Sxl Expression Research Articles

Sex-lethal regulates back-splicing and generation of the sex-differentially expressed circular RNAs.

Conversely to canonical splicing, back-splicing connects the upstream 3' splice site (SS) with a downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely identified and have regulatory functions in eukaryotic gene expression. However, sex-specific back-splicing in Drosophila has not been investigated and its regulation remains unclear. Here, we performed multiple RNA analyses of a variety sex-specific Drosophila samples and identified over ten thousand circular RNAs, in which hundreds are sex-differentially and -specifically back-spliced. Intriguingly, we found that expression of SXL, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene that is only spliced into functional proteins in females, promoted back-splicing of many female-differential circRNAs in the male S2 cells, whereas expression of a SXL mutant (SXLRRM) did not promote those events. Using a monoclonal antibody, we further obtained the transcriptome-wide RNA-binding sites of SXL through PAR-CLIP. After splicing assay of mini-genes with mutations in the SXL-binding sites, we revealed that SXL-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing, whereas SXL-binding on the circRNA exons inhibits back-splicing. This study provides strong evidence that SXL has a regulatory role in back-splicing to generate sex-specific and -differential circRNAs, as well as in the initiation of sex-determination cascade through canonical forward-splicing.

Molecular characterization of the Sex-lethal gene in mud crab Scylla paramamosain and its potential role in sexual development

Sex-lethal (Sxl) gene operates as a master switch of sexual differentiation in Drosophila melanogaster. In this study, we cloned and characterized the full-length cDNA of Sxl in mud crab (Scylla paramamosain) (SpSxl). The deduced amino acid sequence of SpSxl contained two RNA-binding motifs (RRM), namely RRM1 and RRM2. The SpSxl mRNA levels were abundant in the androgenic glands of the male crab, implying its potential role in male sexual development. This hypothesis was supported by the RNAi experiment, revealing that the injection of SpSxl dsRNA in vivo caused an increase in the expression of SpIAG, which is a key gene of male sexual differentiation in crustaceans. The interference result of SpTra-2 suggested that SpSxl and SpTra-2 may be involved in sex-differentiation by direct or indirect regulation of SpIAG gene. The eye stalk ablation (ESA) experiments further confirmed that SpSxl could not regulate SpIAG through eyestalk neuropeptide, implying other regulation pathways. In addition, treatment with SpSxl dsRNA had no effects on SpDmrt expression, suggesting that sex determination in S. paramamosain is different from that in D. melanogaster.

Evidence That Runt Acts as a Counter-Repressor of Groucho During Drosophila melanogaster Primary Sex Determination

Runx proteins are bifunctional transcription factors that both repress and activate transcription in animal cells. Typically, Runx proteins work in concert with other transcriptional regulators, including co-activators and co-repressors to mediate their biological effects. In Drosophila melanogaster the archetypal Runx protein, Runt, functions in numerous processes including segmentation, neurogenesis and sex determination. During primary sex determination Runt acts as one of four X-linked signal element (XSE) proteins that direct female-specific activation of the establishment promoter (Pe) of the master regulatory gene Sex-lethal (Sxl). Successful activation of SxlPe requires that the XSE proteins overcome the repressive effects of maternally deposited Groucho (Gro), a potent co-repressor of the Gro/TLE family. Runx proteins, including Runt, contain a C-terminal peptide, VWRPY, known to bind to Gro/TLE proteins to mediate transcriptional repression. We show that Runt’s VWRPY co-repressor-interaction domain is needed for Runt to activate SxlPe. Deletion of the Gro-interaction domain eliminates Runt-ability to activate SxlPe, whereas replacement with a higher affinity, VWRPW, sequence promotes Runt-mediated transcription. This suggests that Runt may activate SxlPe by antagonizing Gro function, a conclusion consistent with earlier findings that Runt is needed for Sxl expression only in embryonic regions with high Gro activity. Surprisingly we found that Runt is not required for the initial activation of SxlPe. Instead, Runt is needed to keep SxlPe active during the subsequent period of high-level Sxl transcription suggesting that Runt helps amplify the difference between female and male XSE signals by counter-repressing Gro in female, but not in male, embryos.

DrosophilaSister-of-Sex-lethal reinforces a male-specific gene expression pattern by controllingSex-lethalalternative splicing

In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. Here, we identify the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern.

Regulation of Notch Signaling by the Heterogeneous Nuclear Ribonucleoprotein Hrp48 and Deltex in Drosophila melanogaster.

Notch signaling is an evolutionarily conserved pathway that is found to be involved in a number of cellular events throughout development. The deployment of the Notch signaling pathway in numerous cellular contexts is possible due to its regulation at multiple levels. In an effort to identify the novel components integrated into the molecular circuitry affecting Notch signaling, we carried out a protein-protein interaction screen based on the identification of cellular protein complexes using co-immunoprecipitation followed by mass-spectrometry. We identified Hrp48, a heterogeneous nuclear ribonucleoprotein in Drosophila, as a novel interacting partner of Deltex (Dx), a cytoplasmic modulator of Notch signaling. Immunocytochemical analysis revealed that Dx and Hrp48 colocalize in cytoplasmic vesicles. The dx mutant also showed strong genetic interactions with hrp48 mutant alleles. The coexpression of Dx and Hrp48 resulted in the depletion of cytoplasmic Notch in larval wing imaginal discs and downregulation of Notch targets cut and wingless Previously, it has been shown that Sex-lethal (Sxl), on binding with Notch mRNA, negatively regulates Notch signaling. The overexpression of Hrp48 was found to inhibit Sxl expression and consequently rescued Notch signaling activity. In the present study, we observed that Dx together with Hrp48 can regulate Notch signaling in an Sxl-independent manner. In addition, Dx and Hrp48 displayed a synergistic effect on caspase-mediated cell death. Our results suggest that Dx and Hrp48 together negatively regulate Notch signaling in Drosophila melanogaster.

Primary Sex Determination in Drosophila melanogaster Does Not Rely on the Male-Specific Lethal Complex.

It has been proposed that the Male Specific Lethal (MSL) complex is active in Drosophila melanogaster embryos of both sexes prior to the maternal-to-zygotic transition. Elevated gene expression from the two X chromosomes of female embryos is proposed to facilitate the stable establishment of Sex-lethal (Sxl) expression, which determines sex and represses further activity of the MSL complex, leaving it active only in males. Important supporting data included female-lethal genetic interactions between the seven msl genes and either Sxl or scute and sisterlessA, two of the X-signal elements (XSE) that regulate early Sxl expression. Here I report contrary findings that there are no female-lethal genetic interactions between the msl genes and Sxl or its XSE regulators. Fly stocks containing the msl3(1) allele were found to exhibit a maternal-effect interaction with Sxl, scute, and sisterlessA mutations, but genetic complementation experiments showed that msl3 is neither necessary nor sufficient for the female-lethal interactions, which appear to be due to an unidentified maternal regulator of Sxl. Published data cited as evidence for an early function of the MSL complex in females, including a maternal effect of msl2, have been reevaluated and found not to support a maternal, or other effect, of the MSL complex in sex determination. These findings suggest that the MSL complex is not involved in primary sex determination or in X chromosome dosage compensation prior to the maternal-to-zygotic transition.

Hrp48 attenuates Sxl expression to allow for proper notch expression and signaling in wing development.

Different signaling pathways are deployed in specific developmental contexts to generate sexually dimorphic traits. Recently, Sex-lethal (Sxl), the female determinant in Drosophila melanogaster, was shown to down-regulate Notch (N) signaling to accomplish sex-specific patterning. Paradoxically, however, both Sxl and N are ubiquitously expressed in all of the female cells. This raises a key question as to how, during monomorphic female development, N signaling escapes the negative impact of Sxl. Here, we uncover a regulatory loop involving Hrp48, an abundant Drosophila hnRNP, Sxl and N. Phenotypic consequences of the partial loss of hrp48 resemble that of N but are more pronounced in females than in males. Likewise, N levels are drastically diminished only in females. Interestingly, monomorphic female tissues including wing, eye and antennal discs display considerable increase in Sxl amounts. Finally, female-specific attenuation of N signaling is rescued upon simultaneous removal of Sxl. Thus, our data demonstrate that in monomorphic contexts, Hrp48 functions as a moderator of Sxl expression to achieve adequate levels of N receptor production and signaling. We propose that it is critical to modulate the activities of the master determinant underling sexual dimorphism, to ensure that it does not function inappropriately in monomorphic tissues and disrupt their development.

PPS, a large multidomain protein, functions with sex-lethal to regulate alternative splicing in Drosophila.

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.

Maternal Groucho and bHLH repressors amplify the dose-sensitive X chromosome signal in Drosophila sex determination

In Drosophila, XX embryos are fated to develop as females, and XY embryos as males, because the diplo-X dose of four X-linked signal element genes, XSEs, activates the Sex-lethal establishment promoter, SxlPe, whereas the haplo-X XSE dose leaves SxlPe off. The threshold response of SxlPe to XSE concentrations depends in part on the bHLH repressor, Deadpan, present in equal amounts in XX and XY embryos. We identified canonical and non-canonical DNA-binding sites for Dpn at SxlPe and found that cis-acting mutations in the Dpn-binding sites caused stronger and earlier Sxl expression than did deletion of dpn implicating other bHLH repressors in Sxl regulation. Maternal Hey encodes one such bHLH regulator but the E(spl) locus does not. Elimination of the maternal corepressor Groucho also caused strong ectopic Sxl expression in XY, and premature Sxl activation in XX embryos, but Sxl was still expressed differently in the sexes. Our findings suggest that Groucho and associated maternal and zygotic bHLH repressors define the threshold XSE concentrations needed to activate SxlPe and that they participate directly in sex signal amplification. We present a model in which the XSE signal is amplified by a feedback mechanism that interferes with Gro-mediated repression in XX, but not XY embryos.

Indirect Effects of Ploidy Suggest X Chromosome Dose, Not the X:A Ratio, Signals Sex in Drosophila

In the textbook view, the ratio of X chromosomes to autosome sets, X:A, is the primary signal specifying sexual fate in Drosophila. An alternative idea is that X chromosome number signals sex through the direct actions of several X-encoded signal element (XSE) proteins. In this alternative, the influence of autosome dose on X chromosome counting is largely indirect. Haploids (1X;1A), which possess the male number of X chromosomes but the female X:A of 1.0, and triploid intersexes (XX;AAA), which possess a female dose of two X chromosomes and the ambiguous X:A ratio of 0.67, represent critical tests of these hypotheses. To directly address the effects of ploidy in primary sex determination, we compared the responses of the signal target, the female-specific SxlPe promoter of the switch gene Sex-lethal, in haploid, diploid, and triploid embryos. We found that haploids activate SxlPe because an extra precellular nuclear division elevates total X chromosome numbers and XSE levels beyond those in diploid males. Conversely, triploid embryos cellularize one cycle earlier than diploids, causing premature cessation of SxlPe expression. This prevents XX;AAA embryos from fully engaging the autoregulatory mechanism that maintains subsequent Sxl expression, causing them to develop as sexual mosaics. We conclude that the X:A ratio predicts sexual fate, but does not actively specify it. Instead, the instructive X chromosome signal is more appropriately seen as collective XSE dose in the early embryo. Our findings reiterate that correlations between X:A ratios and cell fates in other organisms need not implicate the value of the ratio as an active signal.

Male or Female? The Answer Depends on When You Ask

Male or Female? The Answer Depends on When You Ask

Misregulation of Sex-Lethal and Disruption of Male-Specific Lethal Complex Localization in Drosophila Species Hybrids

A major model system for the study of evolutionary divergence between closely related species has been the unisexual lethality resulting from reciprocal crosses of Drosophila melanogaster and D. simulans. Sex-lethal (Sxl), a critical gene for sex determination, is misregulated in these hybrids. In hybrid males from D. melanogaster mothers, there is an abnormal expression of Sxl and a failure of localization of the male-specific lethal (MSL) complex to the X chromosome, which causes changes in gene expression. Introduction of a Sxl mutation into this hybrid genotype will allow expression of the MSL complex but there is no sequestration to the X chromosome. Lethal hybrid rescue (Lhr), which allows hybrid males from this cross to survive, corrects the SXL and MSL defects. The reciprocal cross of D. simulans mothers by D. melanogaster males exhibits underexpression of Sxl in embryos.

The TAGteam DNA motif controls the timing ofDrosophilapre-blastoderm transcription

The Drosophila sex-determination switch gene Sex-lethal (Sxl) and the X-chromosome signal element genes (XSEs) that induce the female-specific expression of Sxl are transcribed extremely early in development when most of the genome of this organism is still silent. The DNA sequence CAGGTAG had been implicated in this pre-cellular blastoderm activation of sex-determination genes. A genome-wide computational search, reported here, suggested that CAGGTAG is not specific to early sex-determination genes, since it is over-represented upstream of most genes that are transcribed pre-cellular blastoderm, not just those involved in sex determination. The same search identified similarly over-represented, one-base-pair degenerate sequences as possible functional synonyms of CAGGTAG. We call these heptamers collectively, the TAGteam. Relevance of the TAGteam sequences to pre-cellular blastoderm transcription was established through analysis of TAGteam changes in Sxl, scute (an XSE), and the ;ventral repression element' of the pattern-formation gene zerknüllt. Decreasing the number of TAGteam sites retarded the onset of pre-blastoderm transcription, whereas increasing their number correlated with an advanced onset. Titration of repressors was thought to be the rate-limiting step determining the onset of such early transcription, but this TAGteam dose effect shows that activators must also play an important role in the timing of pre-blastoderm gene expression.

A Theoretical Model for the Regulation of Sex-lethal, a Gene That Controls Sex Determination and Dosage Compensation in Drosophila melanogaster

Cell fate commitment relies upon making a choice between different developmental pathways and subsequently remembering that choice. Experimental studies have thoroughly investigated this central theme in biology for sex determination. In the somatic cells of Drosophila melanogaster, Sex-lethal (Sxl) is the master regulatory gene that specifies sexual identity. We have developed a theoretical model for the initial sex-specific regulation of Sxl expression. The model is based on the well-documented molecular details of the system and uses a stochastic formulation of transcription. Numerical simulations allow quantitative assessment of the role of different regulatory mechanisms in achieving a robust switch. We establish on a formal basis that the autoregulatory loop involved in the alternative splicing of Sxl primary transcripts generates an all-or-none bistable behavior and constitutes an efficient stabilization and memorization device. The model indicates that production of a small amount of early Sxl proteins leaves the autoregulatory loop in its off state. Numerical simulations of mutant genotypes enable us to reproduce and explain the phenotypic effects of perturbations induced in the dosage of genes whose products participate in the early Sxl promoter activation.

Masters change, slaves remain.

Sex determination offers an opportunity to address many classic questions of developmental biology. In addition, because sex determination evolves rapidly, it offers an opportunity to investigate the evolution of genetic hierarchies. Sex determination in Drosophila melanogaster is controlled by the master regulatory gene, Sex lethal (Sxl). DmSxl controls the alternative splicing of a downstream gene, transformer (tra), which acts with tra2 to control alternative splicing of doublesex (dsx). DmSxl also controls its own splicing, creating an autoregulatory feedback loop that ensures expression of Sxl in females, but not males. A recent paper has shown that in the dipteran Ceratitis capitata later (downstream) steps in the regulatory hierarchy are conserved, while earlier (upstream) steps are not. Cctra is regulated by alternative splicing and apparently controls the alternative splicing of Ccdsx. However, Cctra is not regulated by CcSxl. Instead it appears to autoregulate in a manner similar to the autoregulation seen with DmSxl.

A host parasite interaction rescues Drosophila oogenesis defects.

The cytoplasmically inherited bacterium Wolbachia pipientis is a widespread parasite of arthropods that manipulates the reproductive biology of its hosts, often to their detriment, in order to foster its own transmission through egg cytoplasm. Here we report that infection by Wolbachia restores fertility to Drosophila melanogaster mutant females prevented from making eggs by protein-coding lesions in Sex-lethal (Sxl), the master regulator of sex determination. Suppression of sterility by Wolbachia discriminates markedly among similar germline-specific Sxl alleles, and is not observed for mutations in other genes that produce similar 'tumorous ovary' phenotypes, including one that blocks Sxl germline expression. This allele and gene specificity indicates that suppression probably results from a specific interaction with Sxl protein, rather than from a bypass of the normal germline requirement for this developmental regulator or from an effect on Sxl expression. The Sxl-Wolbachia interaction provides a rare opportunity to explore host-parasite relationships at the molecular level in a model insect. Furthermore, demonstration that a parasite infection can counteract the deleterious effects of mutations in host genes illustrates how hosts might become dependent on parasites.

Molecular identification of virilizer, a gene required for the expression of the sex-determining gene Sex-lethal in Drosophila melanogaster.

Sex-lethal (Sxl) is a central switch gene in somatic sexual development of Drosophila melanogaster. Female-specific expression of Sxl relies on autoregulatory splicing of Sxl pre-mRNA by SXL protein. This process requires the function of virilizer (vir). Besides its role in Sxl splicing, vir is essential for male and female viability and is also required for the production of eggs capable of embryonic development. We have identified vir molecularly and found that it produces a single transcript of 6 kb that is ubiquitously expressed in male and female embryos throughout development. This transcript encodes a nuclear protein of 210 kD that cannot be assigned to a known protein family. VIR contains a putative transmembrane domain, a coiled-coil region and PEST sequences. We have characterized five different alleles of vir. Those alleles that affect both sexes are associated with large truncations of the protein, while alleles that affect only the female-specific functions are missense mutations that lie relatively close to each other, possibly defining a region important for the regulation of Sxl.

Cell-autonomous and somatic signals control sex-specific gene expression in XY germ cells of Drosophila.

When XX germ cells develop in a testis they become spermatogenic. Thus, somatic signals determine the sex of genetically female germ cells. In contrast, XY germ cells experimentally transferred to an ovary do not differentiate oogenic cells. Because such cells show some male characteristics when analyzed in adults, it was assumed that XY germ cells autonomously become spermatogenic. Recently, however, evidence showing that a female soma feminizes XY germ cells was reported. The conclusion was drawn that the sex determination of XY germ cells is dictated by the sex of the soma. We monitored the fate of XY germ cells placed in a female environment throughout development. Here we report that such germ cells respond to both cell-autonomous and somatic sex-determining signals, depending on the developmental stage. Analyzing the expression of sex-specific molecular markers, we first detected autonomous male-specific gene expression in XY germ cells embedded in female embryos and larvae. At later stages, however, we found that sex-specific regulation of gene expression within XY germ cells is influenced by somatic gonadal cells. After metamorphosis, XY germ cells developing in a female soma start expressing female-specific and male-specific markers. Transcription of female-specific genes is maintained, while that of male-specific genes is later repressed. We show that in such XY germ cells, the female-specific gene Sex-lethal (Sxl) is activated. Within the germline, Sxl expression is required for the activation of a further female-specific gene and the repression of male-specific genes. We thus report for the first time the existence of downstream targets of the gene Sxl in the germline.

Histone Acetylation and Gene Expression Analysis of Sex lethal Mutants in Drosophila

The evolution of sex determination mechanisms is often accompanied by reduction in dosage of genes on a whole chromosome. Under these circumstances, negatively acting regulatory genes would tend to double the expression of the genome, which produces compensation of the single-sex chromosome and increases autosomal gene expression. Previous work has suggested that to reduce the autosomal expression to the female level, these dosage effects are modified by a chromatin complex specific to males, which sequesters a histone acetylase to the X. The reduced autosomal histone 4 lysine 16 (H4Lys16) acetylation results in lowered autosomal expression, while the higher acetylation on the X is mitigated by the male-specific lethal complex, preventing overexpression. In this report, we examine how mutations in the principal sex determination gene, Sex lethal (Sxl), impact the H4 acetylation and gene expression on both the X and autosomes. When Sxl expression is missing in females, we find that the sequestration occurs concordantly with reductions in autosomal H4Lys16 acetylation and gene expression on the whole. When Sxl is ectopically expressed in Sxl(M) mutant males, the sequestration is disrupted, leading to an increase in autosomal H4Lys16 acetylation and overall gene expression. In both cases we find relatively little effect upon X chromosomal gene expression.

Recombination and disjunction in female germ cells of Drosophila depend on the germline activity of the gene sex-lethal.

Gametogenesis in males and females differs in many ways. An important difference in Drosophila is that recombination between homologous chromosomes occurs only in female meiosis. Here, we report that this process relies on the correct functioning of Sex-lethal (Sxl) which is primarily known as the master gene in somatic sex determination. Certain alleles of this gene (Sxl(fs)) disrupt the germline, but not the somatic function of Sxl and cause an arrest of germ cell development during cystocyte proliferation. Using dominant suppressor mutations that relieve this early block in Sxl(fs) mutant females, we discovered additional requirements of Sxl for normal meiotic differentiation of the oocyte. Females mutant for Sxl(fs) and carrying a suppressor become fertile, but pairing of homologous chromosomes and formation of chiasmata is severely perturbed, resulting in an almost complete lack of recombinants and a high incidence of non-disjunction events. Similar results were obtained when germline expression of wild-type Sxl was compromised by mutations in virilizer (vir), a positive regulator of Sxl. Ectopic expression of a Sxl transgene in premeiotic stages of male germline development, on the other hand, is not sufficient to allow recombination to take place, which suggests that Sxl does not have a discriminatory role in this female-specific process. We propose that Sxl performs at least two tasks in oogenesis: an 'early' function in formation of the egg chamber, and a 'late' function in progression of the meiotic cell cycle, suggesting that both events are coordinated by a common mechanism.