RNA on the X-1

DEVELOPMENTAL BIOLOGY

Embryonic

Expression of roX1 is initally observed in blastoderm-stage embryos of both sexes, about 2.5 hours after egg laying. Expression becomes stronger during gastrulation and is especially marked in the elongated germband [Images]. Rows of neuroblast precursors stain prominently. Patterns seen during germband elongation are observed in both male and female embryos, indicating that they are specific to developmental stage rather than sex. At about 8 hours of development, cells migrate inwards to form the CNS, which becomes heavily loaded with transcript. There is prominent expression in newly formed brain hemispheres as well as in a row of cells in the ventral midline. It is during germband retraction, about 8 to 9 hrs after the start of development, that the first clear sexual dimorphism in expression is seen. Transcripts can be detected in nonneural tissues in males but not in females. Staining disappears from the female CNS, first from the ventral cord then from the brain (Meller, 1997).

The roX1 and roX2 genes of Drosophila produce non-coding transcripts that localize to the X-chromosome. In spite of their lack of sequence similarity, they are redundant components of an RNA/protein complex that up-regulates the male X-chromosome, contributing to the equalization of X-linked gene expression between males and females. roX1 is detected at 2 h AEL, prior to formation of the complex, and is present in both sexes. Maternally provided MLE (Maleless) is required for roX1 stability. By contrast, roX2 is male-specific and is first observed at 6 h. Either of the two roX transcripts can support X-localization of the complex, but localization is delayed in roX1 mutants until roX2 expression. These results support a model for the ordered assembly of the complex in embryos (Meller, 2003).

roX1 has been reported to be male-limited in embryos, but expression in both sexes has also been observed. An intriguing possibility is that these conflicting reports are attributable to the presence of antisense transcripts in females. These would have been detected by the double stranded DNA probes used in the initial study, but would not have been detected by the single stranded riboprobes subsequently used. To address this possibility, in situ analysis was repeated using sense and antisense RNA probes to the roX1 region. No roX1 signals were detected in preblastoderm embryos. Antisense roX1 probes detect transcript in embryos at the blastoderm stage. Male and female embryos may be distinguished by the use of a female-specific LacZ reporter. Following X-Gal staining, both male and female embryos expressing roX1 were readily identified. No transcripts overlapping the roX1 gene were detected using sense riboprobes. Although roX1 RNA is transcribed in embryos of both sexes, roX1 expression is strikingly male-preferential in larvae and adults. Loss of the roX1 transcript from females mid way through embryogenesis has been noted, and the onset of sex-specific roX1 expression is consistent with the reduction in signal from an antisense roX1 probe at 10 h AEL (Meller, 2003).

By contrast, roX2 is much less strongly expressed in embryos and is undetectable before 6 h. In wild type populations, 50% of older embryos have roX2 signal and these are invariably male, as revealed by in situ hybridization to embryos carrying the LacZ reporter. Although the roX1 and roX2 probes used in these studies produce signals of similar strength when hybridized to salivary glands from third instar male larvae, roX2 staining of embryos is much weaker than roX1 staining. This indicates that the relative levels of roX1 and roX2 transcripts shift as development proceeds, with roX1 prominent during embryogenesis and the two roX RNAs becoming more equivalent in the third larval instar (Meller, 2003).

Localization of the MSL proteins to the salivary gland X-chromosome requires formation of the intact complex, and hence the presence of all the MSL proteins. At least one roX RNA is similarly required for localization in larvae, and a prior study using roX1^mb710 combined with an embryonic lethal deletion removing roX2 suggests that one roX gene is also required for localization in male embryos. However, this study used deletions also removed the largest subunit of RNA polymerase II (RPII215), which is less than 10 kb distal to roX2. The resulting disruption in zygotic gene expression may affect dosage compensation non-specifically. The requirement for roX transcripts is therefore determined in embryos mutated for each of the roX genes, but otherwise fully viable (Meller, 2003).

Previous studies have documented the male-specificity of MSL2 immunoreactivity in embryos, and have established that MSL2 can first be detected localizing to the X-chromosome at the end of the blastoderm stage, about 3 h AEL. X-localization, which is dependent on the presence of all of the MSL proteins, precedes the onset of dosage compensation as detected by enrichment of H4Ac16. X-chromosome localization produces a characteristic punctate MSL immunostaining pattern that is readily distinguished from non-localized immunoreactivity. As expected, anti-MSL2 antibodies labeled ~50% of gastrulating wild type embryos, and the appearance of punctate staining indicative of X-chromosome localization could be clearly observed in males older than 3 h AEL. Populations of embryos in which all males were of identical genotype were produced, thus simplifying the scoring of stained preparations. One-third of the developing embryos from these collections are male, and MSL2 immunoreactivity is detected in the anticipated proportion of embryos. Embryos in which MSL2 was detected with HRP were scored for developmental stage and localized staining. When males carry wild type roX genes, the appearance of punctate MSL2 immunoreactivity is first detected at 3 h and is observed in approximately one-third of older embryos. Df(1)52 is a deficiency of less than 30 kb that removes roX2 and several closely linked essential genes, including RPII215. Male embryos carrying the Df(1)52 chromosome produce MSL2, but its localization is delayed by >2 h. Viability can be restored to Df(1)52 flies by supplying cosmid [w⁺4Delta4.3], which carries all essential genes removed by the deletion but lacks roX2. [w⁺4Delta4.3] restores the normal timing of MSL2 localization in Df(1)52 males. The roX2 gene is therefore unnecessary for the initial formation of the MSL complex or its localization. However, disruption of development by deletion of the region surrounding roX2 delays the onset of MSL2 localization in a manner unrelated to the presence of the roX2 gene (Meller, 2003).

The timing of MSL2 localization suggests that roX1 could be necessary for initial X-localization of the dosage compensation complex. This was tested by determining the timing of MSL2 localization in roX1^ex6 males. The roX1^ex6 allele was created by an imprecise excision removing 1.4 kb from the 5' end of the gene. roX1 RNA is not detected in larvae or adults carrying this allele. Although MSL2 could be detected in one-third of the developing embryos from these collections, it did not appear in a punctate pattern until 7 h. Therefore, in roX1^ex6 males there is a 4 h lag in localization of the complex, which now follows shortly after roX2 expression. X-chromosomes mutated for both roX genes were used to determine the ability of MSL2 to localize in the absence of any wild type roX RNA. MSL2 expression is detected in these embryos, but the strong foci normally observed upon localization of the MSL complex to the X-chromosome are not observed in roX1^ex6 roX2^- males. However, weak foci of MSL2 staining could be detected in some mutant embryos during germ band retraction, and these are more apparent when detected by immunofluorescence. Although differing from the more consistent and intense foci observed in wild type males, the presence of weak foci in roX1^ex6 roX2^- embryos indicates that MSL2 retains some ability to localize within the nucleus, even in the absence of a wild type roX gene. This is consistent with the observation that, although strikingly reduced, some X-localization of MSL2 is retained on polytene preparations from roX1^ex6 roX2^- males (Meller, 2003).

During larval and adult stages, roX1 is highly unstable in the absence of an intact dosage compensation complex. The embryonic transcription of roX1 precedes expression of MSL2 and formation of the complex, yet roX1 appears stable in embryos. In addition, until 10 h AEL roX1 transcripts are detected in female embryos, which lack MSL2. With the exception of MSL2, the MSL proteins are maternally provisioned and support the formation of the initial dosage compensation complexes. To test the hypothesis that one or more of these maternal proteins is responsible for roX1 stability, roX1 was examined in embryos from mothers homozygous for mutations in each of the msl genes. As anticipated, the maternal genotype with respect to the missense msl2¹ mutation has no effect. Early roX1 expression is also unchanged in embryos from females homozygous for a null allele of male-specific lethal 1 (msl1^L60, a 2 kb deletion removing most of the coding region), male-specific lethal 3 (msl3²), and males absent on first (mof¹ and mof², missense and nonsense mutations, respectively). By contrast, although an initial burst of roX1 expression is detected in blastoderm stage embryos produced by mothers homozygous for the nonsense mle¹ mutation, roX1 disappears upon gastrulation. Between 4 and 5 h, roX1 can once more be detected. MLE is a member of the DExH family of helicases, and has RNA and DNA helicase activity in vitro. Interestingly, a similar lack of roX1 stability was detected in embryos expressing the mutated MLE^DQIH protein that lacks helicase activity, indicating that this activity is required for roX1 stability. MLE has also been linked to the ability of the roX transcripts to travel from their sites of synthesis on the polytene X-chromosome. In embryos from mle¹ mothers, spots of transcription within blastoderm nuclei are particularly apparent, and embryos displaying either one or two sites of synthesis per nucleus are readily observed. This suggests that MLE is also required to move roX1 from its site of synthesis in embryos. These results demonstrate that none of the MSL proteins are required for initial roX1 transcription, but maternal stores of MLE contribute to roX1 RNA stability in early embryos (Meller, 2003).

roX2, but not roX1, can move to many sites on polytene X-chromosomes in the absence of MSL3, suggesting that these transcripts have distinct functionality in assembly or localization of the complex. The differences between roX1 and roX2 expression during embryogenesis further imply that these RNAs might fulfil different functions during the establishment of dosage compensation. Removal of the primary early source of roX RNA by the roX1^ex6 mutation delays the onset of a punctate MSL2 staining pattern, which indicates X-localization, until roX2 is transcribed several hours later. Therefore, roX2 can support a delayed initiation of dosage compensation, and will do so if roX1 is unavailable. The temporal linkage of roX2 RNA expression to the localization of MSL2 in roX1 mutants, and the more profound disruption of MSL2 localization by mutation of both RNAs, support assertions that one of these RNAs is required for correct targeting of the dosage compensation complex to the male X-chromosome in embryos and larvae. In spite of the striking differences in the timing, amount and sex-specificity of roX1 and roX2 during embryogenesis, these transcripts appear interchangeable in their ability to direct the initiation of dosage compensation (Meller, 2003).

The occasional observation of weak foci of MSL2 staining in older male embryos carrying X-chromosomes mutated for both of the roX genes suggests either that roX RNA is not absolutely essential for localization of the dosage compensation complex, or that the roX1^ex6 allele, a partial excision, retains some ability to produce functional transcripts, or that other genes produce transcripts which can partially support complex assembly and localization. All of these theories have been previously raised to account for a low level of developmentally delayed adult male escapers that are roX1^ex6 roX2^- (Meller, 2003 and references therein).

The findings of this study will support a model for the assembly of dosage compensation complexes during embryogenesis. roX1, highly expressed in all blastoderm stage embryos, is transcribed in advance of MSL2 translation and is likely to form an initial complex with MLE. It is possible that several of the MSL proteins must contact roX1 during assembly of the complex. In all, three members of the dosage compensation complex, MLE, MSL3 and MOF, have been reported to have RNA binding activity in vitro, or to be removed from the X-chromosome by RNase A digestion. With the exception of MSL2, all of the MSL proteins are present upon initial transcription of roX1 at 2 h AEL. The onset of dosage compensation has been linked to the male-limited production of MSL2 about 3 h AEL. This sequence of events suggests that MSL2 may complete a complex that is already organized by several RNA-binding proteins and the roX1 transcript. The proposed primary association between roX1 and MLE could reflect a need for the MLE helicase activity to disrupt incorrect base pairing or RNA/protein interactions preventing the large roX1 transcripts from correctly assembling with the other RNA-binding proteins of the dosage compensation complex (Meller, 2003).

Larval

Expression is seen in the salivary gland of third-instar larval males (Amrein, 1997). Staining is clearly nuclear, confined to discrete compartments in the large polyploid cells of the gastric ceca, the ring gland and the nephrocytes, and in the polytene salivary glands. There is a complete overlap of staining in salivary glands labeled by double staining with roX1 probes and MSL-1 antibody (see MSL-2), indicating that the roX1 transcript binds the male X chromosome (Meller, 1997).

Adult

roX1 transcripts are present in cells of the central nervous system of male but not female flies. Most or all cells in the brain and thoracic ganglia stain strongly. Weaker signals are observed in the gut, in parts of the reproductive tract, especially the ejaculatory bulb, and in fat cells (Meller, 1997). roX1 is about 4-fold more abundant than roX2. The two male-specific genes are expressed in the cell bodies of the central brain and the mushroom bodies, the neurons of the optic lobe (the lamina and medulla), and the interneurons of the antennal lobes. Both genes are also expressed in peripheral neurons, notably the photoreceptor cells, as well as the olfactory neurons in the third antennal segment. Expression is observed in the thoracic gland and in a small region of the digestive tract. No expression is observed in larval or adult females (Amrein, 1997).

Effects of mutation or deletion

Mutant flies are without any obvious defects in viability or fertility. Two imprecise excisions of the gene that remove several kilobases of the transcribed roX1 RNA are without obvious defects (Meller, 1997).

The roX1 and roX2 genes of Drosophila produce male-specific non-coding RNAs that co-localize with the Male-Specific Lethal (MSL) protein complex. This complex mediates up-regulation of the male X chromosome by increasing histone H4 acetylation, thus contributing to the equalization of X-linked gene expression between the sexes. Both roX genes overlap two of ~35 chromatin entry sites, DNA sequences proposed to act in cis to direct the MSL complex to the X chromosome. Although dosage compensation is essential in males, an intact roX1 gene is not required by either sex. Flies have been generated lacking roX2 and this gene is also found to be non-essential. However, simultaneous removal of both roX RNAs causes a striking male-specific reduction in viability accompanied by relocation of the MSL proteins and acetylated histone H4 from the X chromosome to autosomal sites and heterochromatin. Males can be rescued by roX cDNAs from autosomal transgenes, demonstrating the genetic separation of the chromatin entry and RNA-encoding functions. Therefore, the roX1 and roX2 genes produce redundant, male-specific lethal transcripts required for targeting the MSL complex (Meller, 2002).

The X-localization of roX transcripts, their male-specificity and regulation of roX RNA accumulation by the MSL proteins all have suggested a role for these RNAs in dosage compensation. However, failure to detect a phenotype in males mutated for roX1 and the absence of precise mutations in roX2 has previously precluded formal tests of this possibility. Furthermore, slight differences between the roX RNAs, such as the ability of transcripts from roX2, but not roX1, to move to all entry sites on the X chromosome in a fly mutated for msl3, and the identification of flies and cell lines lacking roX1, but none lacking roX2, have supported the notion that roX2 might be an essential gene in males. However, the mutation strategy used in this study, generation of a lethal deletion and restoration of essential gene functions with a genomic cosmid, has produced healthy males lacking roX2 RNA. When the roX2 deletion was combined with a roX1 mutation, male-specific lethality was revealed. Heat shock-driven expression of either roX RNA restores the viability of doubly mutated males, indicating that the male-specific phenotype is due to lack of roX RNA only. These experiments show that roX2 is not essential in males, and the synthetic lethality detected when both roX genes are mutated eliminates the possibility of undetected roX transcripts with similar function. The roX2 deletion removes the roX2-associated chromatin entry site at 10C, and all but 40 bp of the roX1 site is removed in roX1 ^ex6. Because male viability is rescued by roX RNA in trans, male lethality is consequential to loss of the roX transcripts and not to mutation of the chromatin entry sites. It is concluded that the RNA products of the roX genes are redundant male-specific lethals. The roX2 deletant that was generated is still complex, since two genes flanking roX2 are disrupted by Df(1)52 and not restored by cosmid [w ⁺4Delta4.3]. The rescue of male viability by roX cDNAs also demonstrates that male lethality is not an unintended consequence of other mutations associated with the Df(1)52 chromosome or the rescuing [w ⁺ 4Delta4.3] transgene (Meller, 2002).

The involvement of the roX RNAs in dosage compensation can be functionally demonstrated by the rescue of females forced to express msl2. Production of the MSL2 protein triggers the formation of intact MSL complexes that bind to the X chromosome and enhance the transcription of X-linked genes. Female expression of msl2 leads to an inappropriate up-regulation of both X chromosomes resulting in a high level of lethality that can be blocked by mutations in another of the protein-coding msl genes. Removal of both roX RNAs similarly rescues msl2-expressing females. Partial rescue of female lethality by elimination of only one of the roX RNAs is consistent with previous results indicating that females expressing msl2 from the [w ⁺ H83M2-6I] transgene are a sensitized genetic background in which changes in the level of components of the MSL complex may be detected. These studies specifically demonstrate a block of dosage compensation when both roX RNAs are eliminated (Meller, 2002).

In contrast to the absence of escapers from mutations in the protein-coding msl genes, the doubly mutant X chromosomes used in this study did allow a low number of escaper males. This is a surprising finding in light of the disruption in MSL localization and loss of H4Ac16 enrichment on the X chromosome. It suggests that in spite of the clear importance of the roX RNAs for histone acetylation, compensation of the X chromosome is not completely compromised in the roX ^- males that were generated. It is possible that the roX RNAs are peripheral to formation of the MSL complex. Residual MSL2 binding on the X chromosome of roX ^- males would be consistent with this. Partial complexes with reduced activity could also form in the absence of the roX RNAs, and the simultaneous enrichment at the chromocenter of MSL1, MSL2 and H4Ac16 in roX ^- males supports the idea that some or all of the MSL proteins may still assemble into a complex capable of acetylating histone H4. It is also possible that the available roX1 mutations are not complete loss-of-function alleles (Meller, 2002).

Two lines of evidence support the idea that the MSL proteins, in the absence of the roX RNAs, can act to regulate gene expression. (1) MSL1, MSL2 and H4Ac16 are similarly redistributed in roX ^- males, suggesting that a protein complex able to direct histone acetylation still forms in the absence of roX RNA, although its localization is disrupted. (2) The sites of autosomal accumulation of the MSL proteins in roX ^- males are often puffed, suggestive of locally high rates of transcription. Autosomal sites of MSL3 binding have been reported in wild-type males, and MSL2 has been detected at several sites, including three specifically noted as binding MSL3. The one focused upon, 21B, displays fairly weak MSL2 staining in wild-type males but is often strongly stained and puffed in preparations from roX ^- males. It is suggested that an MSL complex lacking roX transcripts normally functions at a few autosomal sites in wild-type males. It is plausible that these represent a handful of genes with sex-specific expression. Removal of roX RNA releases the bulk of the MSL proteins from the X chromosome, and these are consequently available at high titers for association with the autosomal sites. Puffing of these sites suggests that elevated levels of the MSL proteins can hyper-activate transcription (Meller, 2002).

Alternatively, it is possible that all targeting of the MSL proteins to chromatin requires an RNA cofactor, but only roX1 and roX2 serve to direct the MSL proteins to the X chromosome. This would evoke the presence of other roX-like RNAs that specify a more restricted set of targets, perhaps a few autosomal genes. The potential utility of a system of gene activation that can be redirected by deploying an assortment of transcripts is quite attractive; however, in this instance the restriction to a single sex would limit the range of target genes considerably (Meller, 2002).

Mislocalization of MSL2 is strikingly different in roX ^- males than in those lacking mle, msl3 or mof, where residual MSL2 is observed binding only to ~35 chromatin entry sites. This points to a role for roX RNA in correct targeting of the intact complex to the X chromosome. The mechanism driving relocation of the MSL proteins to heterochromatin is at this point speculative. Two of the components of the MSL complex, MSL3 and MOF, contain variant chromodomain motifs. Chromodomains are involved in targeting HP1 to heterochromatin, and localization depends on the interaction of this domain with histone H3 methylated on lysine 9, a modification found in heterochromatic regions. The variant chromodomains of MSL3 and MOF have been shown to bind RNA in vitro, and it is likely that the roX RNAs are their normal ligands. Removal of roX transcripts could allow these domains to engage in inappropriate protein-protein interactions that target the remaining members of the MSL complex to heterochromatin (Meller, 2002).

Functional redundancy within roX1, a noncoding RNA involved in dosage compensation

Drosophila males dosage-compensate by twofold upregulation of the expression of genes on their single X chromosome. This process requires at least five proteins and two noncoding RNAs, roX1 and roX2, which paint the male X chromosome. A deletion analysis was used to search for functional RNA domains within roX1, assaying RNA stability, targeting of the MSL proteins to the X, and rescue of male viability in a roX1^- roX2^- mutant background. Deletion of 10% segments of the RNA did not dramatically reduce function in most cases, suggesting extensive internal redundancy. The 3' 600 nt of roX1 were most sensitive to mutations, affecting proper localization and 3' processing of the RNA. Disruption of an inverted repeat predicted to form a stem-loop structure was found partially responsible for the defects observed (Stuckenholz, 2003).

The ~3.7-kb roX1 RNA is predicted to make up over half the mass of the MSL complex. Yet, surprisingly, most of the roX1 is dispensable for function, as demonstrated by the observation that constructs lacking almost any 10% of the sequence still rescue roX1^- roX2^- double-mutant male lethality similar to a wild-type roX1 cDNA. Comparison of a 900-bp deletion construct (3' roX1), with the overlapping 300- to 400-nt deletions roX1Delta1-roX1Delta3 show that this region contains at least two redundant elements. It is possible that several additional redundant elements are located within the region of roX1 defined by deletions roX1Delta4-roX1Delta9. The finding that most small domains are dispensable is consistent with the absence of primary sequence homology between the functionally interchangeable roX1 and roX2 RNAs. Whatever roX RNAs do, their functions likely depend on multiple, complex tertiary folds, not primary nucleotide sequence. The one short 30-nt sequence element shared between roX1 and roX2 could be deleted without obvious consequence. This finding is similar to results obtained with Xist, where conserved sequence elements were not necessarily important for RNA function. The deletion in roX1Delta4 is identical to a deletion called roX1DeltaDHS that removes a male-specific DNaseI hypersensitive (DHS) site in the roX1 gene. The DHS site is the principal DNA-binding site for the MSL complex within the roX1 gene, but the sequence has been found to be dispensable in the roX1 RNA (Stuckenholz, 2003).

In addition to functional redundancy in the 5' end of roX1, the 3' end is most important for full function of the RNA. Deleting sequences within the last 600 nt of the RNA resulted in markedly lowered rescue efficiency and abnormal RNA and MSL complex localization. A predicted stem-loop structure in this region is partially responsible for the loss of rescue activity, but it is suspected that a second, partially redundant element is removed by the roX1Delta10 deletion (Stuckenholz, 2003).

roX1 RNA may not undergo typical polyadenylation, but these studies do not address this in detail. Whether roX1 RNA is polyadenylated or is enriched on oligo(dT) columns through internal A-rich stretches is not known. The absence of consensus polyadenylation sites raises the possibility that an unusual mechanism might be employed to terminate the RNA. This mechanism might be important for keeping the RNA in the nucleus and thus for localization of both the MSL proteins and the RNA to the X chromosome (Stuckenholz, 2003).

No physical information about the structure of the MSL RNA-protein complex is available. It is not known whether both roX1 and roX2 are present in one MSL complex or if two different complexes exist, one with roX1 and one with roX2. It is not known whether roX1 RNA is flexible or adopts a rigid structure in the MSL complex, possibly stabilized by many weak RNA-protein interactions. However, the results are compatible with a model in which the RNA is more flexible and perhaps surrounds an MSL protein core. In one view of dosage compensation, the histone-modifying enzymes account for the altered chromatin found on the male X and the resulting twofold increase in transcription. The main function of the roX RNAs might therefore be to restrict MSL action to the X chromosome in vivo. This is consistent with the observations that roX1Delta10 and roX1Delta11 fail to rescue well and mislocalize the mutant MSL complexes to many autosomal sites. It might also account for the presence of escaper roX1^- roX2^- males: since MSL complexes still bind some sites on the X chromosome in the absence of any roX RNAs, enzymatically active MSL proteins might weakly act on the entire genome. Weak H4Ac16 histone acetylation has been observed in roX1^- roX2^- males; therefore, rare males might have sufficient levels of histone modifications on the X chromosome without fatal levels of increased autosomal activity (Stuckenholz, 2003).

The finding of extensive redundancy within the roX1 RNA is reminiscent of a recent study examining Xist using a similar deletion strategy. This study also found multiple redundancies within the Xist RNA (Wutz, 2002). Additional examples from other areas of RNA biology include redundant RNA localization signals in the 3' untranslated region of bicoid in Drosophila and of Vg1 in Xenopus and interactions between HIV Rev and its cognate RNA element, the RRE. The interaction between Rev and the RRE may prove relevant: the Rev protein binds at a bulge in the RRE, a large RNA structure. Once the first Rev molecule is bound to the RRE, however, additional Rev molecules can load onto the same RNA. It is possible that the region within roX1Delta10 is required for the efficient binding of a first MSL protein to the RNA. Once this first protein is bound, more MSL proteins can bind to the RNA and make it fully functional. In this model, most of the RNA would consist of secondary MSL-binding sites of lower affinity, which could result in inefficient loading of the MSL complex to the roX1Delta10 mutant RNA (Stuckenholz, 2003).

roX1 and roX2 are redundant with each other and functional redundancy has been demonstrated within roX1. If both RNAs exert their function through a common tertiary structure, this structure must be able to form despite completely different primary sequences. There are examples of proteins that fold into highly similar structures despite their lack of sequence identity. The versatility in intramolecular interactions within RNAs may be instrumental in allowing them to assume many varied tertiary structures, but most RNA structures are still completely unknown. Presumably RNAs as different as roX1 and roX2 fold into a common tertiary structure necessary for dosage compensation in Drosophila (Stuckenholz, 2003).

The severity of roX1 mutations is predicted by MSL localization on the X chromosome

Dosage compensation equalizes the expression of sex-linked genes between males and females. Most genes on the X chromosome of male Drosophila are transcribed at an increased level, contributing to compensation. The roX1 and roX2 genes produce non-coding transcripts that localize along the X-chromosome of male flies. Although lacking sequence similarity, they are necessary but redundant components of a system that up-regulates gene expression. Simultaneous mutation of both roX genes disrupts the X-limited distribution of proteins that modify chromatin to enhance gene expression. Loss of function roX1 alleles have been generated and characterized that display a continuum of activity. Those that support intermediate male survival have strikingly reduced RNA accumulation, while alleles with minor contributions to male viability typically lack detectable transcript accumulation. Severely mutated roX1 alleles retain some ability to direct modifying proteins to the X chromosome. This ability predicts the level of male survival that each allele supports. This points to a peripheral or transient role for roX in the RNA and protein complex that binds to and regulates the X chromosome (Deng, 2005).

The need to accurately target modifications to an entire chromosome is shared by organisms that compensate the gene dosage of their sex chromosomes. The roX genes occupy a central position in the targeting of MSL complexes to the single X chromosome of Drosophila males. The surprising finding that even molecularly severe roX1 mutations with dramatically reduced transcript accumulation, such as roX1^ex7B and roX1^ex33A, or no detectable accumulation, such as roX1^ex6, retain a low level of roX activity raises questions about the role of roX transcripts in this process. The roX genes appear capable of directing the MSL complex to the X chromosome in two distinct ways. Both roX genes are able to direct compensation to chromatin in cis. This activity appears to reside, at least in part, in short DNA sequences overlapping roX1 and roX2. These sequences attract residual MSL proteins in msl3 mutants and form DNase hypersensitive sites (DHS) in males. roX1^ex40A, deleted for the DHS, supports full male viability when roX2 is also deleted. This indicates that neither roX-associated DHS is necessary for effective dosage compensation. However, these experiments do not eliminate the possibility that other cis-acting sequences associated with the roX genes are capable of activity (Deng, 2005).

In addition to the speculative importance of cis-acting sequences, roX RNA is necessary for ensuring exclusive localization of the MSL proteins to the X chromosome. Proteins similar to the MSLs are found in organisms from yeast to humans, suggesting that dosage compensation in Drosophila originated with the recruitment of preexisting regulatory factors to the X chromosome. Silencing of the mammalian X chromosome similarly depends on recruitment of conserved regulatory factors, including the Polycomb Group (Pc-G) proteins, by Xist. The shared reliance on non-coding RNA to direct chromatin-modification complexes to specific chromosomes is likely to reflect the origin of dosage compensation (Deng, 2005).

In cells that dosage compensate normally, roX transcripts are immunoprecipitated with the MSL proteins. This has been taken as an indication that roX association with the complex is constant and likely to be the factor that ensures X-localization. However, roX1 mutations that support intermediate levels of MSL1 localization to the X chromosome, such as roX1^ex7B and roX1^ex33A, do so with strikingly diminished levels of mutant transcript. One possibility is that mutated roX transcripts transiently associate with the MSL proteins during targeting of the complex. roX1^ex7B transcription appears normal, supporting the idea that mutated transcripts may transiently associate with the MSL proteins. The association of MSL proteins with chromatin, once established, may be stable even in the absence of roX transcript. It is also possible that the requirement for MSL proteins to bind the X chromosome is itself transient. One of the primary functions of the MSL complex is believed to be histone H4 acetylation. A subsequent sex-specific remodeling of X chromatin depends on enrichment for H4Ac16. This suggests that compensation of the male X chromosome is comprised of an ordered series of chromatin modifications. In spite of the long term association of the MSL complex with the male X chromosome, it may only be crucial for initial steps. This model is similar to that for the function of Xist in recognition and modification of the X chromosome of mammalian females. Xist production is required for initiation of compensation. An early event is Xist spreading from its site of synthesis to coat one of the two X chromosomes (reviewed by Wutz, 2002). Xist recruits Pc-G complexes that are responsible for methylation of H3 (Plath, 2003, Plath, 2004; Silva, 2003). Subsequent modifications, including DNA methylation, render the X chromosome refractory to reactivation even after Xist removal (Deng, 2005).

The most prominent accumulation of roX1^ex7B and roX1^ex33A transcripts in eye-antennal imaginal discs is in the morphogenetic furrow and posterior to the furrow. These cells are exiting the cell cycle and undergoing terminal differentiation. Although the current studies do not differentiate between tissue-specific factors increasing roX1 transcription and differential transcript stability, they do reveal that local levels of mutated roX transcripts may be highly variable. In spite of a dramatic, overall reduction of roX1 in the roX1^ex7B and roX1^ex33A mutants, regions of local accumulation may support targeting of the MSL complex at critical developmental stages, allowing the ultimate recovery of male escapers (Deng, 2005).

When increasingly severe roX1 mutations are the only roX source, the amount of MSL1 localized to the X chromosome is decreased and ectopic MSL1 binding to autosomal sites is observed. Two potential sources of gene misregulation may consequently contribute to the poor survival of roX1 roX2 males. Failure to direct MSL complex to X-linked genes is believed to cripple dosage compensation, leading to under expression of most of the X chromosome. This is presumed to be the source of lethality when an msl gene is mutated. However, roX1 roX2 males also display ectopic binding of the MSL complex at autosomal sites, and it is plausible that this results in misregulation of autosomal genes. Although roX1 alleles with intermediate levels of male survival direct considerable MSL protein to the X chromosome, they fail to ensure exclusive X-localization. Reduced male survival could thus be due to reduction in X-linked gene expression, misregulation of autosomal genes, or both. Simultaneous over expression of MSL2 and MSL1 increases the survival of roX1 roX2 males in spite of the fact that this treatment increases binding of the MSL proteins to ectopic autosomal sites as well as to the X chromosome. This points to a deficit in MSL protein binding to the X chromosome, rather than mislocalization to ectopic sites, as the primary source of mortality in roX1 roX2 mutant males (Deng, 2005).

The nature of the X-linked sites that retain MSL proteins in different mutant backgrounds is a continual source of speculation. In severe roX1 roX2 mutants sites retaining MSL1 on the X chromosome appear superficially similar to those visualized in msl3 backgrounds. However, the high affinity sites visualized in msl3 mutants are bright and fairly uniform, but residual MSL1 sites in roX1 roX2 flies are quite variable in intensity. Some sites retaining MSL1 in roX1 roX2 males also appear puffed. A comparison between sites observed in msl3 and roX1 roX2 mutants reveals that about half the msl3 high affinity sites are cytologically indistinguishable from sites that retain MSL1 in roX1 roX2 flies. Eight sites are detected only in msl3 larvae, two of these being the roX1 and roX2 genes themselves. The DNA sequences that form the roX-associated sites are deleted by the roX mutations used. An additional 8 sites are detected only in roX1 roX2 larvae. The heterogeneity of msl3 high affinity sites was established by identification of a site not associated with a roX gene. The current study suggests an even greater level of diversity. This may reflect differing strategies for capturing the dosage compensation machinery employed by chromatin domains or individual genes (Deng, 2005).

The level of MSL1 protein recruitment to the X chromosome appears linked to the severity of each roX1 mutation. roX1^ex6 and roX1^mb710 support a greater enrichment of MSL1 on the X chromosome than the more severe alleles roX1^ex84A and roX1^SMC17A. Although over expression of MSL1 and MSL2 partially rescues all roX1 roX2 males, the level of male rescue remains linked to the severity of the roX1 allele being tested. These observations provide evidence for male benefits derived from roX1^ex6 and roX1^mb710. The ability of these severely mutated roX1 genes to continue to direct some MSL protein to the X chromosome, although surprising, accounts for escaper males that each supports. By contrast, the severity of the roX1^SMC17AroX2 phenotype approaches that for mutation of a protein-coding msl gene. The nature of the roX1^SMC17A mutation, introduction of a transcriptional stop signal between two essential regions of roX1, suggests that inability to produce a partially functional transcript underlies the severity of this particular mutation. The severity of the roX1^SMC17AroX2 phenotype discounts the possibility that unidentified genes with weak roX function contribute to the support of escaper males (Deng, 2005).

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).

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date revised: 23 April 2019

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