Sxl Transcripts Research Articles

Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction.

Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In Drosophila melanogaster, PGCs from embryos maternally compromised for germ cell-less (gcl) misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. Here we demonstrate that the somatic determinant of female fate, Sex-lethal (Sxl), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso (torsoDeg) can activate Sxl transcription in PGCs, whereas simultaneous loss of torso-like (tsl) reinstates the quiescent status of gcl PGCs. Intriguingly, like gcl mutants, embryos derived from mothers expressing torsoDeg in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction.

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.

Cooperative and Antagonistic Contributions of Two Heterochromatin Proteins to Transcriptional Regulation of the Drosophila Sex Determination Decision

Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. Here we report a similar phenotype for the telomeric partner of HP1, HOAP, and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, SxlPe, is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (SxlPm) in both sexes. HOAP mutants show inappropriate SxlPe firing in males and the concomitant inappropriate splicing of SxlPm-derived transcripts, while females show premature firing of SxlPe. HP1 mutants, by contrast, display SxlPm splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with SxlPe sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to SxlPe are severely compromised. Our genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at SxlPe.

Male or Female? The Answer Depends on When You Ask

Male or Female? The Answer Depends on When You Ask

Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination.

With a focus on Sex-lethal (Sxl), the master regulator of Drosophila somatic sex determination, we compare the sex determination mechanism that operates in the germline with that in the soma. In both cell types, Sxl is functional in females (2X2A) and nonfunctional in males (1X2A). Somatic cell sex is determined initially by a dose effect of X:A numerator genes on Sxl transcription. Once initiated, the active state of SXL is maintained by a positive autoregulatory feedback loop in which Sxl protein insures its continued synthesis by binding to Sxl pre-mRNA and thereby imposing the productive (female) splicing mode. The gene splicing-necessary factor (snf), which encodes a component of U1 and U2 snRNPs, participates in this RNA splicing control. Here we show that an increase in the dose of snf+ can trigger the female Sxl RNA splicing mode in male germ cells and can feminize triploid intersex (2X3A) germ cells. These snf+ dose effects are as dramatic as those of X:A numerator genes on Sxl in the soma and qualify snf as a numerator element of the X:A signal for Sxl in the germline. We also show that female-specific regulation of Sxl in the germline involves a positive autoregulatory feedback loop on RNA splicing, as it does in the soma. Neither a phenotypically female gonadal soma nor a female dose of X chromosomes in the germline is essential for the operation of this feedback loop, although a female X-chromosome dose in the germline may facilitate it. Engagement of the Sxl splicing feedback loop in somatic cells invariably imposes female development. In contrast, engagement of the Sxl feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature to conclude that Sxl is not a switch gene in germ cells for at least some sex-specific aspects of their differentiation. Ironically, the testis may be an excellent organ in which to study the interactions among regulatory genes such as Sxl, snf, ovo and otu which control female-specific processes in the ovary.

Sex-lethal interacts with splicing factors in vitro and in vivo.

The Drosophila sex determination gene Sex-lethal controls its own expression and the expression of downstream target genes such as transformer by regulating RNA splicing. Genetic and molecular studies have established that Sxl requires the product of another gene, snf, to autoregulate the splicing of its own transcripts. snf has recently been shown to encode a Drosophila U1 and U2 small nuclear ribonucleoprotein particle protein. In the work reported here, we demonstrate that the Sxl and Snf proteins can interact directly in vitro and that these two proteins are part of an RNase-sensitive complex in vivo which can be immunoprecipitated with the Sxl antibody. Unlike bulk Snf protein, which sediments slowly in sucrose gradients, the Snf protein associated with Sxl is in a large, rapidly sedimenting complex. Detailed characterization of the Sxl-Snf complexes from cross-linked extracts indicates that these complexes contain additional small nuclear ribonucleoprotein particle proteins and the U1 and U2 small nuclear RNAs. Finally, consistent with the RNase sensitivity of the Sxl-Snf complexes, Sxl transcripts can also be immunoprecipitated by Sxl antibodies. On the basis of the physical interactions between Sxl and Snf, we present a model for Sxl splicing regulation. This model helps explain how the Sxl protein is able to promote the sex-specific splicing of Sxl transcripts, utilizing target sequences that are distant from the regulated splice sites.

Transposon insertions causing constitutive Sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing.

Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle "on" vs. "off" reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. "Male-lethal" (SxlM) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, SxlM haplo-X animals (chromosomal males) die because of improper dosage compensation, and SxlM chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Five independent spontaneous SxlM alleles were shown previously to be transposon insertions into what was subsequently found to be the region of regulated sex-specific Sxl RNA splicing. We show that these five alleles represent three different mutant types: SxlM1, SxlM3, and SxlM4. SxlM1 is an insertion of a roo element 674 bp downstream of the translation-terminating male-specific exon. SxlM3 is an insertion of a hobo transposon (not 297 as previously reported) into the 3' splice site of the male exon, and SxlM4 is an insertion of a novel transposon into the male-specific exon itself. We show that these three gain-of-function mutants differ considerably in their ability to bypass the sex determination signal, with SxlM4 being the strongest and SxlM1 the weakest. This difference is also reflected in effects of these mutations on sex-specific RNA splicing and on the rate of appearance of SXL protein in male embryos. Transcript analysis of double-mutant male-viable SxlM derivatives in which the SxlM insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these SxlM alleles is a consequence of an alteration of the structure of the pre-mRNA that allows some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of SxlM alleles appears to depend on the mutant alleles' responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce.

A bZIP protein, sisterless-a, collaborates with bHLH transcription factors early in Drosophila development to determine sex.

Sexual identity in Drosophila is determined by zygotic X-chromosome dose. Two potent indicators of X-chromosome dose are sisterless-a (sis-a) and sisterless-b (sis-b). Genetic analysis has shown that a diplo-X dose of these genes activates their regulatory target, the feminizing switch gene Sex-lethal (Sxl), whereas a haplo-X dose leaves Sxl inactive. sis-b encodes a transcriptional activator of the bHLH family that dimerizes with several other HLH proteins required for the proper assessment of X dose. Here, we report that sis-a encodes a bZIP protein homolog that functions in all somatic nuclei to activate Sxl transcription. In contrast with other elements of the sex-determination signal, the functioning of this transcription factor in somatic cells may be specific to X-chromosome counting. Using in situ hybridization, we determined the time course of sis-a, sis-b, and Sxl transcription during the first few hours after fertilization. The pattern of sis-a RNA accumulation is very similar to that for sis-b, with a peak in nuclear cycle 12 at about the time of onset of Sxl transcription. Considered in the context of other studies, these results suggest that the ability to distinguish one X from two is attributable to combinatorial interactions between bZIP and bHLH proteins and their target, Sxl, as well as to positive and negative interactions with maternally supplied and zygotically produced proteins.

The primary sex determination signal of Drosophila acts at the level of transcription

For Drosophila, the choice between male and female development is made by the switch gene, Sxl, in response to the X:A ratio. Once Sxl is turned on in females, it actively maintains the determined state, independent of the X:A signal, by a positive autoregulatory feedback loop in which Sxl proteins direct the female-specific splicing of Sxl transcripts. In this paper we have investigated the mechanism controlling pathway initiation. Our results suggest a two-step model for the initial activation of Sxl in females. In the first step, a special class of Sxl mRNAs is expressed in female embryos from an early promoter that responds to the genes signaling the X:A ratio. The proteins produced from these early mRNAs then initiate the autoregulatory loop by directing the female-specific processing of transcripts from the late Sxl promoter.

Genetic and molecular analysis of new female-specific lethal mutations at the gene Sxl of Drosophila melanogaster.

We have isolated three female-specific lethal mutations at the gene Sex-lethal (Sxl): Sxlfb, Sxlfc and Sxlfd. We have carried out the complementation analysis between these mutations and other previously reported Sxlf mutations. It is possible to classify the alleles tested in this report into two complementation groups: the bc group defined by Sxlfb, and Sxlfc, and the LS group defined by SxlfLS. The other alleles tested affect both complementation groups albeit with different degrees. Contrary to what happens with mutations at the LS group, mutations at the bc group do not affect sex determination, nor late dosage compensation nor oogenesis. Both Sxlfb and Sxlfc present a DNA insertion of at least 5 kb between position -10 and -11 on the molecular map, within the fourth intron. On the contrary, Sxlfd, a strong mutation affecting all Sxl functions, is not associated to any detectable DNA alteration in Southern blots, so that it seems to be a "point" mutation. In agreement with their phenotypes, both Sxlfc/SxlfLS and Sxlfc homozygous female larvae express only the late Sxl transcripts characteristic of females, while females homozygous for SxlfLS express only the late Sxl transcripts characteristic of males. Moreover, Sxlfc presents a lethal synergistic interaction with mutations at either da or the X:A ratio, two signals that define the initial activity state of Sxl, while SxlfLS do not. These data suggest that the two complementation groups are related to the two sets of early and late Sxl transcripts, which are responsible for the early and late Sxl functions, respectively: Sxlfb and Sxlfc would affect the early functions and SxlfLS would affect the late Sxl functions.

The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides.

Sex-lethal (Sxl), a key sex determination gene in Drosophila melanogaster, is known to express a set of three early transcripts arising during early embryogenesis and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs were distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). We have now analyzed the structures of the late Sxl transcripts by cDNA sequencing, Northern (RNA) blotting, primer extension, and RNase protection. The late transcripts appear to use a common 5' end but differ at their 3' ends by the use of alternative polyadenylation sites. Two of these sites lack canonical AATAAA sequences, and their use correlates in females with the presence of a functional germ line, suggesting possible tissue-specific polyadenylation. Besides the presence of the male-specific exon, no additional sex-specific splicing events were detected, although a number of non-sex-specific splicing variants were observed. In females, the various forms of late Sxl transcript potentially encode up to six slightly different polypeptides. All of the protein-coding differences occur outside the previously defined ribonucleoprotein motifs. One class of Sxl mRNAs also includes a second long ORF in the same frame as the first ORF but separated from it by a single ochre codon. The function of this second ORF is unknown. Significant amounts of apparently partially processed Sxl RNAs were observed, consistent with the hypothesis that the regulated Sxl splices occur relatively slowly.

The Complex Set of Late Transcripts from the Drosophila Sex Determination Gene Sex-lethal Encodes Multiple Related Polypeptides

Sex-lethal (Sxl), a key sex determination gene in Drosophila melanogaster, is known to express a set of three early transcripts arising during early embryogenesis and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs were distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). We have now analyzed the structures of the late Sxl transcripts by cDNA sequencing, Northern (RNA) blotting, primer extension, and RNase protection. The late transcripts appear to use a common 5' end but differ at their 3' ends by the use of alternative polyadenylation sites. Two of these sites lack canonical AATAAA sequences, and their use correlates in females with the presence of a functional germ line, suggesting possible tissue-specific polyadenylation. Besides the presence of the male-specific exon, no additional sex-specific splicing events were detected, although a number of non-sex-specific splicing variants were observed. In females, the various forms of late Sxl transcript potentially encode up to six slightly different polypeptides. All of the protein-coding differences occur outside the previously defined ribonucleoprotein motifs. One class of Sxl mRNAs also includes a second long ORF in the same frame as the first ORF but separated from it by a single ochre codon. The function of this second ORF is unknown. Significant amounts of apparently partially processed Sxl RNAs were observed, consistent with the hypothesis that the regulated Sxl splices occur relatively slowly.

Positive autoregulation of Sex-lethal by alternative splicing maintains the female determined state in Drosophila

Sex-lethal is a binary switch gene that controls all aspects of Drosophila sexual dimorphism. It must be active in females and inactive in males. The on/off regulation reflects alternative RNA splicing in which full-length proteins are produced only in females. Here we investigate the role of Sxl in maintaining sexual pathway commitments. By ectopic expression of a female Sxl cDNA in transgenic male flies, we show that Sxl protein induces a rapid switch from male- to female-specific splicing. The ectopically expressed Sxl protein will trans-activate an endogenous wild-type Sxl gene. This establishes a feedback loop in which Sxl proteins induce their own synthesis by directing the female-specific splicing of Sxl transcripts. We conclude that the female determined state is maintained by Sxl through positive autoregulation, while the male determined state is maintained by default.

The Drosophila melanogaster fl(2)d gene is needed for the female-specific splicing of Sex-lethal RNA.

In Drosophila melanogaster, sex determination and dosage compensation are under the control of the Sex-lethal (Sxl) gene. We have identified a gene, female-lethal-2-d (fl(2)d), located in the second chromosome, that interacts with Sxl. fl(2)d homozygous clones, induced during the larval stage of fl(2)d/+ females, develop male structures instead of female ones. fl(2)d homozygous females hypertranscribe their two X chromosomes, as measured by comparing the level of the X-linked sgs-4 transcript, which is dosage compensated, with that of the autosomal sgs-3 transcript. Thus, with respect to the processes of sex determination and dosage compensation, loss-of-function mutations at the fl(2)d and at the Sxl genes are equivalent. Moreover, fl(2)d homozygous female larvae express the Sxl transcripts characteristic of males. These results indicate that the fl(2)d gene is needed for the sex-specific splicing pattern of the Sxl RNA that occurs in females, thus suggesting the involvement of the fl(2)d gene in the positive autoregulatory pathway of Sxl.

The Drosophila female-specific sex-determination gene, Sex-lethal, has stage-, tissue-, and sex-specific RNAs suggesting multiple modes of regulation.

For proper sexual development of females, the Sex-lethal (Sxl) gene must be activated early in development and remain on during the rest of the life cycle. Conversely, in males, Sxl must remain functionally off through development. Here, we show that the Sxl transcription unit spans a DNA segment of greater than 20 kb and encodes at least 10 distinct, but overlapping, RNA species. These RNAs range in size from 4.4 to 1.7 kb and exhibit sex, stage, and tissue specificity. Six RNAs, three female specific and three male specific, are first detected by midembryogenesis and persist through the adult stage: Their expression reflects the on/off regulation of Sxl's activity at the level of sex-specific alternate splicing. Four Sxl RNAs are found in adult females. Two of these RNAs are dependent on the presence of a functional germ line and may be relevant to Sxl's role in adult germ-line development. All four are present in unfertilized eggs. Finally, three Sxl RNAs are found only transiently during very early embryogenesis; we suggest that the expression of these RNAs may reflect an early regulation of Sxl at the level of transcription and that these transcripts are involved in the initial selection of the Sxl activity state in response to the primary sex-determination signal, the X/A ratio.