consuming RNA remodeling step, during which MLE recognizes the SLroX structure and converts it into a more extended form. Accordingly, the stable, low-energy SL conformation that roX2 adopts in vitro represents an inactive, closed conformation, which needs to be actively opened to expose binding sites for MSL proteins (Maenner, 2013).

This study has found that the specific interaction of MSL2, the key identifier of the MSL dosage compensation complex, with roX RNA depends on the prior remodeling of a prominent stem-loop structure in the 3' end of roX2. This hairpin, together with a related structure in roX1, is indispensable for roX function in flies. Previous failures to detect specific MSL interactions with SLroX2 are now at least in part explained by the observation that SLroX2 itself does not serve as a binding site but has to be converted into an alternative, open conformation through the ATP-dependent remodeling action of MLE. The data describe a function for a nuclear helicase in lncRNA conformation. The occurrence and functional importance of the proposed conformational switch during organismal development remains to be tested in future studies (Maenner, 2013).

A role for MLE in resolving long dsRNA stems was already suggested more than a decade ago (Reenan, 2000). A mutation in MLE, the mle^napts allele, causes the failure to express the para-encoded Na+ channel. Para transcripts are subject to consecutive RNA editing and splicing. Adenosine-to-inosine editing is guided by the formation of a stable dsRNA secondary structure that needs to be resolved for subsequent splicing. The mutated helicase binds to and stabilizes the stem structure rather than unwinding it, as it would normally do to allow access of the splicing machinery (Reenan, 2000). Interestingly, the ds editing structure of the para transcript also contains two A-U motifs in the vicinity of the edited sequence, which resemble the roX-box motif. It is suggested that MLE functions more generally to unwind long base-paired RNA structures to expose ssRNA sequences for downstream interactions (Maenner, 2013).

The data show that MLE preferentially binds to RNA containing an SLroX even in the absence of ATP and that this interaction is improved by increasing the stability of the SL. In the presence of ATP, the specificity of interaction with this particular binding site is reinforced. Because the MLE^GET mutant that binds nucleotides with reduced affinity does not show the effect, it is assumed that the ATP-dependent disruption of the SL strengthens the MLE interaction with roX. The SLroX structures share a conserved sequence, the roX-box, which contributes to MLE affinity. The four consecutive A-U pairs may facilitate the unwinding of the stem and/or provide a binding site for MLE and/or other MSL subunits in their single-stranded conformation (Maenner, 2013).

It is hypothesized that RNA remodeling by MLE physically connects the two male-specific principles that are key to the formation of the MSL-DCC: the male-specific MSL2 protein and the roX RNAs. Numerous observations already suggested a close interdependency of their functions. In the absence of MSL2, roX RNAs are unstable and, conversely, any MSL2 that is not in a complex with roX will be degraded (Lim, 2012 and Meller, 2000). The enrichment of MLE on the X chromosome depends on MSL2 and MSL1. MSL2 may physically connect the MLE-roX module to the MSL1-MSL3-MOF module (Hallacli, 2012 and Morales, 2004). If MSL1 is mutated to abolish the MSL2 interaction, roX RNAs are no longer incorporated into the MSL-DCC (Hallacli, 2012). It is currently unclear whether MLE would remain part of a stable MSL-DCC complex once it has remodeled the roX RNA. Earlier findings that MLE readily dissociates during purification of MSL complexes indicate that MLE was rather loosely bound. The helicase may only fulfill a transient function during the biogenesis of the complex. The striking enrichment of MLE at 240 HAS on the X chromosome in direct neighborhood of MSL2 identifies these loci as primary sites of MLE action (Straub, 2013) (Maenner, 2013).

In summary, it is proposed that the active remodeling of roX RNA by the ubiquitous RNA helicase MLE constitutes an obligatory, regulated step in the biogenesis of the MSL-dosage compensation complex. This study paves the way for a mechanistic dissection of this fundamental conformational switch. Lessons learned from the dosage compensation system will undoubtedly enhance the understanding of other lncRNA-containing chromatin regulators (Maenner, 2013).

Tandem stem-loops in roX RNAs act together to mediate X chromosome dosage compensation in Drosophila

Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. By using UV crosslinking followed by deep sequencing, this study shows that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem-loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, different roX RNA domains were shown to have overlapping function, since only combinatorial mutations in the tandem stem-loops result in severe loss of dosage compensation and consequently male-specific lethality. It is proposed that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes (Ilik, 2014).

Identification of functional domains in lncRNAs is an important step toward understanding how they work in vivo. This study characterizes roX1 and roX2 as the most significant RNAs associated with MLE and MSL2. roX1 and roX2 contain common, conserved, and distinct structural domains, which form the binding platform for these proteins. Interestingly, regions of lncRNAs that lie outside of these MLE/MSL2 interaction domains appear to be unstructured and not conserved. These data also provide evidence on how roX1 and roX2 RNAs can be functionally redundant by showing that MLE-MSL2-interacting regions are present in multiple copies in both RNAs (Ilik, 2014).

Earlier work on roX RNAs has identified short stretches of RNA that are shared between these lncRNAs that are conserved throughout evolution, called the roX boxes. This study shows that all three roX boxes at the 3' end of roX2 RNA and one of the three roX boxes in roX1 form stable helices in vitro. Moreover, these elements represent binding sites for MLE and MSL2 proteins in vivo. Detailed analysis of MLE individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) data revealed that there are other RNA elements in both roX1 and roX2 that resemble the roX box sequence, and also serve as binding sites for MLE. These elements were named roX box-like sequences. Together roX box and roX box-like sequences uncover a consensus binding site for MLE in roX RNAs. No other RNAs were identified in the Drosophila transcriptome that contained the roX box/roX box-like motif other than the roX RNAs, adding to the evidence that it is likely that there are no more roX-like RNAs that can function in Drosophila dosage compensation (Ilik, 2014).

The results expose the logic of a roX RNA: stem-loops containing RBL elements at the 5' end and roX boxe (RB) containing helical structures joined by a flexible, single-stranded spacer region. In roX2, these elements are repeated on a very small scale with perfect copies of RBL/RB elements. In contrast, in roX1, these sequence motifs deviate from the ideal consensus more than roX2, but roX1 probably compensates for this by containing multiple, autonomous interaction domains that form a much larger RNA, which is about six times as big as roX2 (Ilik, 2014).

Endogenously expressed MLE and affinity-tagged MLE proteins have the ability to differentiate between the two stem-loop clusters in the two halves of roX2 exon-3 (1-280 versus 281-504). Interestingly, addition of ATP led to the specific interaction of endogenously expressed MLE with the second stem-loop cluster of roX2 exon-3 that contains roX box-containing helical structures. Furthermore, it was observed that the N-terminal dsRBDs are not only required for the ATP-independent interactions with the 5' end of roX2 exon-3 but are also required for the ATP-dependent interactions of MLE with the 3' end of roX2 exon-3. A similar dichotomy was observed in High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) and iCLIP experiments. In HITS-CLIP, the reads accumulated around the first stem-loop cluster of roX2 exon-3, concentrating on R2H1, whereas in iCLIP the balance was shifted toward the second cluster (Ilik, 2014).

MLE is a member of the RHA/DEAH family of RNA helicases, which can remodel RNA and RNPs. Interestingly, biochemical work on the closely related DEAD box RNA helicases shows that these enzymes have varying degrees of affinity toward their RNA substrates during their ATPase cycles. Since the GRNA chromatography experiments reveal that the second half of roX2 gains MLE binding when supplemented with ATP in vitro, it is possible that the quaternary complex between MLE, roX2, and ADP-Pi is being scored for in these experiments, and enzyme is being caught on its way to eventually remodeling of this part of the RNA (Ilik, 2014).

The ATP-independent binding of MLE to the first half of roX2 RNA via its N-terminal dsRBDs could be an initial regulatory step as roX RNAs constitute important tethers for MLE within the MSL complex. However, the in vivo analysis revealed that at high expression levels this cluster 'A' is dispensable for dosage compensation. The second hairpin cluster appears to recruit MLE through the helicase domain in a way that still requires N-terminal dsRBDs, thus providing a dynamic platform for rerecruitment and spreading of MLE along the roX RNA. The function of this cluster 'B' can only be partially compensated by higher expression, suggesting that it is functionally distinct from 'A.' It is proposed that the dynamic interaction of MLE with roX RNAs may ensure that different regions of roX RNAs are exposed such that they can be used in a redundant or cooperative manner for the interaction with the MSL complex members providing plasticity, as clearly detected for MSL2 binding on roX RNAs in the iCLIP data in vivo. However, it can be excluded that integration of roX RNAs with the 'core' MSL complex may also follow a more direct route involving independent MSL-RNA interactions that need not require MLE as a mediator (Ilik, 2014).

roX genes have a dual function in Drosophila dosage compensation: they are sites of roX transcription, but they also contain two high-affinity sites (HASs) that can recruit the MSL complex independent of the roX RNAs (Kelley, 1999). Moreover, roX RNAs can travel from their sites of synthesis to the X chromosome when placed as a transgene to an autosomal site. This suggests that the holo- MSL complex, containing the core components (MSL1-3 and MOF) and MLE together with roX RNAs, can form on chromatin at roX transcription sites and spread in cis on the X chromosome, but it may also form in solution and be targeted to X-chromosomal sites in trans. It has been recently shown that MSL2 interacts with a dimer of MSL1 and is itself present as a dimer within the MSL complex (Hallacli, 2012). Interestingly, this study found that roX RNAs do not interact with each other in vivo, suggesting that there could be one roX RNA species per holo-MSL complex (Ilik, 2014).

Notably, iCLIP methodology utilizing UV crosslinking provides a snapshot of a pool of interactions that are present at the instant of irradiation. Although iCLIP data clearly show that MLE and MSL2 bind to the same domains, it is possible that they occupy different stem-loops on different molecules of roX RNAs rather than occupying the same structure at the same time. roX RNAs, by evolving multiple interaction platforms, can indeed support such combinatorial binding events, thus facilitating spreading along the X chromosome. It is proposed that roX RNAs, by virtue of being able to interact with MLE and the MSL complex, play a central role in the assembly of the holo- MSL complex containing the 'core' (MSL1, MSL2, MSL3, MOF) and MLE. These complexes may form in solution or on chromatin such as on sites of roX transcription. Such configuration thus brings different enzymatic activities together (ATPase/ helicase activity of MLE; acetyltransferase activity of MOF and ubiquitin-ligase activity of MSL2) for the X chromosome-specific dosage compensation process. Directing the assembly of such a complex and by providing plasticity through its multiple stem-loops, roX RNAs could thus facilitate local spreading of the MSL complex from HAS into neighboring chromatin including low-affinity sites (LASs) (Ilik, 2014).

Domains identified in roX1 and roX2 could be defined as 'information units' within these lncRNAs, which organizationally resemble stem-loops strung together like 'beads on a string'. It is tempting to hypothesize that one could compare the 'information unit' within roX lncRNAs to be the counterpart of codons which provide the basic information units in mRNAs. For roX lncRNAs, the information unit is a bit larger (11-15 nt) and involves both primary sequence and secondary structure. Another important aspect is that the domains are repeated in the lncRNA but do not appear to have a strict requirement of spacing or order relative to each other (i.e., no polarity and reading frame), so functional versions may be relatively easy to evolve during evolution (Ilik, 2014).

In summary, these data suggest that lncRNAs may utilize discrete repetitive motifs for distinct protein-RNA or even RNADNA interactions to achieve functional specificity in vivo. Such a mechanism may also be used for other global epigenetic phenomenon such as the X-inactivation in mammalian cells. Future analysis of other lncRNAs that combine CLIP methods and RNA structural analysis will be crucial in identifying structural domains within these RNAs and understanding their precise roles in the regulation of gene expression (Ilik, 2014).

RNA-on-X 1 and 2 in Drosophila melanogaster fulfill separate functions in dosage compensation

In Drosophila melanogaster, the male-specific lethal (MSL) complex plays a key role in dosage compensation by stimulating expression of male X-chromosome genes. It consists of MSL proteins and two long noncoding RNAs, roX1 and roX2, that are required for spreading of the complex on the chromosome and are redundant in the sense that loss of either does not affect male viability. However, despite rapid evolution, both roX species are present in diverse Drosophilidae species, raising doubts about their full functional redundancy. Thus, this study investigated consequences of deleting roX1 and/or roX2 to probe their specific roles and redundancies in D. melanogaster. Anew mutant allele of roX2 was created, and roX1 and roX2 were shown to have partly separable functions in dosage compensation. In larvae, roX1 is the most abundant variant and the only variant present in the MSL complex when the complex is transmitted (physically associated with the X-chromosome) in mitosis. Loss of roX1 results in reduced expression of the genes on the X-chromosome, while loss of roX2 leads to MSL-independent upregulation of genes with male-biased testis-specific transcription. In roX1 roX2 mutant, gene expression is strongly reduced in a manner that is not related to proximity to high-affinity sites. These results suggest that high tolerance of mis-expression of the X-chromosome has evolved. It is proposed that this may be a common property of sex-chromosomes, that dosage compensation is a stochastic process and its precision for each individual gene is regulated by the density of high-affinity sites in the locus (Kim, 2018).

The dosage compensation machinery involving rox1 and rox2 RNAs provides a valuable model system for studying the evolution of lncRNA-genome interactions, chromosome-specific targeting and gene redundancy. LncRNAs differ from protein coding genes and are often less conserved at the level of primary sequence, as expected due to their lack of protein-coding restrictions. Like those encoding other lncRNAs, rapid evolution, i.e., low conservation of the primary sequences of roX genes has complicated comparative studies. Despite their differences in length and primary sequences, rox1 and rox2 have also been considered functionally redundant in Drosophila melanogaster. However, remarkably considering their rapid evolution and apparent redundancy, orthologs for both rox1 and rox2 have been found in all of 26 species within the Drosophila genus with available whole genome assemblies. Models that explain evolutionarily stable redundancy have been proposed suggesting that the presence of both rox1 and rox2 in these diverged species may be attributable to differences in targets, affinities and/or efficiency or additional functions (Kim, 2018).

On polytene chromosomes, binding patterns of rox1 and rox2 are more or less indistinguishable, except in region 10C where rox2 is almost exclusively present. In the rox2 mutant, genes located in the 10C bin are on average downregulated, but similar downregulation of genes in many other bins is observed, so the effect cannot be directly attributed to loss of rox2. In wildtype 1st instar larvae, levels of rox1 RNA are much higher than levels of rox2 RNA. Interestingly, in rox1 mutant larvae the absolute amount of rox2 RNA increases, but only to ~10% of wildtype levels of total roX RNA. This appears sufficient to avoid lethality, but still causes a significant decrease in X-chromosome expression. However, despite the huge difference in amounts, not only in number but even more considering the size of the two roX RNAs, the staining intensities of roX RNA on rox1 mutant and wildtype polytene chromosomes seem to be roughly equal. On mitotic chromosomes rox1 RNA is only observed in the MSL complexes bound to the distal X-chromosome and this binding is not redundant. This indicates that just after cell division rox1 RNA will be the dominating variant in assembled MSL complexes. Taken together, the results suggest that rox2 RNA has higher affinity than rox1 RNA for inclusion in MSL complexes. Moreover, varying amounts of the two species with different affinities at given cell cycle stages may support proper transmission, spreading of assembled MSL complexes and maintenance of appropriate levels of the complexes (Kim, 2018).

It should be noted that some male rox1 rox2 mutants escaped, so loss of roX is not completely male-lethal, unlike loss of mle, msl1, msl2, msl3 or mof. The complete male lethality in these mutants is attributed to reductions in dosage compensation that have been measured in several studies and observed not only in msl mutants but also following RNAi-mediated depletion of MSL proteins. Notably, the average reduction of X-chromosome expression, relative to wildtype levels, calculated in these cases has varied from ca. 20 to 30%; substantially less than the 35% reduction observed in the rox1 rox2 mutant. Some of the reported differences may be due to use of different techniques and bioinformatics procedures (including use of different cut-offs for expression and developmental stages). However, the reasons why some males can survive the very dramatic imbalance observed in expression of a large portion of the genome are unclear. Furthermore, the reduction in expression of X-chromosome genes observed in the rox1 mutant is not accompanied by any reported phenotypic changes, indicating that D. melanogaster has high intrinsic ability to cope with significant imbalances in X-chromosome expression. It is speculated that in parallel with a compensation mechanism that addresses dosage imbalances the fly has evolved a high degree of tolerance to mis-expression of the X-chromosome (Kim, 2018).

Evolutionary studies have shown that sex chromosomes do not always represent terminal stages in evolution-in fact, the 4th chromosome was ancestrally an X-chromosome that reverted to an autosome. Moreover, the fly shows high and unusual tolerance to dosage differences and mis-expression of the 4th chromosome (although much smaller than the tolerance to those of the X-chromosome). These observations suggest that tolerance of mis-expression is a common outcome in the evolution of sex-chromosomes and this property has been retained with respect to the 4th chromosome, even after its reversion to an autosome. It is proposed that high tolerance of mis-expression in the absence of full functional dosage compensation may be selected for during evolution of sex-chromosomes. This is because gradual degeneration of the proto-Y chromosome will be accompanied by an increasing requirement to equalize gene expression between a single X- (in males) and two X-chromosomes (in females), but changes in genomic location of highly sensitive genes will be favored during periods of incomplete (or shifting) dosage compensation. On transcript level, responses to reductions in dosages of X-chromosome genes have been found to be similar to those of autosomal genes. Thus, potential mechanisms for the higher tolerance are post-transcriptional compensatory mechanisms or selective alterations in gene composition (changes in genomic locations), similar to those proposed for the observed demasculinization of the Drosophila X-chromosome (Kim, 2018).

Prompted by the strong relationship between orchestration of the X- and 4th chromosomes by the MSL complex and POF system, respectively, this study also measured effects of roX suppression on chromosome 4 expression in roX mutants. Weak but significant reduction of expression were observed in the rox2 mutant, but the cause of this reduction remains elusive. In rox2 mutant transcriptional upregulation of X-chromosome genes classified as having low expression levels was also oberved, late replication and weak MSL complex-binding. The loss of rox2 resulting in MSL complexes only including rox1 RNA might alter the spreading properties. It is therefore hypothesized that the observed upregulation might be caused by mis-targeting of the MSL complex in the absence of rox2. However, the ChIP experiment revealed no enrichment of MSL complexes on these genes, and results rather suggest that rox2 directly or indirectly restricts expression of these male-biased genes independently of its role in the MSL complex (Kim, 2018).

It is well known that roX RNAs are important for spreading of the MSL complex in regions between high affinity sites (HAS). It is therefore surprising that loss of roX causes a relatively even reduction in expression of X-chromosomal genes and the decrease is not more dramatic with larger distances, as would be expected for reductions in spreading capacity. Indeed, observed reductions in expression were smaller for genes located far from HAS than for closer genes. A possible explanation is that expression of these genes is compensated by an MSL-independent mechanism. It has been previously shown that most genes on the X-chromosome are dosage-compensated, but a subset are not bound by the MSL complex and do not respond to its depletion. The results corroborate these findings since loss of roX RNA in the rox1 rox2 mutant had little effect on the expression of genes classified as having weak MSL complex binding, clearly indicating that at least one other mechanism is involved. The results further show that high-affinity sites, as defined by MSL-targets in the absence of rox1 and rox2, are highly correlated to genes with the highest MSL binding levels. Therefore, sites targeted in the absence of roX provide a more stringent definition of HAS, with stronger correlation to genes bound by high levels of MSL complex, than targets in the absence of mle, mof or msl3 (Kim, 2018).

The increase in expression mediated by the MSL complex is considered a feed-forward mode of regulation, and appears to be more or less equal (ca. 35%) for all MSL-bound genes. Evidently, highly expressed genes need a stronger increase in transcription than weakly-expressed genes. These results suggest that dosage compensation is a stochastic process that depends on HAS distribution and is correlated with expression levels. Evolutionary analysis has shown that newly formed X-chromosomes acquire HAS, putatively via rewiring of the MSL complex by transposable elements and fine-tuning of its regulatory potential. Such a dynamic process may be required for constant adaptation of the system. Highly expressed genes tend to accumulate HAS in their introns and 3'UTRs, and thus bind relatively high amounts of MSL complex, thereby stimulating the required increase in expression. This also implies that the gene organization on X-chromosomes is under more constraints than autosomes (Kim, 2018).

This study presents the first high-throughput sequencing data and analysis of transcriptomes of rox1, rox2 and rox1 rox2 mutant flies. The results reveal that rox1 and rox2 fulfill separable functions in dosage compensation in D. melanogaster. The two RNA species differ in both transcription level and cell-cycle regulation (Kim, 2018).

In third instar larvae, rox1 is the more abundant variant and the variant that is included in MSL complexes transmitted physically associated with the X-chromosome in mitosis. Loss of rox1, but not loss of rox2, results in decreased expression of genes on the X-chromosome, albeit without apparent phenotypic consequences. Loss of both roX species leads to a dramatic reduction of X-chromosome expression, but not complete male lethality. Taken together, these findings suggest that high tolerance for mis-expression of X-chromosome genes has evolved. It is speculated that it evolved in parallel with dosage compensation mechanisms and that it may be a common property of current and ancient sex-chromosomes (Kim, 2018).

The roX RNAs are important for spreading of the MSL-complex from HAS, but the reduction of X-chromosome expression in rox1 rox2 mutant is not affected by the need for spreading, i.e., distance from HAS. In addition, the genes targeted by the MSL complex in the rox1 rox2 mutant also show strongly reduced expression. The results suggest that the function of the MSL complex which is still present at HAS is compromised in the rox1 rox2 mutant and that the dosage of distant genes is compensated by an alternative, unknown, mechanism. It is proposed that dosage compensation is a stochastic process that depends on HAS distribution. Creation and fine-tuning of binding sites is a dynamic process that is required for constant adaptation of the system. Highly expressed genes will accumulate and be selected for strong HAS (and thus bind more MSL complex) since they require high levels of bound MSL complex for the required increases in expression (Kim, 2018).

Structure of the RNA helicase MLE reveals the molecular mechanisms for uridine specificity and RNA-ATP coupling

The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. This study identified a stable MLE core comprising the DExH helicase module and two auxiliary domains: a dsRBD and an OB-like fold. MLE_core is an unusual DExH helicase that can unwind blunt-ended RNA duplexes and has specificity for uridine nucleotides. The 2.1 Å resolution structure of MLE_core bound to a U10 RNA and ADP-AlF₄ was determined. The OB-like and dsRBD folds bind the DExH module and contribute to form the entrance of the helicase channel. Four uridine nucleotides engage in base-specific interactions, rationalizing the conservation of uridine-rich sequences in critical roX substrates. roX2 binding is orchestrated by MLE's auxiliary domains, which is prerequisite for MLE localization to the male X chromosome. The structure visualizes a transition-state mimic of the reaction and suggests how eukaryotic DEAH/RHA helicases couple ATP hydrolysis to RNA translocation (Prabu, 2015).

It is concluded that the roX RNAs are a paradigm for how lncRNAs function to assemble ribonucleoprotein complexes. The results provide a structural underpinning to the observation of interdependence between MLE and roX RNA for X chromosome targeting and lay the foundation for a future mechanistic analysis of roX RNA remodeling by MLE. It is envisioned that MLE may load on the helical regions of the roX RNAs in an ATP-independent manner via the exposed surface of dsRBD2 and then thread the 3' end of the RNA into the helicase core, rationalizing why the roX boxes are found near the 3' end of the roX RNAs. The finding that MLE also binds uridine-rich sequences at the 5' end of the roX RNAs might be due to the accumulation of the helicase as it progresses from the 3' to the 5' end of the transcript or due to direct loading by a partial opening of the DExH ring. In addition to the canonical helicase interactions with the sugar-phosphate backbone of the RNA, the OB-like fold of MLE can recognize uridine-rich sequences by base-specific interactions. The pattern of uridine-specific interactions observed in the structure (UxUUU) does not exactly match the sequence motif of the roX boxes (UUUU). Imperfectly matching pockets might tune RNA binding strength and the efficiency of translocation, which are inversely correlated. Along these lines, slowed unwinding as MLE interacts with the uridine-rich sequences could provide a window of opportunity for recruiting MSL proteins near the base of helicase, where the 3' end of the unwound strand of the roX RNA emerges, thus contributing to subsequent steps of complex assembly (Prabu, 2015).

Besides revealing the roles of the auxiliary domains in imparting specific uridine-binding and dsRNA-unwinding properties to MLE, the structure reported in this study provides the snapshot of a eukaryotic DExH helicase in the transient 'on' state, showing how the nucleotide and RNA ligands are coupled. The structure of the Ski2-like protein Hel308 showed that unwinding is achieved by a β-hairpin in the RecA2 domain and suggested that translocation is achieved by a ratchet helix in the HA2 domain. MLE operates with different mechanisms, because it has different auxiliary domains and different features that line the unwinding entrance of the helicase channel. The interpretation from the structural data on MLE is that global and local changes in RecA2 propel the movement in the 3'-5' direction, with a loop jointed on RecA2 acting as the pawl in the ratcheting mechanism. It is proposed that a conceptually similar mechanism for 3'-5' directional translocation might be at play in other eukaryotic RHA/DEAH helicases (Prabu, 2015).

High-affinity sites form an interaction network to facilitate spreading of the MSL complex across the X chromosome in Drosophila

Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, this study shows that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNAs target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. It was shown that the MSL complex regulates nucleosome positioning at HAS, therefore acting locally rather than influencing the overall chromosomal architecture. The study proposes that the sex-independent, three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males (Ramírez, 2015).

This study provides a first step toward understanding the role of chromosome conformation in dosage compensation in D. melanogaster. HAS, the landing regions of the MSL complex on the X chromosome, frequently reside in proximity to TAD boundaries. HAS are enriched in Hi-C contacts to each other and to other X chromosomal regions and that this organization remains comparable between male and female cells (Ramírez, 2015).

This analysis revealed that HAS are characterized by a combination of DNA sequence (MREs), chromatin state (active), and gene architecture, which drives the specificity of the MSL complex toward the X chromosome. The data suggest that when the MSL complex binds to HAS, it then spreads (either via an active mechanism or via diffusion) to spatially close regions to place the histone H4 lysine 16 acetylation (H4K16ac) mark on active genes. A 'conformation-based affinity' model is proposed based on the strategic location of HAS at highly interconnected regions of the D. melanogaster X chromosome that efficiently distribute the MSL complex over the X chromosome by attracting the MSL complex to cis-interacting HAS on the X chromosome. This system ensures that only this chromosome is specifically and globally targeted. By spreading from those HAS over short (3D) distances, all active genes on the X chromosome are then reached and acetylated without influencing the autosomes. It is suggested that this system is resilient to major perturbations, exemplified by the large autosomal insertion from chromosome 3L and the ectopic expression of the roX genes that produce viable cells and flies, respectively (Ramírez, 2015).

MNase-seq analysis shows a direct effect of the MSL complex on nucleosome organization specifically on HAS and not on the TSS, despite prominent binding of MSL1/2 to promoter regions. The MSL complex may act similar to a pioneer DNA binding protein to establish nucleosome patterns at HAS and may act on neighboring active regions rather than modifying TAD boundaries. This system may be unique to flies because the Drosophila dosage compensation evolved a fine-tuning transcription activation mechanism rather than a complete shutdown of gene transcription as seen in mammalian X chromosome inactivation. It would be very interesting to see how nucleosome positioning is affected upon Xist binding in mammals (Ramírez, 2015).

Although many factors, including the CCCTC-binding factor (CTCF) as well as tRNA and housekeeping genes, have been shown to be enriched at boundaries, by dissecting the targeting and spreading activity of the MSL complex for the X chromosome, this study offers a plausible explanation behind the advantages of HAS localization. HAS are enriched at the X chromosomal boundaries and not at autosomal boundaries, where all other boundary factors will bind indiscriminately. Furthermore, it was found that the few HAS that are not near a boundary also occupy locations of an elevated number of long-range contacts, indicating that HAS form interaction hubs for the spreading of the MSL complex (Ramírez, 2015).

Hi-C as well as in vivo immunofluorescence show that active roX genes have more contacts and are closer to each other than inactive regions. These observations are in line with previous reports showing that active chromatin compartments interact more often with each other and that active chromatin localizes to the interface of the chromosomal territory. The results imply that different transcriptional programs in each cell line or tissue are likely to be associated with a particular arrangement of long-range contacts, suggesting that the dosage compensation must be flexible to act over such diverse conformations without disturbing them. This idea is consistent with the observation that the chromosome conformation remains unchanged after knockdown of the MSL complex, and stays in contrast to mammalian X inactivation, which involves chromatin condensation, gene inactivation, and alterations in chromosome conformation (Ramírez, 2015).

Dosage compensation mechanisms in flies and mammals lead to opposite outcomes; namely, gene activation versus gene repression. However, both systems use lncRNAs transcribed from the dosage-compensated X chromosome. roX1 and roX2 RNA are expressed from the male hyperactivated X chromosome in D. melanogaster, whereas Xist is expressed from the inactivated X chromosome in mammalian females. Recent work has shown that Xist spreads to distal sites on the X chromosome. Interestingly, this spreading is dependent on the spatial proximity of sites distal to the Xist gene. This is further exemplified by ectopic expression of Xist from chromosome 21, where Xist spread only in cis on this chromosome. In this study, ectopic insertion of roX transgenes on autosomes demonstrated that the roX/MSL complex can reach the X chromosome and rescue male lethality. Therefore, acting in trans is a special feature of roX RNAs (in conjunction with the MSL complex) not observed for Xist, indicating that the two systems utilize the respective lncRNAs differently. In both systems, however, the lncRNAs need to be functional because the stem loop structures of the roX RNAs are required for dosage compensation in D. melanogaster, whereas Xist needs the "A repeat domain" to induce mammalian X chromosome inactivation. The distinct mechanisms utilized by the Xist and roX RNAs exemplify the great versatility by which lncRNAs can be involved in the global regulation of single chromosomes and might reflect important differences between the two systems. In mammals, only one of the two X chromosomes needs to be inactivated. Therefore, a trans action of Xist RNA on the sister X chromosome would be detrimental to the organism. In contrast, the dosage-compensated X chromosome is present singularly in males in Drosophila. However, because the roX RNAs can act in trans, it may be disadvantageous to target the activating MSL complex to active genes on autosomes, hence the need for specific target regions (the HAS) unique to the X chromosome (Ramírez, 2015).

To fully understand the occurrence of HAS at sites with extensive long-range interactions on the X chromosomes, it could be helpful to consider evolutionary models proposing that X chromosomes tend to evolve faster than autosomes (faster X effect). Under the faster X effect, traits only beneficial for males can introduce significant changes specific to the X chromosome on a short evolutionary timescale. Based on these and other observations suggesting that the X chromosome in flies is different from autosomes, it is assumed that selective pressures on males favored the occurrence of HAS at regions of increased interactions, like TAD boundaries. Future analyses of different Drosophila species will open exciting opportunities to study the evolutionary changes of HAS in the context of X chromosomal architecture. Moreover, conformation-based affinity could be a generic mechanism for other regulatory elements to exert their functions. It remains to be seen in which contexts the in cis versus in trans action of different lncRNAs is essential for their function and how chromosome conformation, long-range contacts, HAS, and regulation of transcription have co-evolved for dosage compensation (Ramírez, 2015).

UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation

Dosage compensation is a regulatory process that balances the expression of X-chromosomal genes between males (XY) and females (XX). In Drosophila, this requires non-coding RNAs and RNA-binding proteins (RBPs) whose specific functions remain elusive. This study shows that the Drosophila RBP UNR promotes the targeting of the activating male-specific-lethal complex to the X-chromosome by facilitating the interaction of two crucial subunits: the RNA helicase MLE and the long non-coding RNA roX2 (Militti, 2014).

In Drosophila, dosage compensation involves the binding of the MSL dosage compensation complex (MSL-DCC) to hundreds of sites on the single male X-chromosome and the subsequent twofold hypertranscription of active genes. The MSL-DCC contains five proteins: MSL1, MSL2, MSL3, Maleless (MLE) and Males-absent-on-first (MOF), and two long non-coding RNAs, RNA on X (roX) 1 and 2, which differ in size and sequence but display redundant functions. MSL2 is the limiting subunit of the MSL-DCC and, together with MSL1, nucleates complex formation at specific X-chromosomal sites known as high-affinity sites (HAS). The RNA helicase MLE colocalizes with MSL2 at HAS and facilitates the incorporation of roX into the complex. The RNA is important for the distribution of the MSL-DCC along the X-chromosome, where it activates target genes (Militti, 2014).

Upstream of N-Ras (UNR) is a conserved RNA-binding protein (RBP) containing five cold-shock domains (CSD) that regulates mRNA translation and stability by interacting with single-stranded RNA. It has been shown previously that Drosophila UNR performs sex-specific opposing roles in dosage compensation. In females, UNR inhibits MSL-DCC assembly by repressing the synthesis of MSL2. In males, UNR promotes the targeting of the MSL-DCC to the X-chromosome by a poorly understood mechanism that does not involve translational regulation of MSL proteins . UNR-dependent regulation can be recapitulated in male S2 cells, which express only roX2. This work describes efforts to determine whether UNR interacts with the DCC assembly and targeting machinery in more specific ways. Primed by the finding of a preferred RNA-binding element for UNR close to the known roX remodelling site of the helicase MLE (Maenner, 2013), this study systematically explored the relationship between UNR and MLE. UNR was found to facilitates the binding of MLE to its target at limiting MLE concentrations. These biochemical analyses approximate the physiological conditions to a good extent, since depletion of UNR also diminishes the MLE-roXinteraction and reduces the association of MLE with HAS in dosage-compensating cells. These results identify UNR as a general RBP with specific roles in dosage compensation (Militti, 2014).

To assess whether recombinant UNR could directly bind to roX2, an electrophoretic mobility shift assay (EMSA) was used. UNR binds full-length roX2 RNA and several roX2-derived fragments. UNR interacted most strongly with a roX2 fragment spanning nucleotides 316-379. Interestingly, this purine-rich region is located within stem-loop 6 (SL6) just upstream of a prominent, conserved stem-loop structure (SLroX2 or SL7) that, when multimerized, is sufficient to restore the X-chromosomal targeting defects of a roX null mutant. Pull-down assays using MS2-tagged RNA as bait confirmed that UNR bound with highest affinity to roX2 fragments containing SL6 (Militti, 2014).

Because of the roles of UNR in RNA metabolism, whether UNR binding affected roX2 levels, nucleocytoplasmic distribution or splicing of its major isoforms in S2 cells was assessed. UNR depletion showed no effect on any of these features. To gain insight into UNR-roX2 interactions, enzymatic and chemical footprinting was performed. Addition of UNR protected unpaired nucleotides of SL6, in particular the terminal loop (nts 367-374) and the internal bulge (nts 352-357), suggesting UNR binding to these regions. UNR also protected to a lower extent the terminal loop of SL7 and the single-stranded region between SL7 and SL8 but did not protect efficiently the terminal loop of SL8. These results confirmed that UNR interacts preferentially with SL6. Mutational analysis showed that UNR recognizes the purine-rich stretches in the loops. Interestingly, nucleotides 417-419 and 497 were rendered more reactive upon the addition of UNR. These positions form a 7bp-extended version of SL7, which maybe disrupted by UNR. These results suggest a role for UNR as a roX2 chaperone (Militti, 2014).

In bacteria, CSD-containing RNA chaperones associate with RNA helicases to promote RNA remodelling . It was thus, asked whether UNR interacts with the RNA helicase MLE. Co-immunoprecipitation experiments with recombinant proteins demonstrated a weak interaction between MLE and UNR. Interestingly, this interaction was strongly stimulated by roX2 but not by an unrelated control RNA of similar length. The interaction persisted after efficient RNase treatment, monitored by measuring the presence of trace-labelled roX2 in the pellet, indicating that roX2 promotes strong direct interactions between MLE and UNR, or that any connecting RNA within the complex is protected from RNase digestion. To test whether these interactions occurred in vivo, UNR was immunoprecipitated from nuclear extracts of S2 cells. Although UNR is primarily cytoplasmic, a small amount can be found in the nucleus that interacts with MLE, but not with MSL3, indicating that the interaction is specific. Consistent with the in vitro data and with the presence of roX2 in the nucleus, the interaction of endogenous MLE and UNR is resistant to RNase treatment after formaldehyde crosslinking, suggesting that interactions involve protein-protein crosslinks and not merely protein-RNA crosslinks. Altogether, the data suggest that UNR, MLEand roX2 form a complex. Indeed, a ternary complex is detected by EMSA when roX2 or its 3' half fragment (SL678) is incubated with recombinant UNR and MLE (Militti, 2014).

Intriguingly, the base of the extended SL7 of roX2 that is potentially remodelled by UNR serves as a binding site for MLE, which disrupts SL7 thereby promoting subsequent interactions with MSL2. Thus, melting of the base of SL7 by UNR and exposure of the corresponding nucleotides could facilitate MLE interaction with roX2. To test this hypothesis, binding of recombinant UNR and MLE to roX2 derivatives was monotored using RNA pull-down assays. MLE on SL67 was titrated in the presence or absence of UNR. At high concentrations, MLE was able to bind SL67. MLE, however, was unable to efficiently bind the target RNA at reduced concentrations. Remarkably, under those conditions the interaction of MLE with SL67 was strongly facilitated by adjacent UNR binding. To test the correlation between MLE and UNR binding, the roX2 derivatives SL2345 and SL67 were employed. UNR bound to both RNAs in the absence of MLE and, consistent with the EMSA, it bound with higher affinity to SL67. MLE bound to roX2 fragments after UNR addition in a manner that correlated with the strength of UNR binding. In addition, MLE bound strongly to full-length roX2 in the presence of UNR, and this binding was reduced upon deletion of either SL6 or SL7. These results indicate that UNR promotes the association of MLE with roX2 in vitro (Militti, 2014).

To test whether UNR promotes the association of MLE with roX2 in vivo, UNR from was depleted from S2 cells. Ablation of UNR diminished the amounts of roX2 associated with the endogenous helicase. This effect could neither be attributed to variations in the amounts of MLE or roX2 upon UNR depletion nor to differences in the efficiency of MLE immunoprecipitation. To explore whether the reduced MLE-roX2 interaction affects the association of MLE with its chromosomal targets, the HAS, chromatin immunoprecipitation (ChIP) was used. Notably, it was found that UNR depletion decreased the MLE association to four different HAS. As controls, UNR depletion did not affect the association of MLE to promoters within the same genes, or the association of MOF to HAS or to an autosomal gene. Altogether, these results demonstrate a role for UNR in facilitating the binding of the RNA helicase MLE with its target sites on roX2 RNA in vitro and in vivo (Militti, 2014).

It is concluded that in the absence of MLE (and roX), MSL2 and MSL1 can associate with HAS but no functional DCC is assembled that distributes the activating histone acetylation to the target genes . ChIP experiments show prominent, specific interaction of MLE at HAS in S2 cells. The current data now suggest that the general RBP UNR facilitates this interaction. Remarkably, UNR also binds and regulates the activity of msl2 mRNA together with the sex determination switch SXL , suggesting that sex determination and dosage compensation have co-evolved to employ similar factors from the large portfolio of RBPs. UNR, thus, extends the small list of RBPs involved in dosage compensation. Such factors include hnRNPU and YY1, which tether the lncRNA Xist to the inactive X-chromosome during mammalian dosage compensation. Rather than a molecular tether, however, the role of UNR seems more transient and directed to facilitate initial steps of MSL-DCC assembly through the modulation of RNA conformation. UNR, therefore, acts as a 'catalyst' of the MLE-roX interaction. RNA structural transitions are at the basis of many fundamental post-transcriptional processes . The current results illustrate the emerging concept that lncRNA structural dynamics may contribute to chromatin organization, and indicate that general RBPs, such as UNR, can be harnessed to contribute key molecular events in the assembly of specialized machineries (Militti, 2014).

The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females

Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X (roX) RNAs that are transcriptionally activated by the MSL complex. Targeting of the MSL complex to the X-chromosome has been shown to be dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, the CRISPR/Cas9 genome editing system was used to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. clamp null females were found to die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, it was found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. These results provide new insights into sex-specific gene regulation by an essential transcription factor (Urban, 2017).

Many species employ a sex determination system that generates an inherent imbalance in sex chromosome copy number, such as the XX/XY system in most mammals and some insects. In this system, one sex has twice the number of X-chromosome-encoded genes compared to the other. Therefore, a mechanism of dosage compensation is required to equalize levels of X-linked transcripts, both between the sexes and between the X-chromosome and autosomes. Dosage compensation is an essential mechanism that corrects for this imbalance by coordinately regulating the gene expression of most X-linked genes (Urban, 2017).

In Drosophila melanogaster, transcription from the single male X-chromosome is increased 2-fold by recruitment of the male-specific lethal (MSL) complex. The MSL complex is composed of two structural proteins, MSL1 and MSL2, three accessory proteins, MSL3, males absent on the first (MOF), and maleless (MLE), and two functionally redundant non-coding RNAs, RNA on the X (roX1) and roX2. Previous work has shown that recruitment of the MSL complex to the X-chromosome requires the zinc finger protein chromatin-linked adapter for MSL proteins (CLAMP) (Soruco, 2013; Urban, 2017 and references therein).

In addition to its role in male MSL complex recruitment, it was suggested that CLAMP has an additional non-sex-specific essential function because targeting of the clamp transcript by RNA interference results in a pupal lethal phenotype in both males and females (Soruco, 2013). Further understanding of CLAMP function in the context of the whole organism required a null mutant. However, due to the pericentric location of the clamp gene, no deficiencies or null mutations were available. Using the CRISPR/Cas9 system, a frameshift mutation was introduced in the clamp gene, leading to an early termination codon before the major zinc finger binding domain. This frameshift mutation generated the clamp² allele, which eliminates detectable CLAMP protein production and is therefore a protein null allele. The majority of clamp² mutant males die prior to the third instar stage. On the other hand, females die at the third instar stage, suggesting sex-specific functions for CLAMP. Furthermore, CLAMP regulates the roX genes in a sex-specific manner, activating their accumulation in males and repressing their accumulation in females. Overall, we present a new tool for studying dosage compensation and suggest that CLAMP functions to assure that roX RNA accumulation is sex specific (Urban, 2017).

Previous work demonstrated that CLAMP has an essential role in MSL complex recruitment to the male X-chromosome (Soruco, 2013). However, it was not possible to perform in vivo studies to further investigate CLAMP function because there was no available null mutant line. The current work present a CLAMP protein null mutant and determine that this protein is essential in both sexes. This allele will provide a key tool for future in vivo studies on the role of CLAMP in dosage compensation, as well as identification of the essential function of CLAMP in both sexes (Urban, 2017).

The initial characterization of the clamp² protein null allele revealed sexually dimorphic roles for CLAMP in regulation of the roX genes. CLAMP was seen to promotes roX2 transcription in males but represses transcription of both roX genes in females. It is likely that recruitment of the MSL complex to the roX2 locus by CLAMP promotes roX2 expression in males. In females, where the MSL complex is not present, CLAMP may function to repress these loci as an additional mechanism to ensure that dosage compensation is male-specific. Additionally, it was determined that most clamp² homozygous males die earlier in development than clamp² homozygous females. Earlier lethality in males is likely due to a misregulation of the dosage compensation process as a result of the loss of CLAMP-mediated MSL complex recruitment. However, CLAMP is enriched at the 5' regulatory regions of thousands of genes across the genome. Therefore, it is likely that other non-sex-specific regulatory pathways are disrupted resulting in female lethality (Urban, 2017).

Furthermore, CLAMP is an essential protein because our CRISPR/Cas9-generated protein null clamp allele is homozygous lethal in both males and females. These results indicate that CLAMP has a previously unstudied non-sex-specific role that is essential to the viability of both males and females. An interesting observation that arose from this characterization is that polytene chromosome organization is disrupted in clamp² mutant females, suggesting that CLAMP may play a role in regulation of genome-wide chromatin organization of interphase chromosomes. A function in regulating chromatin organization provides one possible explanation for how CLAMP performs sexually dimorphic functions. For example, CLAMP may repress roX expression in females by promoting the recruitment of a repressive chromatin-modifying factor in the absence of the MSL complex. In contrast, CLAMP may activate roX2 in males by creating a chromatin environment permissive for MSL complex recruitment in males. Although roX1 and roX2 are functionally redundant, the results suggest that CLAMP specifically activates roX2 but not roX1 in males. Interestingly, Villa (2016) recently reported that roX2, but not roX1, is likely to be an early site of MSL complex recruitment (Villa, 2016), suggesting that CLAMP may function early in the process of dosage compensation (Urban, 2017).

Overall, the newly generated clamp² protein null allele provides an important tool to study how the essential CLAMP protein regulates its many target genes in vivo. The generation of the clamp² allele will facilitate future studies that will reveal a mechanistic understanding of how a single transcription factor can promote different sex-specific functions within an organism (Urban, 2017).

Targeting of Painting of fourth to roX1 and roX2 proximal sites suggests evolutionary links between dosage compensation and the regulation of the fourth chromosome in Drosophila melanogaster

In Drosophila melanogaster, two chromosome-specific targeting and regulatory systems have been described. The male-specific lethal (MSL) complex supports dosage compensation by stimulating gene expression from the male X-chromosome, and the protein Painting of fourth (POF) specifically targets and stimulates expression from the heterochromatic 4^th chromosome. The targeting sites of both systems are well characterized, but the principles underlying the targeting mechanisms have remained elusive. This study presents an original observation, namely that POF specifically targets two loci on the X-chromosome, PoX1 and PoX2 (POF-on-X). PoX1 and PoX2 are located close to the roX1 and roX2 genes, which encode noncoding RNAs important for the correct targeting and spreading of the MSL-complex. The targeting of POF to PoX1 and PoX2 is largely dependent on roX expression, and a high-affinity target region was identified that ectopically recruits POF. The results presented support a model linking the MSL-complex to POF and dosage compensation to regulation of heterochromatin (Lundberg, 2013).

High-affinity sites have been characterized for the MSL-complex and there are several published examples of short regions, including the roX1 and roX2 loci, that are capable of recruiting this complex when presented as transgenes. In contrast, until now no high-affinity sites for POF targeting have been identified. Translocated 4^th chromosomes will not be targeted by POF, unless the proximal heterochromatic region is present and under conditions that favor heterochromatin formation. The characterization of POF targeting to PoX1 and PoX2 in females thus provides a unique opportunity to study the targeting of POF to a nonheterochromatic target and to further understanding of the evolution of these two targeting systems (Lundberg, 2013).

Considering the evolutionary relationship between POF and the MSL-complex (reviewed by Stenberg, 2011), it was intriguing to find POF targeting to two distinct regions on the X-chromosome, i.e., X:3E and X:10E-F. The apparent spreading of POF targeting in these two regions (resembling the spreading of the MSL-complex when it is targeted to roX transgenes) and the close location of these regions to roX1 and roX2 suggested a link with the MSL-complex and dosage compensation. It has been hypothesized that POF originated as a dosage compensation system, since POF targets the male X-chromosome in, for example, D. busckii and D. ananassae and in those species POF colocalizes with H4K16ac and the MSL-complex, respectively (Larsson, 2004; Stenberg. 2011). However, the targeting of POF to endogenous PoX1 and PoX2 in D. melanogaster is restricted to females. This sex-specific targeting is not caused by sex-specific expression of the targeted genes, since comparable expression levels of >RE64691 as well as SelG and CG1840 are consistently found in male and female salivary glands (Lundberg, 2013).

Not only are the two targeted loci, PoX1 and PoX2, located in close proximity to roX1 and roX2, the targeting is also largely dependent on roX, because losses of roX1 alone or of roX1 and roX2 cause a clear decrease in the frequency of PoX targeting. Importantly, in all roX mutant conditions tested, complete loss of POF binding to the PoX sites was never found. Therefore, roX is not absolutely required for PoX targeting but rather it enhances or stabilizes the interaction. The dependence of targeting on roX is not caused by the close proximity of the PoX loci to the corresponding roX loci, because in the duplications tested the PoX1 and PoX2 are located on another chromosome, i.e., chromosome arm 3L, and the roX genes are not included in the duplicated region. Despite this, two of the duplications show targeting by POF, comparable to that to the endogenous loci. Furthermore, targeting to these transgenic regions was found to be largely dependent on roX, which indicates that roX can act in trans to enhance or stabilize POF targeting. The most parsimonious model to explain these observations is that it is the roX ncRNA species that enhance or stabilize targeting of POF to these non-chromosome 4 targets. This model is supported by the fact that roX2 overexpression seems to further increase the frequency of targeting. However, it should be stressed that endogenous roX expression in females is reported to be at very low levels or absent. In females, roX1 RNA has been reported in early embryos but it appears to be lost midway through embryogenesis, whereas in males expression is maintained through adulthood. roX2 RNA first appears a few hours after roX1, but only in male embryos (Lundberg, 2013).

No high-affinity sites for POF targeting have previously been identified. It therefore came as a surprise that a short (6-kb) region from PoX2 functions as a strong ectopic target for POF in both males and females. The nonsex-specific targeting of POF to the P[w⁺ SelG CG1840] transgene, in contrast to Dp(1;3)DC246 and endogenous PoX, may be explained by a competition of targeting between POF and the MSL-complex in males. This competition will be more pronounced at the endogenous PoX sites and the duplications as these are also targets for the MSL-complex in males. This finding is supported by the fact that on polytene chromosomes, Dp(1;3)DC246 is targeted by MSL-complex in males while the P[w⁺ SelG CG1840] transgene is not targeted. A competition in targeting is also supported by the reduction frequency of PoX1 and PoX2 targeting by POF observed in females expressing a partial MSL-complex, i.e. w; P[w⁺ hsp83:msl2] msl3 females. It is important to note that the targeting of POF to the P[w⁺ SelG CG1840] transgene was not caused by genomic location of this transgene since the same attP docking site (3L:65B2) was used as for the duplications of the PoX1 and PoX2 loci. The lack of targeting of POF to translocated parts of the 4^th chromosome and the strong targeting to the PoX2 transgene suggest that the PoX regions may be POF targets that are functionally separable from the 4^th chromosome genes. Since both Setdb1 and HP1a are detected on the transgene, it appears likely that POF recruitment leads to, or is connected with, the formation of GREEN (HP1a and H3K9me enriched) chromatin structure (Filion, 2010) (Lundberg, 2013).

The targeting of POF to the 4^th chromosome depends on its well-characterized heterochromatic nature and on the presence of HP1a and Setdb1. It is therefore important to note that links between the MSL-complex, roX1 and roX2 and heterochromatic regions have been reported previously, though they remain to be understood. It is known that in roX1 roX2 mutant males, the MSL-complex is still detected on the X-chromosome, albeit at a reduced number of sites, but binding is also found in the chromocenter and at a few reproducible sites on the 4^th chromosome. In contrast to the X-chromosome, where the MSL-complex is believed to stimulate gene expression, loss of roX RNA reduces expression from genes located in the chromocenter and on the 4^th chromosome. It has been suggested that roX RNAs participate in two distinct regulatory systems, the dosage compensation system and a system for the modulation of heterochromatin. Although the mechanism by which roX RNAs enhance binding of POF to PoX loci remains elusive, the observation supports a model linking dosage compensation to modulation of heterochromatin. Additional factors supporting a model linking heterochromatin to dosage compensation are the proposed binding of HP1a to the male X-chromosome and the fact that a reduction in the histone H3S10 kinase JIL-1 results in the spreading of heterochromatic markers (such as H3K9me2 and HP1a) along the chromosome arms, with the most marked increase taking place on the X-chromosomes. The JIL1 kinase, which is believed to counteract heterochromatin formation, is highly enriched on the male X-chromosome and is reported to be loosely attached to the MSL-complex). It is noteworthy that POF, which targets genes in a heterochromatic environment, i.e., on the 4^th chromosome, has an intrinsic ability to target the male X-chromosome, as seen in, e.g., D. ananassae, and the targeting to X-chromosome sites reported in this study is dependent on roX RNAs. At the same time the MSL-complex, which binds to and stimulates expression of genes on the male X-chromosome, has an intrinsic ability to target heterochromatin as seen in the roX1 roX2 mutant background. The link between these two systems is intriguing and promises to increase understanding of balanced gene expression (Lundberg, 2013).

High-affinity targeting to the PoX1 and PoX2 loci therefore provides a novel system for further studies on targeting mechanisms involved in chromosome-wide gene regulation, the evolutionary relationship between POF and dosage compensation and the evolution of balanced gene expression, and the results favor a model involving not only the X-chromosome but also balance to heterochromatin (Lundberg, 2013).

Imprinting of the Y Chromosome Influences Dosage Compensation in roX1 roX2 Drosophila melanogaster

Drosophila melanogaster males have a well-characterized regulatory system that increases X-linked gene expression. This essential process restores the balance between X-linked and autosomal gene products in males. A complex composed of the male-specific lethal (MSL) proteins and RNA is recruited to the body of transcribed X-linked genes where it modifies chromatin to increase expression. The RNA components of this complex, roX1 and roX2, are functionally redundant. Males mutated for both roX genes have dramatically reduced survival. This study shows that reversal of sex chromosome inheritance suppresses lethality in roX1 roX2 males. Genetic tests indicate that the effect on male survival depends upon the presence and source of the Y chromosome, revealing a germ line imprint that influences dosage compensation. Conventional paternal transmission of the Y chromosome enhances roX1 roX2 lethality, while maternal transmission of the Y chromosome suppresses lethality. roX1 roX2 males with both maternal and paternal Y chromosomes have very low survival, indicating dominance of the paternal imprint. In an otherwise wild-type male, the Y chromosome does not appreciably affect dosage compensation. The influence of the Y chromosome, clearly apparent in roX1 roX2 mutants, thus requires a sensitized genetic background. It is believed that the Y chromosome is likely to act through modulation of a process that is defective in roX1 roX2 mutants: X chromosome recognition or chromatin modification by the MSL complex (Menon, 2009).

Reversal of sex chromosome inheritance is a potent suppressor of roX1 roX2 male lethality. Males carrying a paternal roX1 roX2 chromosome and a maternal Y chromosome have dramatically higher survival than males that inherit identical sex chromosomes conventionally. Surprisingly, this effect can be attributed solely to the presence, and parent of origin, of the Y chromosome. A maternally transmitted Y chromosome suppresses roX1 roX2 lethality, a paternally transmitted Y chromosome enhances roX1 roX2 lethality, and absence of the Y chromosome produces an intermediate level of male survival. Males with both maternal and paternal Y chromosomes have very low survival, suggesting that the effect of the paternal Y chromosome is dominant. In spite of the widely held view that the Y chromosome has little genetic information or importance, Y chromosomes from different Drosophila strains have unexpectedly large effects on expression throughout the genome, particularly the expression of male-biased genes. However, the Y chromosome is not necessary for dosage compensation and is not believed to influence this process in otherwise normal males. The effect observed thus requires a roX1 roX2 mutant background. A dose-sensitive X-linked reporter and quantitative reverse transcription-PCR (qRT-PCR) of X-linked genes reveals higher expression in roX1 roX2 males with a maternal Y chromosome than with a paternal Y chromosome. It is concluded that a maternally imprinted Y chromosome suppresses roX1 roX2 lethality through a process that culminates in increased expression of X-linked genes (Menon, 2009).

Regulation of histone H4 Lys16 acetylation by predicted alternative secondary structures in roX noncoding RNAs

Despite differences in size and sequence, the two noncoding roX1 and roX2 RNAs are functionally redundant for dosage compensation of the Drosophila melanogaster male X chromosome. Consistent with functional conservation, this study found that roX RNAs of distant Drosophila species could complement D. melanogaster roX mutants despite low homology. Deletion of a conserved predicted stem-loop structure in roX2, containing a short GUb (GUUNUACG box) in its 3' stem, resulted in a defect in histone H4K16 acetylation on the X chromosome in spite of apparently normal localization of the MSL complex. Two copies of the GUb sequence, newly termed the 'roX box,' were functionally redundant in roX2, as mutants in a single roX box had no phenotype, but double mutants showed reduced H4K16 acetylation. Interestingly, mutation of two of three roX boxes in the 3' end of roX1 RNA also reduced H4K16 acetylation. Finally, fusion of roX1 sequences containing a roX box restored function to a roX2 deletion RNA lacking its cognate roX box. These results support a model in which the functional redundancy between roX1 and roX2 RNAs is based, at least in part, on short GUUNUACG sequences that regulate the activity of the MSL complex (Park, 2008).

This study found that roX RNAs of distantly related Drosophila species had sufficient function in D. melanogaster to target the MSL complex and H4K16ac to the X chromosome. This indicates that roX RNAs in diverse species contain common functional domains for dosage compensation of the X chromosome, in spite of a considerable lack of conservation in overall sequence. By mutating the conserved stem-loop region of roX2 RNA and 'roX box' sequences in both roX RNAs, these regions were found to be essential for normal levels of enzymatic activity of the MSL complex. However, other short, conserved sequences in roX2 were not essential upon deletion, suggesting internal redundancy, as shown in other noncoding RNAs (roX1 and Xist). One possibility is that these conserved regions are functionally redundant with each other, as shown for the roX box sequences. Another possibility is that overexpression of these constructs from the constitutive hsp83 promoter can overcome defects in these RNAs. Expression of WT roX2 RNA from the hsp83 promoter results in approximately threefold higher amounts of stable roX2 RNA than of the endogenously expressed RNA. However, the stem-loop deletion and mutant roX box constructs resulted in a defective phenotype even when they were expressed from the hsp83 promoter (Park, 2008).

These results strongly suggest that the roX box sequences in roX2 RNA are functionally redundant in forming a stem-loop structure at the 3' end of the RNA. Mutation of the roX2-box1 sequence (in mX1 RNA) resulted in more-than-normal accumulation of a longer RNA including the roX2-box2 sequence. It is proposed that the roX2-box2 sequence could form a stem-loop structure with the 5' stem sequence when the roX2-box1 sequence is mutated. Alternatively, it is possible that the roX2-box2 sequence can make its own stem-loop structure within the 3' extension (67 nt). There is an additional potential 5' stem sequence upstream of the roX2-box2 sequence that shows evolutionary conservation (Park, 2008).

In 3' RACE analysis of endogenous and WT transgenic (H83MeroX1) roX1 RNAs, two 3' processing sites were found, located 3' of roX1-box2 and roX1-box3. These were consistent with reported 3' ends of roX1 cDNAs, with mapping on Flybase, and with 3' ends determined by RNase protection assay. Similar to roX2, mature roX1 RNAs display the roX box at their 3' ends. These results strongly suggest that roX1 and roX2 RNAs utilize roX box sequences at the 3' end for normal function of the MSL complex. The alternative 3'-end processing and the locations and functions of the two roX box sequences are very similar in roX1 and roX2. Most importantly, when it was expressed as a hybrid RNA (H-X2), the roX box region from roX1 was able to compensate for the loss of a roX box segment from roX2. Therefore, the presence of roX boxes in both roX1 and roX2 explains at least part of the functional redundancy between roX RNAs (Park, 2008).

At this point, it is not know if the roX1-box1 sequence in roX1 has the same function as the roX1-box2 and roX1-box3 sequences. When the alignment of roX1 genes from eight Drosophila species was carefully analyzed, an evolutionarily conserved sequence was found upstream (135 nt) of the roX1-box1 sequence, which might function as a 5' stem. However, the possibility cannot be excluded that the roX1-box2 and roX1-box3 sequences in roX1 RNA function as primary sequences rather than in secondary structures (Park, 2008).

These results uncouple X chromosome targeting of the MSL complex from histone acetylation activity. The stem-loop region in the 3' end of roX2 RNA and the roX box sequences in the 3' ends of roX1 and roX2 RNAs are critical for full histone acetylation by the MSL complex. It was reported that the histone acetyltransferase activity of the MOF protein is stimulated by the interaction of MOF with MSL1 and MSL3 in vitro. However, in vivo histone acetyltransferase activity of MOF is low in a roX⁻ mutant, suggesting that roX RNA is required for the optimum activity of MOF in vivo. MOF and MSL3 proteins have RNA binding activity in vitro and are dissociated from the MSL complex by RNase treatment in vivo. In future analyses, it will be very interesting to determine whether roX RNAs directly or indirectly stimulate the acetylation activity of the MSL complex (Park, 2008).

An evolutionarily conserved domain of roX2 RNA is sufficient for induction of H4-Lys16 acetylation on the Drosophila X chromosome

The male-specific lethal (MSL) complex, which includes two noncoding RNA on X (roX)1 and roX2 RNAs, induces histone H4-Lys16 acetylation for twofold hypertranscription of the male X chromosome in Drosophila melanogaster. To characterize the role of roX RNAs in this process, evolutionarily conserved functional domains of roX RNAs were identified in several Drosophila species (eight for roX1 and nine for roX2). Despite low homology between them, male-specific expression and X chromosome-specific binding are conserved. Within roX RNAs of all Drosophila species, conserved primary sequences, such as GUUNUACG, were found in the 3' end of both roX1 (three repeats) and roX2 (two repeats). A predicted stem-loop structure of roX2 RNA contains this sequence in the 3' stem region. Six tandem repeats of this stem-loop region (72 nt) of roX2 were enough for targeting the MSL complex and inducing H4-Lys16 acetylation on the X chromosome without other parts of roX2 RNA, suggesting that roX RNAs might play important roles in regulating enzymatic activity of the MSL complex (Park, 2007).

Sequence-specific targeting of Drosophila roX genes by the MSL dosage compensation complex

MSL complexes bind the single male X chromosome in Drosophila to increase transcription approximately 2-fold. Complexes contain at least five proteins and two noncoding RNAs, roX1 and roX2. The mechanism of X chromosome binding is not known. A 110 bp sequence in roX2 has been identified, characterized by high-affinity MSL binding, male-specific DNase I hypersensitivity, a shared consensus with the otherwise dissimilar roX1 gene, and conservation across species. Mutagenesis of evolutionarily conserved sequences diminish MSL binding in vivo. MSL binding to these sites is roX RNA dependent, suggesting that complexes become competent for binding only after incorporation of roX RNAs. However, the roX RNA segments homologous to the DNA binding sites are not required, ruling out simple RNA-DNA complementarity as the primary targeting mechanism. These results are consistent with a model in which nascent roX RNA assembly with MSL proteins is an early step in the initiation of dosage compensation (Park, 2003).

A strong MSL binding site within the roX1 gene has been mapped by a combination of male-specific DNase I hypersensitivity (DHS) assays and transgenic deletion analyses. The binding site was narrowed down to ~200 bp, centrally located within the roX1 transcription unit. Transposons carrying the 200 bp roX1 DHS segment can attract MSL complexes to ectopic sites on autosomes, and are sufficient, at least as a nine-copy multimer, to occasionally nucleate limited spreading of MSL complexes into flanking chromatin. Based on this information, the roX2 gene was checked for male-specific DNase I hypersensitivity and evidence was found for a DHS region at the 3' end of the gene. Although this site is not as prominent as its roX1 counterpart, this 270 bp segment is sufficient to attract MSL complexes to autosomes in multiple independent transgenic lines. Hereafter, 'DHS' and 'MSL binding site' will be used synonymously. Binding of MSL complexes to the ectopic roX2 DHS in polytene chromosomes is robust, but unlike the roX1 multimer, no detectable MSL spreading was observed from roX2 DHS multimer constructs inserted at six different autosomal locations. MSL binding at the roX2 DHS is msl3 independent, which is a hallmark of roX genes and ~33 other proposed 'chromatin entry sites' on the X chromosome (Park, 2003).

Searches for elements necessary to target dosage compensation to the X chromosome have failed to yield cis-acting DNA sequences. Although it has been theoretically possible that MSL complexes might recognize some sequence-independent structural characteristic in chromatin entry sites, a broad domain of small islands of consensus sequences has been found to be important for MSL binding at roX genes. Computer-based comparisons of the roX1 and roX2 sequences failed to identify this region. Only after male-specific DNase I hypersensitive sites were identified within each gene and assayed for MSL binding activity in vivo did the consensus target sequence become apparent. Candidates for the additional ~33 postulated chromatin entry sites could not be identified by searching for sequences similar to the consensus MSL binding sequence in roX genes. This may be due to a failure of the search parameters. Alternatively, this result is consistent with a model in which roX genes are thought to be fundamentally different from other entry sites (Park, 2003).

The most prominent feature of the two roX genes is that they produce noncoding RNA components of MSL complexes. When either is mutant, the other is sufficient for MSL function, but males mutant for both roX RNAs cannot localize their MSL complexes properly. This shows that if any of the other postulated entry sites produce an RNA component of MSL complexes, it is not sufficient to replace these two key components. Several additional lines of evidence now point to the existence of only two roX genes, rather than several dozen. (1) SAGE analysis for sex-specific transcripts in adult heads easily found roX1 and roX2 but no other candidate male-specific noncoding RNAs. (2) The roX genes differ from the other entry sites in being highly MLE dependent. Finally, if the conserved DHS sequence is a signature for roX genes, it occurs only twice in the genome (Park, 2003).

The locations of MSL proteins bound to the X chromosome have not been precisely mapped in roX1 roX2 mutant males due to the poor morphology of the chromosomes, but they resemble the pattern of chromatin entry sites. If true, this would indicate that MSL proteins can bind weakly to numerous sites on the X, but have a strict RNA requirement to bind the roX1 and roX2 genes. Such a roX RNA dependence is consistent with an earlier report that roX genes differ from other entry sites in that they are not bound by any MSL subunit unless the MLE helicase is present. In previous studies, it was found that the cytological locations of chromatin entry sites, visualized as partial MSL complex binding, are very similar in msl3, mle, or mof mutants. While the vast majority of sites are common in the three different genotypes, the roX2 site at 10C is specifically absent in the mle mutant. When a roX1 cDNA transgene was assayed in isolation, it was also found to require mle⁺ for binding. A requirement for roX RNA in complexes that bind these sites would be consistent with this mle requirement, since MLE was previously shown to be critical for roX RNA inclusion in partial MSL complexes (Park, 2003).

Based on these results, it is proposed that roX RNAs can assemble into complexes locally at their sites of transcription. roX RNAs are unstable in the absence of MSL proteins, suggesting that complex assembly must occur rapidly for the RNA to escape destruction. Although it had been considered that the MSL proteins might be prepositioned at roX genes to facilitate capture of nascent transcripts, it was instead found that MSL proteins become competent to bind roX genes only after roX RNA is incorporated into the complex. Only a few other chromatin binding proteins have been shown to require an RNA component for chromatin interaction. HP1, a major constituent of heterochromatin, was shown to require RNA for chromatin association. Heterochromatin silencing in fission yeast may also require an RNA component. Likewise, in plants, dsRNA can lead to gene silencing not only by destruction of cognate RNA through a standard RNAi mechanism, but also by methylating the gene producing the offending RNA. In this case, a large multisubunit complex with an RNA helicase subunit is thought to use a short ~22 nt RNA component to search the genome for sequence homology. The roX DHS sequence is found in the middle of all roX1 RNAs and at the 3' end of some roX2 transcripts. An initial test to determine if the MSL complex uses this segment of roX RNA as a template to search the genome for homology ruled this out. However, the possibility that other short elements within roX RNA might play such a role has not been ruled out. Alternatively, the roX RNA may play some structural role in positioning the MSL proteins so that they can make specific DNA contacts (Park, 2003).

An important clue leading to the identification of the MSL binding sequence was the discovery of male-specific DNase I hypersensitive sites within roX1 and roX2. Nuclease sensitivity is often attributed to mobile or absent nucleosomes exposing DNA to nuclear proteins. The most conspicuous feature within the DHS is three copies of the GAGA sequence separated by conserved distances. Several Drosophila proteins are known to bind similar sequences, including the GAGA factor encoded by the trithorax-like gene and the Pipsqueak protein. GAGA factor is thought to keep the chromatin of regulatory regions, such as promoters and Polycomb response elements, in an accessible DHS configuration, possibly by targeting the nucleosome remodeling factor NURF to these sites. However, the action of GAGA factor is not limited to male flies and hence cannot explain a male-specific DHS. Although the possibility cannot be excluded that the altered chromatin structure in this region precedes MSL binding, it seems more likely that MSL binding to this sequence in males induces the more exposed structure. Simple protein-DNA contacts often cover 10-20 bp, so finding essential MSL recognition elements distributed over several turns of the DNA helix suggests a requirement for multiple factors to create a context for MSL binding. It is not known whether any of the five characterized MSL proteins directly contact DNA, but it is interesting to note that in the absence of roX RNA, most MSL proteins are lost from the X and are instead ectopically bound to centric heterochromatin. Satellite IV sequences are located in the centric heterochromatin, making up over 1% of the diploid genome. It consists of the sequence (AAGAGAG)_n, which resembles conserved elements in the roX DHS (Park, 2003).

Why does MSL binding at roX genes appear to differ from binding of the many MSL targets on the X chromosome? Two functions seem possible. (1) Although the isolated roX2 DHS transgenes do not support ectopic MSL spreading over flanking autosomal chromatin, in the context of complete roX genes, high-affinity MSL binding might facilitate epigenetic MSL spreading. (2) Little is known about the transcriptional control of the roX genes or how dosage compensation causes only a 2-fold upregulation of X-linked genes. Perhaps bound MSL complex contributes to regulation of roX RNA transcription, to provide a precise level of MSL complexes for hypertranscription of the X chromosome (Park, 2003).

Assembly of roX RNAs into MSL complexes

In the male Drosophila, the X chromosome is transcriptionally upregulated to achieve dosage compensation, in a process that depends on association of the MSL proteins with the X chromosome. A role for non-coding RNAs has been suggested in recent studies. The roX1 and roX2 RNAs are male-specific, non-coding RNAs that are produced by, and also found associated with, the dosage-compensated male X chromosome. Whether roX RNAs are physically part of the MSL complex has not been resolved. roX RNAs are found to colocalize with the MSL proteins and are highly unstable unless the MSL complex is coexpressed, suggesting a physical interaction. roX2 RNA could be precipitated from male tissue-culture cells with antibodies to the proteins Msl1 and Mle, consistent with an integral association with MSL complexes. Localization of roX1 and roX2 RNAs in mutants indicates an order of MSL-complex assembly in which roX2 RNA is incorporated early in a process requiring the Mle helicase. It was also found that the roX2 gene, like roX1, is a nucleation site for MSL complex spreading into flanking chromatin in cis. These results support a model in which MSL proteins assemble at specific chromatin entry sites (including the roX1 and roX2 genes); the roX RNAs join the complex at their sites of synthesis; and complete complexes spread in cis to dosage compensate most genes on the X chromosome (Meller, 2000).

Wild-type males have complete MSL complexes bound to hundreds of bands along the X chromosome in a highly reproducible pattern where they mediate hypertranscription. Genetic analysis of debilitated MSL complexes lacking either Mle, Msl3 or Mof subunits, or containing enzymatically dead Mof acetyltransferase or Mle helicase, has shown that partial MSL complexes can bind to only ~35 bands that have been termed chromatin entry sites (formerly called high-affinity sites). It has been proposed that these sites direct dosage compensation to the X chromosome by serving as sites of assembly and subsequent spreading of the MSL complex into flanking chromatin in cis. roX2 has been identified as a chromatin entry site based on the ability of roX2 transgenes to attract the MSL complex to autosomal insertion sites, even in the absence of Msl3. To test whether roX2 could also support spreading of MSL complexes into flanking autosomes, multiple lines of transgenic larvae were created carrying genomic roX2 transgenes. It was found that the roX2 chromatin entry site also supports variable spreading of MSL complexes into flanking chromatin. The spreading displays the same characteristics as has been reported for a roX1 transgene: bidirectionality, dependence on insertion site, and substantial variability even from nucleus to nucleus in the same animal. These results strongly support the idea that most or all chromatin entry sites will act as nucleation points for MSL spreading in cis (Meller, 2000).

To determine whether the roX RNAs and MSL proteins are associated in the same binding pattern on the X chromosome, the immunostaining protocol was adapted to allow detection of roX RNAs by in situ hybridization to spread polytene chromosomes. It was found that the MSL proteins, roX1 and roX2 RNAs are precisely colocalized along the male polytene X chromosome. In addition, a subtle enrichment for roX1 RNA has been consistently detected in several bands surrounding its site of synthesis (Meller, 2000).

MSL proteins are found at decreased levels when they are unable to assemble into a wild-type complex. Therefore, it was asked whether roX RNAs might exhibit the same behavior, indicative of a requirement for complex assembly for their integrity. roX1 RNA was expressed constitutively from an H83roX1 transgene and it was determined whether roX1 RNA could accumulate and perhaps even associate with the X chromosomes in females, which lack MSL complexes. It was found that ectopic roX1 RNA fails to bind the X chromosome in females as assayed by in situ hybridization. To determine whether the roX1 RNA produced from the H83roX1 transgene could be stabilized by the presence of the MSL complex, its behavior was examined in males lacking endogenous roX1, or females that form MSL complexes as a result of ectopic expression of Msl2. It was found that transgenic roX1 RNA associates with the X chromosome in males and in Msl2-expressing females. Thus, roX1 RNA is dependent on the MSL complex for its stability (Meller, 2000).

The idea that the MSL complex assembles at chromatin entry sites rests on the observation that these are the only places partial MSL complexes bind when one subunit is removed by mutation. Therefore, it was determined whether roX RNAs might also be associated with these partial complexes in the absence of spreading into flanking chromatin. To visualize chromatin entry sites, females were used that ectopically express Msl2 but are mutant for msl3 or mle. They assemble partial MSL complexes at the same sites as dying mutant males, but are viable and produce excellent polytene chromosomes. Surprisingly, a difference was found in the localization of roX1 and roX2 RNAs in msl3 mutants. Although roX1 RNA is detected only at cytological position 3F, the site of its synthesis, roX2 is consistently seen at many chromatin entry sites including 3F. This result suggests that roX2 may assemble first into partial MSL complexes and then be exported to other chromatin entry sites. These roX2-containing complexes may subsequently acquire a second roX RNA species (such as roX1) or be present in distinct complexes from roX1 (Meller, 2000).

mle mutants were examined for localization of roX RNAs. It was found that roX2 RNA is no longer able to move to other chromatin entry sites, but is only detected at its site of synthesis, like roX1. This demonstrates that the Mle helicase plays an early role, perhaps in packaging roX2 RNA into growing MSL complexes. Blocking assembly at this point prevents roX2 RNA from reaching other chromatin entry sites (Meller, 2000).

To further test the idea that roX2 assembly may occur at an earlier step than roX1, it was determined whether roX2 RNA assembles correctly in the absence of roX1. It was found that roX2 RNA is localized to the male X chromosome in the absence of roX1, with no apparent perturbation of its staining pattern. It is concluded that roX2 RNA, like the MSL protein complex, is independent of roX1 for localization on the X chromosome (Meller, 2000).

MSL complexes can be immunoprecipitated from Schneider SL2 tissue-culture cells, which exhibit male-like X chromosomal MSL complex and histone H4Ac16 by immunostaining. Therefore, reverse transcription (RT)-PCR analyses was performed to determine whether roX RNA could be coprecipitated with MSL complexes from SL2 cell extracts. Focus was placed on roX2 because the genetic experiments had predicted that it may be included at an early step in MSL complex assembly. RT-PCR products were first confirmed from male larval total RNA, SL2 cell total RNA, and SL2 cell protein lysates, using primers flanking an intron of 141 bp. Surprisingly, a smaller RNA with a 270 bp intron removed is the predominant product detected in both male larvae and SL2 cell total RNA. The intron encompasses the 141 bp intron and utilizes the same 3' splice site. The spliced roX2 RNA is detected in immunoprecipitates from SL2 cells using anti-Msl1 and anti-Mle antibodies. It is concluded that the MSL proteins are physically associated with the spliced form of roX2 RNA (Meller, 2000).

Until recently, it had been assumed that X-chromosome-specific cis-acting sequences would be associated with most genes bound by the MSL proteins. However, studies of roX1 have suggested that, if provided with a chromatin entry site (a roX1 transgene), the MSL complex could be attracted in cis to genes on autosomes that were never before dosage-compensated. The complex could not normally gain access to the autosomes in wild-type males because the MSL chromatin entry sites are found only on the X chromosome. Here, it has been shown that roX2 transgene can also function as a chromatin entry site for spreading of MSL complexes into autosomal chromatin (Meller, 2000).

In addition to roX genes functioning as chromatin entry sites, it is thought that the RNAs themselves may play roles in MSL complex assembly or function on the X chromosome. Although large ribonucleoprotein machines are well known in the form of ribosomes, spliceosomes and telomerases, RNAs have not been considered important factors in transcriptional regulation. There have been recent reports, however, of transcriptional control by non-coding RNAs in several diverse systems. A prominent example is the Xist RNA, which has been shown to play a central role in silencing most of the genes along one of the two X chromosomes in mammals. Another example is SRA, a novel non-coding RNA discovered as a coactivator of steroid hormone receptors. Individually, roX RNAs do not appear essential for MSL complex assembly on the X chromosome, but the phenotype of mutant embryos that lack roX1, roX2 and an unknown number of neighboring genes suggest that assembly of the MSL complex is delayed or abolished when both RNAs are removed (Meller, 2000 and references therein).

These data are consistent with assembly of roX RNAs into MSL complexes: (1) roX2 RNA can be immunoprecipitated from male tissue-culture cells, using anti-Msl1 or anti-Mle antibodies; (2) the roX RNAs are highly unstable in the absence of MSL proteins even if their synthesis is driven by a strong constitutive or inducible promoter; (3) movement of roX2 RNA from its site of synthesis to other chromatin entry sites requires Mle helicase. These observations are most easily understood if the MSL proteins physically contact the roX RNAs, protecting them from cytoplasmic export or degradation and targeting them instead to the X chromosome (Meller, 2000).

One clear difference between roX1 and roX2 RNAs is that roX2 RNA can travel to ~35 other chromatin entry sites along the X chromosome in the absence of Msl3. Maturation of the complex is thought to take place at these sites and then to initiate spreading into flanking chromatin. In the same nuclei lacking Msl3 protein, roX1 RNA is found only at its site of transcription. This suggests that the protein and RNA components of the MSL complex may assemble in a stepwise manner at several sites, and that roX2 may play a role in early events (Meller, 2000).

These results support three interrelated roles for roX genes in dosage compensation: (1) to produce RNA components of the MSL complex; (2) to provide sites for complex assembly and, (3) to mark the X chromosome for dosage compensation by acting as nucleation sites for spreading the MSL complex in cis to surrounding genes. A model has been presented for an ordered pathway of MSL-roX assembly, in which roX2 RNA joins partial MSL complexes at an early step and is exported to other chromatin entry sites in a process dependent on the Mle helicase. The roX2-containing complexes may complete assembly at other chromatin entry sites, perhaps by acquiring a second roX RNA species (such as roX1). Only mature complexes are functional to spread and dosage compensate flanking genes on the X chromosome (Meller, 2000).

Search PubMed for articles about Drosophila Rox2

Hallacli, E., Lipp, M., Georgiev, P., Spielman, C., Cusack, S., Akhtar, A. and Kadlec, J. (2012). Msl1-mediated dimerization of the dosage compensation complex is essential for male X-chromosome regulation in Drosophila. Mol Cell 48: 587-600. PubMed ID: 23084835

Ilik, I. A., Quinn, J. J., Georgiev, P., Tavares-Cadete, F., Maticzka, D., Toscano, S., Wan, Y., Spitale, R. C., Luscombe, N., Backofen, R., Chang, H. Y. and Akhtar, A. (2013). Tandem stem-loops in roX RNAs act together to mediate X chromosome dosage compensation in Drosophila. Mol Cell 51: 156-173. PubMed ID: 23870142

Kelley, R. L., Meller, V. H., Gordadze, P. R., Roman, G., Davis, R. L. and Kuroda, M. I. (1999). Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 98: 513-522. PubMed ID: 10481915

Kelley, R. L., Lee, O. K. and Shim, Y. K. (2008). Transcription rate of noncoding roX1 RNA controls local spreading of the Drosophila MSL chromatin remodeling complex. Mech Dev 125: 1009-1019. PubMed ID: 18793722

Kim, M., Faucillion, M. L. and Larsson, J. (2018). RNA-on-X 1 and 2 in Drosophila melanogaster fulfill separate functions in dosage compensation. PLoS Genet 14(12): e1007842. PubMed ID: 30532158

Kugel, J. F. and Goodrich, J. A. (2012). Non-coding RNAs: key regulators of mammalian transcription. Trends Biochem Sci 37: 144-151. PubMed ID: 22300815

Lee, J. T. (2012). Epigenetic regulation by long noncoding RNAs. Science 338: 1435-1439. PubMed ID: 23239728

Lim, C. K. and Kelley, R. L. (2012). Autoregulation of the Drosophila Noncoding roX1 RNA Gene. PLoS Genet 8: e1002564. PubMed ID: 22438819

Lundberg, L. E., Kim, M., Johansson, A. M., Faucillion, M. L., Josupeit, R. and Larsson, J. (2013). Targeting of Painting of fourth to roX1 and roX2 proximal sites suggests evolutionary links between dosage compensation and the regulation of the fourth chromosome in Drosophila melanogaster. G3 (Bethesda) 3: 1325-1334. PubMed ID: 23733888

Maenner, S., Muller, M., Frohlich, J., Langer, D. and Becker, P. B. (2013). ATP-dependent roX RNA remodeling by the helicase maleless enables specific association of MSL proteins. Mol Cell 51: 174-184. PubMed ID: 23870143

Meller, V. H., Gordadze, P. R., Park, Y., Chu, X., Stuckenholz, C., Kelley, R. L. and Kuroda, M. I. (2000). Ordered assembly of roX RNAs into MSL complexes on the dosage-compensated X chromosome in Drosophila. Curr Biol 10: 136-143. PubMed ID: 10679323

Menon, D. U. and Meller, V. H. (2009). Imprinting of the Y Chromosome Influences Dosage Compensation in roX1 roX2 Drosophila melanogaster. Genetic 183(3): 811-820. PubMed Citation: 19704014

Militti, C., Maenner, S., Becker, P. B. and Gebauer, F. (2014). UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation. Nat Commun 5: 4762. PubMed ID: 25158899

Morales, V., Straub, T., Neumann, M. F., Mengus, G., Akhtar, A. and Becker, P. B. (2004). Functional integration of the histone acetyltransferase MOF into the dosage compensation complex. EMBO J 23: 2258-2268. PubMed ID: 15141166

Moran, V. A., Perera, R. J. and Khalil, A. M. (2012). Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs. Nucleic Acids Res 40: 6391-6400. PubMed ID: 22492512

Park, S. W., Kang, Y., Sypula, J. G., Choi, J., Oh, H. and Park, Y. (2007). An evolutionarily conserved domain of roX2 RNA is sufficient for induction of H4-Lys16 acetylation on the Drosophila X chromosome. Genetics 177: 1429-1437. PubMed ID: 18039876

Park, S. W., Kuroda, M. I. and Park, Y. (2008). Regulation of histone H4 Lys16 acetylation by predicted alternative secondary structures in roX noncoding RNAs. Mol Cell Biol 28: 4952-4962. PubMed ID: 18541664

Park, Y., Mengus, G., Bai, X., Kageyama, Y., Meller, V. H., Becker, P. B. and Kuroda, M. I. (2003). Sequence-specific targeting of Drosophila roX genes by the MSL dosage compensation complex. Mol Cell 11: 977-986. PubMed ID: 12718883

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date revised: 12 December 2022

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