Sex lethal

Table of contents

Promoter Structure

Heterodimers between Daughterless (DA) and Sisterless-b (SIS-b, also known as Scute) bind several sites on the Sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Repression by the Deadpan product of da/sis-b dependent activation of Sex-lethal results from specific binding of Deadpan protein to a unique site within the promoter (Hoshijima, 1995).

In the early embryo, the activity of Sxl early promoter (Sxl-Pe) is controlled in a highly dose-sensitive fashion by the genes on the X chromosome that function as numerator elements and by genes located on the autosomes that function as denominator elements. In other words gene products from the X chromosome activate Sxl, while autosomal gene products repress Sxl, and it is the ratio between activator and repressor proteins that determine whether the Sxl promotor is activated. Functional dissection of Sxl-Pe indicates that activating the promoter in females requires the cumulative action of multiple numerator genes which appear to exert their effects through reiterated cis-acting target sites in the promoter. Conversely, maintaining the promoter silent in males requires the repressive activities of denominator genes, and at least one of the denominator genes also appears to function through target sequences within the promoter (Estes, 1995).

Less then three hours after fertilization, SxlPe shuts off and a 'sexual pathway establishment' promoter, SxlPm, comes on in both sexes. In contrast to transcripts from SxlPe, transcripts from SxlPm are only processed into mRNA that encodes full-length Sxl protein if active Sxl protein is already present to direct RNA splicing. In the absence of such protein, SxlPm-derived mRNA includes an exon that prematurely terminates translation, allowing only inactive product to be generated. The transient expression of SxlPe, which occurs only in females, results in a pulse of Sxl protein that triggers the productive splicing of SxlPm transcripts. Productive RNA splicing mode is self-maintained thereafter by the Sxl protein, which arises as a consequence and which interacts directly with Sxl pre-mRNA. Because SxlPe is silent in males, males never engage this postive feedback loop for RNA splicing and only produces Sxl mRNAs tha include the translation-terminating exon (Hager, 1997).

With a focus on Sex-lethal (Sxl), the master regulator of Drosophila somatic sex determination, a comparison has been carried out between the sex determination mechanism and that which 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 mRNA is maintained by a positive autoregulatory feedback loop in which Sxl protein ensures its continued synthesis by binding to SXL pre-mRNA and thereby imposing the productive (female) splicing mode. Ectopic expression of Sxl protein triggers the female-specific Sxl mRNA feedback loop in male germ cells without disrupting spermatogenesis. There is no adverse effect on male viability or fertility. The presence of Sxl protein may sometimes retard the rate of differentiation of spermatocytes, but does not abort the process (Hager, 1997).

The gene splicing-necessary factor (sans fille or snf), which encodes a component of U1 and U2 snRNPs, participates in SXL RNA splicing control. 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. 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. In fact, increased doses of snf+ and Sxl+ can feminize germ cells when germ cells have an altered X chromosome to autosome ratio. snf+ and Sxl+ feminize 2X3A germ cells. Flies with the higher doses of snf+ display a greater proportion of yolky germline cysts and eggs. Somatic sex is important in this feminization as the sexual phenotype of all internal and external somatic dimorphic characters appears to be fully female in 2X3A animals carrying a heat-shock transformer transgene. What then is the role of Snf in the germ-line? It seems likely that Snf acts to boost the autoregulatory effectiveness of very low levels of female Sxl protein, rather than acting directly on its own to influence SXL transcript splicing. 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 (Hager, 1997).

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. runt also functions to activate transcription. This paper documents the direct activation of Sex-lethal transcription by the Drosophila Runt protein. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Male embryos mutant for deadpan show ectopic activation of Sxl expression, preferentially within the central, pre-segmented region of the embryo. Thus, it is possible that a major role for runt in the regulation of Sxl transcription is to counteract repression by dpn. Groucho is required to repress Sxl in male embryos. Thus it is possible that Runt bound to Sxl interacts with Groucho in a manner that blocks Groucho-mediated repression (Kramer, 1999 and references).

In situ hybridization was used to define the earliest effects of runt on transcription from the Sxl early embryonic promoter (Sxl_Pe). Wild-type female embryos containing a SxlPe:lacZ reporter gene begin to express lacZ transcripts during the syncitial nuclear division cycles preceding formation of the cellular blastoderm. Expression at nuclear division cycle 12 is observed in punctate dots distributed throughout the embryo except in pole cells. Later, this expression is seen as uniform staining throughout the embryo except in pole cells. Females homozygous for the amorphic runt^LB5 mutation fail to express the SxlPe:lacZ reporter gene within a broad central region of the embryo. This defect is observed concomitant with the earliest detectable expression of this reporter gene, demonstrating an early requirement for runt in Sxl_Pe activation. The alterations in Sxl expression observed in runt mutants correspond well to the initial expression of runt in a broad central domain of syncitial blastoderm stage embryos. This expression precedes the formation of the seven-stripe pair-rule pattern during cellularization, suggesting that runt’s function in Sxl activation can be temporally separated from its role in segmentation. To test this idea, a temperature-sensitive runt mutation, runt^YP17, was used. Female embryos homozygous for runt^YP17 display normal Sxl_Pe expression when reared and collected at the permissive temperature. At the restrictive temperature of 29°C, these embryos show non-uniform Sxl_Pe expression identical to that observed in embryos deleted for runt. To examine runt’s effects on segmentation, the expression pattern of the segment polarity gene engrailed (en) was examined in these embryos. In runt^YP17 embryos maintained at 18°C, En is expressed in a regular, well-spaced 14-stripe pattern, whereas at 29°C this pattern is disrupted. In collections of embryos aged at the non-permissive temperature for two hours and then shifted to the permissive temperature, female embryos with the abnormal Sxl_Pe expression pattern typical of runt mutants show normal En expression. In reciprocal temperature-shift experiments, female embryos, aged at the permissive temperature to the cellular blastoderm stage and then shifted to the non-permissive temperature, show normal Sxl_Pe expression and abnormal En expression. These results demonstrate that runt’s role in the activation of Sxl_Pe is temporally distinct from and precedes the requirement for runt in segmentation, and provide strong evidence that runt’s role as an activator of Sxl transcription occurs prior to cellularization, during the earlier syncitial blastoderm stages of Drosophila embryogenesis (Kramer, 1999).

Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. The early regulation of Sxl transcription by runt is readily explained if Runt interacts directly with the Sxl early promoter to activate transcription. Previous work has identified a 1.1 kb fragment of the Sxl_Pe promoter that contains sequences essential for sex-specific transcriptional activation. A test was performed for direct interactions between Runt and these DNA sequences. Probes that span this DNA fragment were generated and tested in electrophoretic mobility-shift assays (EMSAs). Runt binds only weakly to each of these DNA fragments. However, upon addition of the Brother partner protein (Bro, a homolog of mammalian PEBP2/CBF beta, a protein unrelated to Runt), multiple complexes are obtained with each of these probes. These complexes are Runt-dependent as they are not detected when only Bro protein is added. Competition with a bona fide CBF-binding site from the Polyoma enhancer prevents detection of these complexes. Competition is not observed when a mutant CBF-binding site is used, indicating that the binding is sequence specific. Recombinant mammalian CBF also recognizes multiple sites within these fragments from the Sxl_Pe promoter. Inspection of the sequence for matches to the consensus CBF-binding sequence TG(T/C)GGT(T/C) has identified ten sites that match this consensus at positions two through five that also match at least one of the three other, less critical positions. Interestingly, no perfect matches to the consensus are found. The presence of multiple binding sites is consistent with the hypothesis that activation of Sxl transcription involves direct interactions between Runt and the Sxl promoter. One prediction of this hypothesis is that Runt’s DNA-binding activity should be required for Sxl activation: an in vitro assay shows this to be true (Kramer, 1999).

The 128 amino acid Runt domain confers sequence-specific DNA binding as well as heterodimerization with Brother, Runt's cofactor. As an initial test of the importance of Runt’s DNA-binding domain, a form of runt that is deleted for its Runt domain, runtdeltaRD was injected into the central region of female homozygous runt^LB5 embryos. No evidence of rescue is seen in runtdeltaRD-injected embryos, indicating that the DNA-binding domain is important for runt’s function as an activator of Sxl_Pe. However, since this is a large deletion, the effects could be attributed to improper folding and/or protein stability. Random- and site-directed mutagenesis experiments have identified several amino acids within the Runt domain that specifically affect DNA binding without disrupting association with the partner protein CBFbeta. Two conserved amino acids in Runt that are important for DNA binding correspond to a cysteine at position 127 and a lysine at position 199. In order to obtain a DNA-binding-defective form of Runt, a protein containing mutations at both of these sites (C127S, K199A) was generated. The DNA-binding activity of this mutant was compared with that of wild-type Runt in EMSAs with the high-affinity CBF-binding site from the Polyoma virus enhancer. The mutant protein, Runt[CK] shows only very low levels of complex formation on this DNA, and this is only in the presence of Brother. Similar experiments with a DNA probe from the Sxl promoter confirm the reduced DNA-binding activity of Runt[CK]. It is estimated that these mutations reduce DNA-binding affinity at least 50-fold. The observation that Brother stimulates DNA binding by Runt[CK] suggests that the two mutations do not disrupt interaction between the Runt and Brother proteins. Thus, these two mutations specifically impair DNA binding without affecting the overall structure of the Runt domain. The mRNA injection assay was used to examine the in vivo activity of this DNA-binding- defective form of Runt, and no evidence for rescue of Sxl_Pe expression was found in runt mutant female embryos. These results are consistent with the hypothesis that Runt activates Sxl transcription by binding to sequences in the Sxl_Pe promoter. Additional experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. A small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo (Kramer, 1999).

Although the truncated reporter gene (Sxl_Pe0.4kb:lacZ), isolated from the proximal 400 basepair fragment of Sxl_Pe, exhibits an abnormal pattern of expression in wild-type females, with higher levels found in the anterior and posterior, the expression is sex-specific. There are several putative Runt-binding sites found within this 400 bp fragment. Deletion of a small 70 bp segment within this fragment, which contains at least two putative binding sites for Runt, results in a loss of SxlPe expression. Conversely, a reporter gene that contains multiple copies of this segment, Sxl_PeGOF:lacZ, is expressed at high levels in WT female embryos. Interestingly, the Sxl_PeGOF:lacZ reporter gene is also expressed in males, however, at much lower levels and not in the anterior regions of the embryo. EMSA with Runt and Brother proteins demonstrates that Runt binds to sequences within this small segment. This interaction is sequence specific as it is competed by a DNA fragment from the Polyoma enhancer containing a wild-type CBF-binding site, but not by a similar DNA fragment with a mutant CBF-binding site. The differential expression in female and male embryos indicates that this reporter gene retains the ability to respond to numerator gene dosage. The observation that this transgene is expressed in males suggests that the activation mediated by multimerization of this small segment of DNA is sufficient to overcome the repression that is normally established in males for the parental Sxl_Pe0.4kb:lacZ reporter gene. Furthermore, the preferential expression within the segmented region of the embryo strongly suggests that this reporter gene is responding to runt. To test this, Sxl_PeGOF:lacZ expression was examined in embryos mutant for runt. Expression is reduced in most, but not all, regions of runt mutant male embryos. Thus, the region that is multimerized in the Sxl_PeGOF:lacZ reporter gene mediates runt-dependent transcriptional activation (Kramer, 1999).

The determination of sexual identity in Drosophila depends upon a system that measures the X chromosome to autosome ratio (X/A). This system relies upon the unequal expression of X-linked numerator genes in 1X and 2X nuclei. The numerators activate a special Sxl promoter, Sxl-Pe, in 2X/2A nuclei, but not 1X/2A nuclei. By multimerizing a conserved Sxl-Pe sequence block, a gain-of-function promoter, Sxl-Pe_GOF, is generated that is inappropriately active in 1X/2A nuclei. GOF activity requires the X-linked unpaired (upd) gene, which encodes a ligand for the Drosophila JAK/STAT signaling pathway. upd also functions as a numerator element in regulating wild-type Sxl-Pe reporters. The JAK kinase, Hopscotch, and the STAT DNA-binding protein, Marelle, are also required for Sxl-Pe activation (Jinks, 2000).

The numerators most important for turning on Sxl are sis-a and sis-b (scute). They are expressed throughout the embryo, and mutations in both can have quite pronounced effects on Sxl-Pe activity. However, neither of these numerators is critical for the gain-of-function activity of the Sxl-Pe_GOF promoter. Instead, the two numerators that contribute most to Sxl-Pe_GOF activity are the segmentation genes runt and upd. At the syncytial blastoderm stage, run is expressed in a broad central domain, and it is in this region that Sxl activation is defective in 2X/2A run mutants. Except for a dorsal crescent in the head, the upd expression domain closely coincides with that of run. It is in this same central run-upd domain that the highest levels of Sxl-Pe_GOF promoter activity are observed. Moreover, in both run and upd mutant males, Sxl- Pe_GOF promoter activity is severely impaired. From these findings, it can be inferred that the multimerized 72 bp fragment contains cis-acting targets for run and upd action (Jinks, 2000).

Since Upd is a secreted ligand, it is unlikely that it interacts directly with sequences in the 72 bp fragment. Instead, the data suggests that Upd acts by turning on a Drosophila JAK/STAT signaling cascade consisting of the Hop protein kinase and the Mrl transcription factor. In this model, the extracellular Upd ligand would activate the Drosophila JAK protein Hop. Hop would in turn phosphorylate the D-STAT homolog Mrl, which would then enter the nucleus and activate Sxl-Pe. That the Mrl protein is critical for the activity of Sxl-Pe_GOF is demonstrated by the dramatic reduction in beta-galactosidase expression seen in both 1X/2A and 2X/2A embryos derived from homozygous mrl^- germline clones (Jinks, 2000).

The 72 bp fragment has a sequence that closely matches the consensus D-STAT-binding site. Hence, a plausible hypothesis is that Sxl-Pe_GOF is activated in 1X and 2X embryos by the binding of multiple Mrl proteins to the reiterated STAT sites in the multimerized fragment. Since there are also potential target sites for Runt in the 72 bp fragment, it is possible that Runt and Mrl collaborate in promoter activation. There are precedents in mammals for synergistic interactions between STAT and other transcription factors. Although a definitive answer will require further study, it is interesting that Sxl-Pe_GOF is not activated in male embryos in the dorsal crescent region of the head where upd but not run is expressed (Jinks, 2000).

Since Sxl-Pe_GOF has regulatory properties not seen in other Sxl-Pe promoter constructs, an obvious question is whether the JAK/STAT signaling pathway is a part of the normal X/A counting system. Several lines of evidence suggest that it is. (1) Genetic studies indicate that the upd gene is an X chromosome-counting element. Deletions that remove upd show female lethal interactions with mutations in the numerator genes sis-a and sis-b, and with Sxl. (2) As would be expected for an X chromosome-counting element, deletion of upd in females heterozygous for either sis-a or sis-b compromises the activity of wild-type Sxl-Pe reporter constructs. (3) The gain-of-function hop^Tum allele enhances the activity of the Sxl-Pe promoter in 2X/2A embryos. Moreover, consistent with the idea that a target site for the JAK/STAT signaling pathway is contained in the multimerized 72 bp fragment, the minimal Sxl-Pe_0.4kb promoter (from which the 72 bp fragment is derived) is activated by the hop^Tum-1 mutation. (4) The Sxl autoregulatory feedback loop is not properly turned on in 2X/2A embryos when the maternally derived mrl gene product is absent. The observed defects in SXL protein expression are regional and for the most part overlap with the domain in which the JAK/STAT signaling pathway would be activated by upd expression. (5) The failure to properly activate the Sxl autoregulatory feedback loop in the absence of maternal mrl appears to be due to a marked reduction in Sxl-Pe activity. For the full-length promoter construct, Sxl-Pe_3.0kb, beta-galactosidase expression is almost completely eliminated except in the very anterior of the embryo. In this context, it should be noted that Sxl-Pe contains two consensus STAT/Mrl-binding sites, in addition to the one found in the minimal 0.4 kb promoter. Conceivably these two upstream sites could provide additional targets for Mrl binding and promoter activation in vivo (Jinks, 2000).

The gene encoding the JAK/STAT ligand, upd, is required in the zygote to activate Sxl-Pe. Hence, like other numerators, it is the dose of the upd gene product produced in 1X and 2X embryos that is critical to the X chromosome-counting mechanism. The JAK kinase, hop, and the STAT transcription factor, mrl, have a different function in the counting process. These experiments show that the mrl gene is required in the mother's germline, not in the zygote. The available evidence suggests that this is also true for the X-linked hop gene. Since the products of these two genes would be deposited in constant amounts in the egg during oogenesis, they correspond to signal transduction elements like da. While the findings indicate that the JAK/STAT pathway plays an important role in the choice of sexual identity, the effects of mutations in the pathway do not seem to be as great as those observed for mutations in other components of the X/A counting system. For example, mutations that disrupt the maternal deposition of DA essentially eliminate both Sxl-Pe activity and SXL protein expression in female embryos. By contrast, when maternal mrl is removed, Sxl-Pe is not completely turned off, and SXL protein expression can still be detected, particularly in the termini. This suggests that the JAK/STAT pathway plays a secondary rather than a primary role in X chromosome counting (Jinks, 2000).

It is now clear that transcription factors involved in many different aspects of development, from segmentation to neurogenesis, have been coopted by the sex determination system in Drosophila. These genes generally have cell-autonomous activities and, consequently, are readily adaptable to a process that requires counting the number of chromosomes in each nucleus. Hence, it is somewhat surprising that a JAK/STAT signaling pathway, which depends upon the production and reception of an extracellular ligand, has also been incorporated into the counting system. Moreover, the apparent ligand, upd, corresponds to the X chromosome-counting element. Since Upd is secreted, it could potentially influence the counting process not only in the nucleus that produced the protein to begin with but also in adjacent nuclei. Supporting this possibility, it has been found that upd mutant cells can generate a normal pattern when adjacent to wild-type cells. Except under special circumstances (e.g., in gynandermorphs where 1X and 2X cells are in close proximity), counting elements that function nonautonomously need not have detrimental consequences and might even offer some advantages. For example, the signaling cascade may respond in a nonlinear fashion to variations in the dose of the ligand. In this case, the JAK/STAT pathway may provide a mechanism for magnifying the initial 2-fold difference in the amount of ligand produced in 1X/2A versus 2X/2A nuclei. In addition, signaling between adjacent nuclei might compensate for stochastic differences in numerator expression and might further amplify the signal by a relay mechanism (Jinks, 2000).

Metazoans use diverse and rapidly evolving mechanisms to determine sex. In Drosophila an X-chromosome-counting mechanism determines the sex of an individual by regulating the master switch gene, Sex-lethal (Sxl). The X-chromosome dose is communicated to Sxl by a set of X-linked signal elements (XSEs), which activate transcription of Sxl through its 'establishment' promoter, Sxl_Pe. A new XSE called sisterlessC (sisC) is described whose mode of action differs from that of previously characterized XSEs, all of which encode transcription factors that activate Sxl _Pe directly. In contrast, sisC encodes a secreted ligand for the Drosophila Janus kinase (JAK) and 'signal transducer and activator of transcription' (STAT) signal transduction pathway and is allelic to outstretched (os, also called unpaired). sisC works indirectly on Sxl through this signaling pathway because mutations in sisC or in the genes encoding Drosophila JAK or STAT reduce expression of Sxl_Pe similarly. The involvement of os in sex determination confirms that secreted ligands can function in cell-autonomous processes. Unlike sex signals for other organisms, sisC has acquired its sex-specific function while maintaining non-sex-specific roles in development, a characteristic that it shares with all other Drosophila XSEs (Sefton, 2000).

The two copies of XSEs present in XX individuals in Drosophila specify female development by transiently activating Sxl_Pe in the young embryo. A positive autoregulatory feedback loop acting on RNA splicing keeps Sxl active in females thereafter. Male development ensues in XY individuals because their single set of XSEs is insufficient to activate Sxl_Pe. Because Sxl controls the vital process of X-chromosome dosage compensation as well as sex determination, sexually inappropriate expression of Sxl is lethal. For example, simultaneous duplication of sisA and sisB kills males, as a female dose of these two XSEs in males causes Sxl to be expressed in its female mode, thereby reducing X-linked gene expression (Sefton, 2000).

Because XSEs act additively, males that would be killed by an excess dose of one group of XSEs can be rescued by compensating mutations that reduce the dose of other XSEs. A genetic screen based on this principle of additivity has generated five new mutations that define a new XSE, sisC. The first four sisC alleles recovered, including an apparent null, sisC¹, have no phenotype by themselves, even in trans to deficiencies of the region. In contrast, sisC ⁵, which is also null for sex-determination, exhibits phenotypes unrelated to sex: variably reduced viability (females more than males), female sterility and tergite defects. All mutations were mapped by recombination and deficiency analysis close to os based on their interactions with mutations in other XSEs (Sefton, 2000).

As is true for other Drosophila XSEs, eliminating sisC activity reduces expression of Sxl_Pe, but less than eliminating sisA or sisB. Like sisA and sisB mutations, but unlike runt mutations, sisC mutations affect Sxl_Pe throughout the embryo. Mutations in the Drosophila JAK/STAT signaling pathway reduce expression of the Sxl 'establishment' promoter Sxl _Pe throughout the embryo. In non-mutant situations, most Sxl_Pe:lacZ females stain darkly and comprise the expected 50% of the progeny. The other 50% are males whose light staining matches that of embryos lacking P {Sxl_Pe:lacZ}. Most Df(1)os/os^- females stain lighter than os ⁺ female controls but darker than males, showing that os ^- generally reduces but does not eliminate Sxl_Pe expression. The reduction is not always uniform across the embryo. Any region could be affected, but anterior expression seemed to be reduced the least. The range of effects is considerable: fewer than 50% of the mutant embryos stained above background; some mutant females may not have expressed Sxl_Pe at all, but Sxl _Pe expression in a few others matched that of their os ⁺ sisters. In contrast to the Df(1)os/os^upd females, Df(1)os/os^sisC-1 females are fully viable, but they show a comparable reduction in Sxl_Pe expression. This observation confirms that os^sisC-1 is near null with respect to sex determination, but still supports normal development (Sefton, 2000).

If os acts on Sxl_Pe indirectly through effects on Drosophila JAK (encoded by hopscotch [hop]) and on Drosophila STAT (encoded by Stat92E), then the effect on Sxl _Pe of eliminating either hop or Stat92E should be the same as eliminating os. This prediction was confirmed. Because only maternal rather than zygotic hop and Stat92E are likely to be relevant at the very early embryonic stage when Sxl_Pe is activated, the maternal contribution of these two genes was eliminated by inducing homozygous mutant germline clones in mothers heterozygous for null alleles. Expression of Sxl_Pe:lacZ in these experimentals was compared with that for control embryos derived from hop^-/+ and Stat92E^-/+ germ cells. Loss of maternal hop⁺ does not eliminate Sxl _Pe expression, but expression is substantially reduced: although 49% of the experimental embryos expressed Sxl_Pe:lacZ , essentially identical to the 50% figure for the controls, 32% of the experimental embryos were in the intermediate staining class compared with only 6% for the controls. The reduction was generally more uniform across the embryos than in the os experiment. Similar results were seen for Stat92E. Sixteen per cent of controls stained in the intermediate range, compared with 45% for the experimentals; thus, Sxl_Pe expression was clearly reduced. Curiously, the fraction of experimental embryos staining above background is greater than 50%, suggesting that although loss of maternal Stat92E decreases Sxl_Pe expression in females, it might also increase Sxl_Pe expression in males. Alternatively, this increase might be due to effects on the lacZ enhancer trap present in Stat92E⁶³⁴⁶. The observation that Drosophila STAT is a regulator of Sxl_Pe is consistent with the finding of STAT binding sites (TTCNNNGAA) 253, 393 and 428 bp upstream of the Sxl_Pe transcription start site. The tandem arrangement of these sites in Sxl would facilitate the kind of cooperative binding of STAT dimers shown to be important in some systems (Sefton, 2000).

With the discovery of sisC, the collection of fly XSEs may be nearly complete. The impression given by this collection is that Drosophila relies on biochemically diverse proteins to assess X-chromosome dose, but they all act on Sxl at the level of transcription. In contrast, the XSEs of Caenorhabditis elegans include both transcriptional and post-transcriptional regulators of their target, xol-1. Characterization of sisC reveals that both C. elegans and Drosophila XSEs seem to include proteins that work extracellularly (Sefton, 2000).

For Drosophila flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless proteins are known to form heterodimers that bind E-box sites and activate transcription. SISB-Da binding at Sxl was examined by using footprinting and gel mobility shift assays and SISB-Da was found to bind numerous clustered sites in the establishment promoter Sxl_Pe. Surprisingly, most SISB-Da sites at SxlPe differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. The noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for Sxl_Pe activity in embryos. Together with deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the Sxl_Pe switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements (Yang, 2001).

Deletion analysis has suggested that two subsegments within the 1.4-kb promoter account for most Sxl_Pe enhancer activity. An upstream segment (-1.4 to -0.8 kb) contributes to the strength of the promoter but is not essential for sex specificity. A proximal segment, including the start site and 390 upstream base pairs, drives a low-level, nonuniform female-specific expression. The sequence conservation between Sxl_Pe in D. melanogaster and Drosophila subobscura correlates well with the functional analysis. There is extensive sequence identity in the proximal region, with more limited matches in the distal segment, and no detectable similarity in the similarly sized central spacer segment. Within the proximal 390 bp, the sequences of all six B/Da binding sites are perfectly conserved. In the distal region, E-box sites 7 and 8 are conserved. Interestingly, while the sequence of site 9 is not conserved, another low-affinity B/Da site, CAGCTTG, is present in the equivalent position in D. subobscura (Yang, 2001 and references therein).

A critical question for sex determination is as follows: how can Sxl_Pe sense the twofold difference in male and female SIS and Runt concentrations and translate that into a strong all-or-nothing response? At some level, Sxl_Pe expression must be related to sex-specific differences in binding site occupancy. This is true whether dose sensitivity arises from cooperative DNA binding, competition with negative regulators, or from the sum of multiple independent interactions between the sex signal elements and the transcription machinery. It appears that sex-specific control of Sxl_Pe occurs largely through the activity of two regulatory regions: a central segment located between 1.4 and -0.8 kb, responsible primarily for promoter strength, and a proximal element, -390 to +44 bp, largely responsible for sex specificity. While these regions appear most important, sequences beyond -1.4 kb also contribute to the promoter, as inferred from the stronger lacZ expression from larger promoter fusions and by the ability of upstream sequences to partially substitute for the loss of the central -1.4 to -0.8 kb region (Yang, 2001).

The 10 B/Da sites identified in vitro are located in the central and proximal promoter elements. In addition, the sequence predicts 11 likely B/Da binding sites of high or moderate binding affinity located in the distal region between -1.6 and -3.7 kb, raising the possibility that there may be 21 or more B/Da sites in the functional Sxl_Pe region. Given a 39% GC content, random sequence would predict only 2.7 matches to the B/Da consensus at Sxl_Pe, suggesting that many of these predicted sites are functional binding sequences. Overall, there is a striking positional gradient of predicted binding affinities of the B/Da sites, with the moderate-affinity sites clustered proximally and the highest-affinity sites positioned distally. The asymmetric distribution of high- and moderate-affinity sites hints that the distal sites may be occupied at both high and low B/Da concentrations, with full occupancy of the proximal sites occurring only in XX embryos. This suggests a model in which the on or off response of Sxl_Pe to X-chromosome dose occurs primarily within the proximal X-counting region (XCR), with the distal segments providing an augmentation function that enhances transcription only when the female-specific XCR complex forms. It is unlikely that the distal high-affinity sites titrate B/Da from the XCR in males, because B/Da is in enormous excess over the Sxl binding sites (Yang, 2001).

A shared enhancer controls a temporal switch between promoters during Drosophila primary sex determination

Sex-lethal (Sxl), the master regulatory gene of Drosophila somatic sex determination, is stably maintained in an on or an off state by autoregulatory control of Sxl premRNA processing. Establishment of the correct Sxl splicing pattern requires the coordinate regulation of two Sxl promoters. The first of these promoters, SxlPe, responds to the female dose of two X chromosomes to produce a pulse of Sxl protein that acts on the premRNA products from the second promoter, SxlPm, to establish the splicing loop. SxlPm is active in both sexes throughout most of development, but nothing is known about how SxlPm is expressed during the transition from X signal assessment to maintenance splicing. This study found that SxlPm is activated earlier in females than in males in a range of Drosophila species, and that its expression overlaps briefly with that of SxlPe during the syncytial blastoderm stage. Activation of SxlPm depends on the scute, daughterless, and runt transcription factors, which communicate X chromosome dose to SxlPe, but is independent of the X signal element sisA and the maternal co-repressor groucho. DNA sequences regulating the response of SxlPe to the X chromosome dose also control the sex-differential response of SxlPm. It is proposed that co-expression of Sxl protein and its premRNA substrate facilitates the transition from transcriptional to splicing control, and that delayed activation of SxlPm in males buffers against the inappropriate activation of Sxl by fluctuations in the strength of the X chromosome signal (González, 2008).

The Drosophila sex determination pathway elegantly illustrates the use of premRNA splicing control in development; however, the establishment of sex-specific splicing ultimately depends on the coordinated activities of two promoters for the master regulatory gene Sxl. This work shows that the switch from the initial assessment of X chromosome dose at SxlPe to the stable autoregulatory control of Sxl premRNA splicing exploits an unexpected level of transcriptional control of the Sxl maintenance promoter. Contrary to the prevailing view, SxlPm responds to the X chromosome dose, and it does so by sharing common X-signal elements and a common enhancer with SxlPe. The switch between Sxl promoters thus serves as a tractable model for exploring the logical circuitry and molecular mechanisms that control the fidelity of developmental switches and coordinate the uses of multiple promoters for a single gene (González, 2008).

A priori, a female embryo must do two things to establish and then remember its sex: It must produce a pulse of Sxl protein by transiently activating SxlPe in response to the XX signal, and it must activate SxlPm, so that its transcripts can be spliced to produce yet more Sxl protein. A male embryo needs only to keep SxlPe off so that no Sxl protein is present when SxlPm is active. The system would seem to impose no requirement for sexually dimorphic expression from SxlPm or even for a temporal overlap in transcription from the two promoters, yet both features are conserved across the breadth of Drosophila species. It is suggested that the resolution of this paradox lies in recognizing that the transition to stable autoregulatory Sxl splicing requires the presence of substantial amounts of Sxl protein rather than being driven by trace quantities of Sxl protein. Given this, it is proposed that overlapping expression from the two promoters ensures that XX cells rapidly engage autoregulatory Sxl splicing, whereas the delayed activation of SxlPm in XY cells buffers against improper Sxl activation due to random variations in regulatory protein concentrations. In effect, it is suggested that robustness is conferred on the system by rapid reinforcement of correct decisions. In XX embryos, strong induction of SxlPe, coupled with early activation of SxlPm, ensures that high levels of Sxl protein and its premRNA substrate are present during the transition to splicing control. In XY embryos, chance fluctuations in XSE or inhibitor concentrations that caused low-level activation of SxlPe would not persist to activate SxlPm, thus preventing amplification of rare mistakes into the fully on state. A logically similar, two-target control process operates in the primary sex determination of Caenorhabditis elegans. There, four XSE proteins exert primary control of the master regulator xol-1 at the level of transcription, and a fifth XSE acts posttranscriptionally to ensure the fidelity of X chromosome counting. The inclusion of multiple regulatory steps may prove to be a general mechanism for conferring robustness on dose-sensitive regulatory switches (González, 2008).

SxlPm appears to be equally active in both sexes after the onset of gastrulation. Before that time, SxlPm is expressed in a graded fashion, becoming active earlier and being expressed more strongly in XX embryos than in XY embryos. Sequences governing the early sexually dimorphic expression of SxlPm are included in the same 1.4-kb DNA segment that controls the on-or-off regulation of SxlPe. Importantly, the 1.4-kb region must work as an enhancer for SxlPm rather than exert an indirect effect in cis via activation of SxlPe, because deletion of the SxlPe core promoter has no effect on SxlPm activity. This, combined with the involvement of the XSEs scute and runt in SxlPm regulation, suggests that SxlPm, like SxlPe, responds directly to the number of X chromosomes present in the embryo. However, the fact that neither loss of the strong XSE sisA nor loss of the potent maternal co-repressor Groucho affects SxlPm suggests that the mechanism of X-counting at SxlPm differs from that at SxlPe, despite their shared common cis- and trans-acting components. It is suspected that additional transcription factors contribute to both early SxlPm activation and the female/male differences in timing (González, 2008).

The existence of a shared regulatory region between SxlPe and SxlPm raises the question of how enhancer activity is directed to the correct promoter at the appropriate time. The 1.4-kb region regulates SxlPe from cycle 12 through early cycle 14, yet the enhancer does not lead to significant expression from SxlPm until cycle 14. Expression from the two promoters overlaps briefly before SxlPe is silenced and SxlPm fully controls Sxl transcription. Two general mechanisms are imagined that might explain how the enhancer can chose between the two promoters. First, an insulator situated between the enhancer and the upstream promoter might block the 1.4-kb region from interacting with SxlPm until the insulating protein is removed from the DNA or its activity is overcome by additional positive signals. Second, promoter choice could be dictated by differences in the transcription machinery at the two promoters or by a temporally restricted transcription factor that recruits the enhancer to one of the two Sxl promoters. The developmentally regulated competition between the promoters of the chicken e-globin and β-globin genes for their common enhancer provides a precedent for the latter mechanism. The rapid changeover from SxlPe to SxlPm coincides with the Drosophila maternal-to-zygotic transition, when expression of the zygotic genome begins in earnest and numerous early mRNAs and proteins are eliminated from the embryo. It would not be surprising if the rapid changes at Sxl were directly connected to more general regulatory events occurring during this dynamic period of development (González, 2008).

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. This study reports a similar phenotype for the telomeric partner of HP1, HOAP (Caravaggio), 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, Sxl_Pe, 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 (Sxl_Pm) in both sexes. HOAP mutants show inappropriate Sxl_Pe firing in males and the concomitant inappropriate splicing of Sxl_Pm-derived transcripts, while females show premature firing of Sxl_Pe. HP1 mutants, by contrast, display Sxl_Pm splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with Sxl_Pe sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl_Pe are severely compromised. These genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at Sxl_Pe (Li, 2011).

The canonical heterochromatin protein HP1 is most commonly associated with constitutive heterochromatin and gene repression. This study reports a critical role for it in regulating one of the earliest decisions in metazoan development, whether to embark on a female or male path of sexual differentiation and dosage compensation. The role of heterochromatin in mammalian dosage compensation has been recognized from early work on the mouse. Although Drosophila utilizes a different mechanism to equalize X-linked gene dose, through hyper-activation of the single male X chromosome via chromatin modification, this study provides the first evidence of a role for heterochromatin proteins in the early events of Drosophila sex determination. HP1, together with its telomere partner HOAP, influence the critical decision in sex determination - activation of Sxl_Pe, the Sxl establishment promoter (Li, 2011).

Reductions in HOAP preferentially compromise male viability. This was observed for two different cav mutant alleles and by reducing HOAP through RNAi. The presence of Sxl_Pm-derived transcripts that have been spliced in the female mode in cav mutant males suggested inappropriate Sxl activation to be responsible for this reduced viability. In situ data indicating inappropriate firing of Sxl_Pe in male embryos from cav²²⁴⁸ heterozygous parents support this view, as does the rescue of the cav²²⁴⁸ male viability defect by Sxl loss of function mutations. The more pronounced male lethality observed from reducing HOAP by RNAi expression driven by maternal, versus paternal, contribution of Actin5C GAL4 is consistent with such an early requirement for HOAP for male viability (Li, 2011).

Previous reports have shown that reducing HP1 by RNAi similarly reduces male viability preferentially. RT-PCR assays of Sxl_Pm transcripts in HP1 mutants, however, suggested a more complex scenario as incorrect sex specific transcripts were observed in both sexes. This pointed to an activation, as well as repressor, role for HP1. Consistent with an activation role, reduction of maternal HP1 severely compromised female viability when the dose of Sxl was also reduced in the progeny, and ChIP assays of embryos from this cross showed recruitment of RNAP II to Sxl_Pe to be impaired. This effect of reducing HP1 on female viability was strictly maternal, as was the antagonizing effect of simultaneously reducing maternal HOAP. Moreover, the partial rescue of the Su(var)205 maternal effect by the C-terminally truncated cav¹ allele, which produces a protein that is compromised for HP1-binding, points to an involvement of HP1 in the antagonizing activity of HOAP. Finally, ChIP assays show a dependence of HP1 on HOAP for its association with Sxl_Pe. Combined, these data indicate both antagonistic and cooperative roles for these heterochromatin proteins in regulating Sxl_Pe, whereby HOAP acts as a repressor and HP1 acts as both an activator and repressor. The reliance of HP1 on HOAP for recruitment to the promoter would suggest HOAP may also have a role in the activation function of HP1 at the promoter, although this was not readily apparent in the assays used in this study (Li, 2011).

Although the data clearly show maternal roles for HOAP and HP1 in regulating the activity of Sxl_Pe, for both HOAP and HP1, RNAi knockdown data indicate a substantial zygotic component in their effects on male viability. These zygotic effects, observed only in progeny carrying both an interference RNA transgene and a GAL4 driver transgene, suggest additional later sex-specific roles for both proteins. Such roles could be related to those observed for HP1 and SU(VAR)3-7 in male dosage compensation. Because the effect of reducing these proteins on the chromosomal distribution of DCC proteins is the opposite of those observed for males that are deficient for DCC proteins, as predicted to occur with inappropriate Sxl_Pe expression, the activities of heterochromatin proteins in dosage compensation appear to be distinct from the early roles of HP1 and HOAP at Sxl_Pe. In addition, there may be zygotic roles for heterochromatin proteins in sex-specific gene expression, as proposed for HP1 (Li, 2011).

Previous analysis of Sxl_Pe indicated that 400 bp immediately upstream of the promoter are sufficient for sex-specific regulation, but distal sequences, extending to -1700 bp, are required for wild type levels of expression, E-box binding sites for antagonistically acting bHLH proteins, which are encoded by zygotically expressed X-linked and autosomal signal elements (XSE and ASE) and direct an X counting mechanism, are distributed throughout both regions (Li, 2011).

Both HP1 and HOAP are enriched in the region proximal to Sxl_Pe which contains binding sites for both positive and negative E-box proteins. Within the Sxl_Pe promoter distal region, HOAP alone is enriched in two peaks where there is a striking relationship with E-box binding sites for positive factors, but those for negative factors appear essentially devoid of HOAP. HOAP may antagonize positive factors but permit negative factors to bind in the Sxl_Pe distal region, in an HP1-independent repressing role. Whereas loss of HOAP de-represses Sxl_Pe in males, the strength and uniformity of expression does not approach that in wild type females. This indicates continued influence from the X counting mechanism in cav mutant males. Sxl_Pe is also expressed prematurely in female embryos. This de-repression by reduced levels of maternal HOAP in both sexes indicates that HOAP is present at Sxl_Pe in both sexes of wild type embryos. However, whether the proximal and distal Sxl_Pe regions have the same or different compositions of HOAP and HP1 in the two sexes cannot be determined from ChIP assays, as the embryos are of mixed sexual identity (Li, 2011).

The interdependency of HOAP and HP1 for their binding to the Sxl_Pe proximal region, most notably the dependence of HP1 on HOAP, also indicates both proteins are in this region in, at least, wild type female embryos. In spite of this interdependency, the genetic data show HOAP repression antagonizes HP1 activation. HOAP repression appears to also be partly HP1-dependent; the mutant HOAP protein from the cav¹ allele which lacks HP1-binding also antagonizes HP1 activation. This combination of antagonistic and cooperative interactions suggests a model in which maternal HOAP and HP1 first cooperate to repress Sxl_Pe prior to its activation. The repressive structure formed by maternal HOAP and HP1 likely serves to reduce the sensitivity of Sxl_Pe to spurious fluctuations in zygotic XSE levels, ensuring it is only activated in females where an effective ratio of activating to repressing transcription factors exists. HP1 is retained at Sxl_Pe during its activation in females, where it presumably switches into an activation role. In early embryos constitutive heterochromatin proteins may be more appropriate for such regulation than the Polycomb Group of facultative heterochromatin proteins, as they would not be subject to cross regulatory signals from body plan specification pathways (Li, 2011).

How HP1 switches over to transcriptional activation mode in the Sxl_Pe proximal region is unclear. Changes in HP1 phosphorylation and/or association with other factors could alter its activity. Several XSE (X-linked element) binding sites are nearby, making them strong candidates. Presumably, this would only occur in females where the XSE dose surpasses a threshold and Sxl_Pe is activated (Li, 2011).

This report provides the most clearly defined role for HP1 in developmental control of a euchromatic gene in a metazoan species, and the first evidence of a bifunctional regulatory role for it in such a context. Prior reports describing HP1 in transcriptional activation have focused on it in the context of transcription elongation. ChIP data at Sxl_Pe, however, show a requirement of it for association of RNAP II with the promoter, more consistent with a role in transcription initiation. A role in initiation is also in keeping with the position of HP1 on the gene; very little HP1 is found elsewhere on the Sxl gene, even during the time of Sxl_Pe activity. This dependence of RNAP II association on HP1 is similar to what is observed in the accumulation of noncoding RNAs at S. pombe centromeric repeats and mating type locus. Nonetheless, it is possible that the loss of RNAP II at Sxl_Pe reflects reduced stability of all RNAP II isoforms as a consequence of an early defect in transcription elongation, rather than a defect in RNAP II recruitment to the promoter (Li, 2011).

Pausing of RNAP II in promoter proximal regions prior to activation has been observed in a high proportion of genes under developmental control in Drosophila embryos, and such pauses have also been implicated in regulation of alternative splicing. While Sxl_Pm appears to have the features of a promoter with paused RNAP II in a genome wide RNAP II ChIP study of 0-4 hr embryos, RNAP II was absent from Sxl_Pe. It is likely that the collection window for this study did not precisely coincide with the time of Sxl_Pe activity. A more narrowly timed collection indicates paused RNAP II at Sxl_Pe, suggesting that, like Sxl_Pm, it is a pre-loaded promoter. A preloaded Sxl_Pe also readily explains how generalized up-regulation of phosphorylation of the RNAP II CTD by the loss of Nanos, causes Sxl_Pe activation in males with an unchanged X:A ratio (Li, 2011).

Finally, the dominant negative activity of the cav²²⁴⁸ allele suggests a role for the partially deleted SRY-like HMG box in HOAP association with Sxl_Pe. ChIP data show HOAP association with the Sxl_Pe proximal region is required for HP1 association. This proposed role for the HMG box of HOAP in Sxl_Pe regulation is of particular interest with regards to a recent report linking HP1 and KAP-1 (TIF1β) to SRY-dependent repression of testis-specific genes in the ovary. Because mammalian sex determination is inextricably linked to gonad sex determination, SRY and HOAP each appear to constitute early decision points in their respective sex determination pathways. There are, perhaps, unexpected parallels between these divergent pathways (Li, 2011).

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